UNIVERSITY OF CALGARY

UNIVERSITY OF CALGARY
Targeting Selective Receptor Tyrosine Kinases in Refractory Embryonal Tumors of Childhood
by
Anjali Singh
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
DEAPRTMENT OF MEDICAL SCIENCES
CALGARY, ALBERTA
MARCH, 2016
© Anjali Singh 2016
Abstract
Embryonal tumors are a collection of biologically heterogeneous malignancies and the
exact cellular origin of these tumors is not known. Neuroblastoma (NB) and atypical
teratoid/rhabdoid tumor (AT/RT) are highly malignant tumors of embryonal origin that primarily
affect infants and young children. Neuroblastoma is the most common type of extra cranial solid
tumor in children. In the case of AT/RT, the survival rate of children affected by this disease is
the lowest when compared to all embryonal tumors. Despite intensifying multimodal treatments,
children affected with refractory AT/RT and NB have unacceptably high treatment failure and
mortality rates.
To improve the clinical outcome of these malignancies, it is important to identify the key
molecules and cellular pathways responsible for tumor progression, survival and invasion. Many
childhood cancers have high activation levels of selective receptor tyrosine kinase signaling
pathways. Activation of these signaling pathways promotes cell proliferation, differentiation and
cell survival. Therefore, receptor tyrosine kinases (RTKs) have become attractive therapeutic
targets and the use of small molecule kinase inhibitors to block their signal transduction
functions has led to the discovery of a number of novel therapeutics agents.
This research presents the relevant background information on two pediatric neoplasms
that we have selected to study and aims to provide the rationale for the development of useful
new therapies for their treatment. Presented in details are the data with respect to the
establishment of a screening approach to identify effective therapeutic agents with information
on target validation and target modulation activities that can be utilized to design future clinical
trials for these cancers.
ii
Acknowledgements
I would like to acknowledge my supervisor and mentor, Dr. Aru Narendran, for providing
me the opportunity to pursue my doctoral studies under his guidance. His patience, support and
guidance have helped me tremendously during my doctoral study. He has provided me freedom
to try my own ideas in his laboratory and guided me to think independently. I would also like to
thank my committee members Dr. Ebba Kurz, Dr. Roman Krawetz and Dr. Nizar Bahlis for their
support and guidance throughout the program. Their valuable advice and suggestions were really
helpful during my PhD. I would like to thank the past and present members of Narendran lab, for
their scientific feedbacks during lab meetings and also, for making it fun to work in the lab. I
would like to thank our neighbor’s lab Dr. Ebba Kurz and Dr. Randal Johnston, for being helpful
and kind through out my PhD. I would like to acknowledge Dr. Narendran and Alberta Cancer
Foundation (ACF) for providing me financial support during my doctoral study.
Special thanks to Dr. Navneet Sharma and Sunita Sharma for providing me guidance,
support and being a family for me from past 10 years. I would like to thank Dr. Maneka
Perinpanayagam for her friendship and support during in and outside of the lab. I would like to
thank my friends for the support and encouragement and I look forward to continuing a special
bond will all of them.
Finally, I would like to thank my parents and family for their constant care,
encouragement and support throughout my life. I would like to thank my uncle Dr. Hari Singh
Nalwa for providing me guidance and support to pursue my dream. Without their love, support
and understanding, I would not have made it this far. I would like to thank my husband for taking
care of our son when I was not around. Last but not the least, my son Ahaan, who has been the
real joy of my life. He has been the shining light in my moments of darkness.
iii
Dedication
To my parents Mr. and Mrs. Jagmer and Satbiri Singh
and my son Ahaan Singh.
Without them none of my success would be possible.
iv
Table of Contents
Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Dedication .......................................................................................................................... iv Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... ix List of Figures and Illustrations ...........................................................................................x List of Symbols, Abbreviations and Nomenclature .......................................................... xii Material & Methods ......................................................................................................... xiii Units of Measure .............................................................................................................. xiv CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Paediatric Cancer and Incidence ................................................................................1 1.2 Embryonal tumors of childhood ................................................................................5 1.2.1 Neuroblastoma ...................................................................................................6 1.2.2 Atypical teratoid/rhabdoid tumor ......................................................................7 1.3 Clinical aspects of NB and AT/RT ............................................................................8 1.3.1 Age at diagnosis ................................................................................................9 1.3.2 Tumor stage .......................................................................................................9 1.3.3 Biological aspects ............................................................................................14 1.3.3.1 Neuroblastoma .......................................................................................14 1.3.3.2 Atypical teratoid/rhabdoid tumor...........................................................16 1.3.4 Therapeutic aspects .........................................................................................18 1.3.4.1 Neuroblastoma .......................................................................................18 1.3.4.2 Atypical teratoid/rhabdoid tumor...........................................................19 1.4 Role of Receptor Tyrosine Kinases in cancer ..........................................................21 1.4.1 EGFR and its role in cancer.............................................................................22 1.4.1.1 Targeting EGFR/HER-2 ........................................................................28 1.4.2 IGF-1R and its role in Cancer .........................................................................29 1.4.2.1 Targeting IGF-1R ..................................................................................32 1.4.3 Role of serine/threonine kinases in cancer ......................................................36 1.4.3.1 The PI3K pathway .................................................................................37 1.4.3.2 The MAPK pathway ..............................................................................38 1.4.4 Lapatinib as an EGFR/HER-2 inhibitor ..........................................................41 1.4.5 Ponatinib as an IGF-1R inhibitor ....................................................................42 1.4.6 Cobimetinib as an MEK1/2 inhibitor ..............................................................43 1.4.7 Cancer cell migration and metastasis ..............................................................44 1.5 Hypothesis ...............................................................................................................46 1.5.1 Specific Aims ..................................................................................................46 CHAPTER TWO: MATERIAL AND METHODS ..........................................................47 2.1 Cell Culture ..............................................................................................................47 2.1.1 Freezing and thawing of cells ..........................................................................47 2.1.2 Passaging of cells ............................................................................................48 2.2 Small molecule kinase inhibitors and chemotherapeutic agents .............................50 2.3 Cell viability assays .................................................................................................50 v
2.3.1 Single drug cell cytotoxicity assay ..................................................................50 2.3.2 Drug combination studies ................................................................................50 2.3.3 Automated inverted microscopy......................................................................51 2.4 Preparation of cellular extracts ................................................................................52 2.5 Immunoblotting .......................................................................................................52 2.6 Antibody Array ........................................................................................................55 2.7 Cell Migration Assay ...............................................................................................56 2.8 Immunocytochemical Assay ....................................................................................56 2.9 Tumor Xenograft Study ...........................................................................................57 2.9.1 Generation of tumor cells stably expressing firefly luciferase and eGFP .......57 2.9.2 In vivo real-time monitoring of tumor growth (bioluminescence imaging)....57 CHAPTER THREE: PROFILING PATHWAY-SPECIFIC NOVEL THERAPEUTICS IN
PRECLINICAL ASSESSMENT FOR CENTRAL NERVOUS SYSTEM ATYPICAL
TERATOID RHABDOID TUMORS (CNS AT/RT): FAVORABLE ACTIVITY OF
TARGETING EGFR- ERBB2 SIGNALING WITH LAPATINIB .........................59 3.1 Abstract ....................................................................................................................60 3.2 Introduction ..............................................................................................................61 3.3 Material and Methods ..............................................................................................63 3.3.1 Cell lines and cell culture ................................................................................63 3.3.2 Antineoplasic agents ........................................................................................64 3.3.3 Cell growth inhibition assays ..........................................................................64 3.3.4 Intracellular signaling studies ..........................................................................65 3.3.5 In vitro cell migration assay ("scratch" test) ...................................................65 3.3.6 Tumor xenograft studies ..................................................................................66 3.3.6.1 Generation of BT16 cells stably expressing firefly luciferase and eGFP66 3.3.6.2 In vivo real-time monitoring of tumor growth (bioluminescence imaging)
.................................................................................................................66 3.4 Results ......................................................................................................................67 3.4.1 Cytotoxicity profiling of a panel of novel therapeutic agents against AT/RT cells
..........................................................................................................................67 3.4.2 Activity of lapatinib against AT/RT cell lines ................................................74 3.4.3 Target validation of lapatinib in AT/RT cells .................................................76 3.4.4 Target modulation effects of lapatinib on AT/RT cells...................................80 3.4.5 Synergistic activity of lapatinib with IGF-IR inhibitors..................................82 3.4.6 Lapatinib inhibits BT16 cell migration in vitro ...............................................85 3.4.7 In vivo activity of lapatinib .............................................................................88 3.5 Discussion ................................................................................................................90 3.6 Acknowledgment .....................................................................................................97 3.7 References ................................................................................................................98 CHAPTER FOUR: IN VITRO SENSITIVITY PROFILING OF NEUROBLASTOMA
CELLS AGAINST A COMPREHENSIVE SMALL MOLECULE KINASE
INHIBITOR LIBRARY TO IDENTIFY AGENTS FOR FUTURE THERAPEUTIC
STUDIES ..................................................................................................................99 4.1 Abstract ..................................................................................................................100 4.2 Introduction ............................................................................................................101 vi
4.3 Material and Methods ............................................................................................103 4.3.1 Cell lines and cell culture ..............................................................................103 4.3.2 Small molecule kinase inhibitors...................................................................103 4.3.3 In vitro cytotoxicity assays ............................................................................104 4.3.4 Human antibody array ...................................................................................104 4.3.5 Western blot analysis for protein and phosphorylated protein detection ......105 4.3.6 In vitro cell migration assay (“scratch” test) .................................................105 4.4 Results ....................................................................................................................106 4.4.1 cytotoxicity profiles of small molecule kinase inhibitors against NB cell lines106 4.4.2 Cytotoxicity profiling of small molecule Bcr-Abl kinase inhibitors against NB
cells ................................................................................................................114 4.4.3 Inhibition of NB cell proliferation by ponatinib............................................114 4.4.4 Target validation of ponatinib in NB cells ....................................................117 4.4.5 Evaluation of ponatinib induced inactivation of IGF-1R and down-stream targets
in NB cells......................................................................................................117 4.4.6 Effect of ponatinib on Src phosphorylation...................................................121 4.4.7 Exogenous IGF-1 overcomes the effect of ponatinib on Src dephosphorylation121 4.4.8 Ponatinib treatment leads to activation of apoptosis in NB cells ..................124 4.4.9 Effect of ponatinib on NB cell migration ......................................................124 4.5 Discussion ..............................................................................................................129 4.6 Acknowledgement .................................................................................................136 4.7 References:.............................................................................................................136 CHAPTER FIVE: TARGETED INHIBITION OF MEK1 BY COBIMETINIB LEADS TO
DIFFERENTIATION AND APOPTOSIS IN NEUROBLASTOMA CELLS. .....137 5.1 Abstract ..................................................................................................................138 5.2 Background ............................................................................................................139 5.3 Materials and Methods...........................................................................................141 5.3.1 Cell lines and cell culture ..............................................................................141 5.3.2 Drug cytotoxicity assays................................................................................141 5.3.3 Human phospho-kinase antibody array .........................................................142 5.3.4 Western blot analysis for protein and phosphoprotein detection ..................142 5.3.5 Annexin V staining for apoptosis ..................................................................143 5.3.6 Treatment with cis-RA ..................................................................................144 5.3.7 Immunocytochemical detection of differentiation markers ..........................144 5.3.8 Statistical analysis .........................................................................................144 5.4 Results ....................................................................................................................145 5.5 Discussion ..............................................................................................................163 5.6 Conclusion .............................................................................................................167 5.7 Acknowledgements ................................................................................................167 5.8 References ..............................................................................................................168 CHAPTER SIX: GENERAL DISCUSSION AND INSIGHTS......................................169 6.1 Summary ................................................................................................................169 6.2 Drug screening to identify potential targets for therapeutics for AT/RT ..............170 6.3 Drug screening to identify potential targets for therapeutics for NB ....................173 6.4 Inhibiting MAP kinase signaling in NB ................................................................177 vii
6.5 Drug combination studies ......................................................................................179 6.6 Future directions ....................................................................................................182 6.7 Conclusion .............................................................................................................185 APPENDIX A: EXAMINATION OF THE EFFECTS OF COBIMETINIB ON RAS
MUTATED CELL LINE ........................................................................................187 REFERENCES ................................................................................................................196 viii
List of Tables
Table 1-1. International NB staging system (INSS) ..................................................................... 12 Table 1-2. International NB Risk Group Staging System (INRGSS) ........................................... 13 Table 1-3. Clinical and biological aspects of NB and AT/RT ...................................................... 17 Table 2-1. Characteristics of NB cell lines investigated in this study .......................................... 49 Table 2-2. Summary of primary antibodies used in immunoblotting studies ............................... 54 Table 3-1. Cytotoxicity profiling of a panel of targeted agents against three AT/RT cell lines. .. 71 Table 3-2. Activity of combined IGF-1R inhibition and lapatinib against AT/RT cells. ............. 84 Table 4-1. Summary of cytotoxicity profiles of small molecule kinase inhibitors against six
NB cell lines. ....................................................................................................................... 110 Table 4-2. Cytotoxicity profiling of Bcr-Abl targeting agents against five neuroblastoma cell
lines. .................................................................................................................................... 115 ix
List of Figures and Illustrations
Figure 1-1. The multistep process involved in cancer development .............................................. 4 Figure 1-2. EGFR pathway and its role in cancer ......................................................................... 26 Figure 1-3. IGF-1R and its role in cancer ..................................................................................... 34 Figure 3-1. Differential sensitivity of AT/RT cell lines against the drug panel. .......................... 73 Figure 3-2. In vitro cytotoxicity of lapatinib against AT/RT cell lines. ....................................... 75 Figure 3-3. Identification of potential targets of lapatinib activity on AT/RT cells. .................... 77 Figure 3-4. Expression and activity of EGFR in AT/RT cells. ..................................................... 79 Figure 3-5. Intracellular signaling and lapatinib activity. ............................................................. 81 Figure 3-6. Drug combination study of Lapatinib with AEW-541 in AT/RT cell lines. .............. 83 Figure 3-7. In vitro cell migration assay. ...................................................................................... 87 Figure 3-8. Lapatinib inhibits the tumor activity in vivo. ............................................................. 89 Figure 4-1. Graphical representation of the IC50 values of small molecule kinase inhibitors
against IMR-5, IMR-32 and SHEP NB cell lines presented in Table 4.1. ......................... 113 Figure 4-2. In vitro cytotoxicity of ponatinib against NB cell lines. .......................................... 116 Figure 4-3. Identification of potential targets of ponatinib activity on NB cells. ....................... 118 Figure 4-4. Inhibition of IGF-1R and its downstream pathways by ponatinb. ........................... 120 Figure 4-5. Inhibition of Src phosphorylation by ponatinib in NB cells. ................................... 122 Figure 4-6. Ponatinib blocks the ligand dependent activation of IGF-1R activity. .................... 123 Figure 4-7. Effect of ponatinib on the markers of apoptosis. ..................................................... 126 Figure 4-8. In vitro cell migration assay. .................................................................................... 128 Figure 5-1. Cobimetinib mediated cytotoxicity against NB cells. .............................................. 146 Figure 5-2. Constitutive phosphorylation status of MEK in NB cells. ....................................... 148 Figure 5-3. Identification of potential targets of cobimetinib activity on NB cells. ................... 150 Figure 5-4. Effect of cobimetinib on MAPK cascade pathway. ................................................. 152 x
Figure 5-5. PARP cleavage induced by cobimetinib. ................................................................. 153 Figure 5-6. The flow cytometric analysis of apoptosis in NB cells using FITC-annexin V and
PI double staining. .............................................................................................................. 154 Figure 5-7. Time course analysis of cobimetinib on MAPK cascade pathway. ......................... 157 Figure 5-8. Drug combination studies of cobimetinib with cis-RA in NB cell lines.................. 159 Figure 5-9. Analysis of cellular differentiation induced by cobimetinib, cis-RA or
combination of the two agents. ........................................................................................... 162 Figure 6-1. A detailed design of the experimental procedure used in this research. .................. 173 Figure 6-2. Targeting selective receptor tyrosine kinases in NB and AT/RT ............................ 186 Appendix A-1 In vitro cytotoxicity of cobimetinib against NB and RD cell lines..................... 188 Appendix A-2. Effect of cobimetinib on MAPK cascade pathway in RD cell line. .................. 189 Appendix A-3. In vitro cell migration assay in SHEP cell line. ................................................. 192 Appendix A-4. In vitro cell migration assay in RAS mutated RD cell line............................... 194 xi
List of Symbols, Abbreviations and Nomenclature
Symbol
AKT
ALK
ALL
AR
AT/RT
ATP
ATRA
BAD
BBB
BCL-2-BIM
BDNF
CML
CNS
EGF
EGFR/ErbB
ES
FBS
FLT3
FOXO3
FOXR1
GBM
GD2
GDP
GH
GPCRs
Grb2
GSK3α
GTP
HB-EGF
HCC
HER-2
HGFR
IGF-1R
IGFBPs
IGFs
INRGSS
INSS
JAK/STAT
Definition
Protein kinase B
Anaplastic lymphoma kinase
Acute lymphocytic leukemia
Amphiregulin
Atypical teratoid/rhabdoid tumor
Adenosine triphosphate
All trans-retinoic acid
BCL2-associated agonist of cell death
Blood–brain barrier
B-cell lymphoma 2-interacting mediator of cell
death
Brain-derived Neurotrophic Factor
Chronic myeloid leukemia
Central nervous system
Epidermal growth factor
Epidermal growth factor receptor
Ewing's sarcoma
Fetal bovine serum
Fms-related tyrosine kinase 3
Forkhead family of transcription factors
Forkhead box R1
Glioblastoma multiforme
Disialoganglioside
Guanosine diphosphate
Growth hormone
G protein coupled receptors
Growth factor receptor-bound protein 2
Glycogen synthase kinase 3α
Guanosine triphosphate
Heparin-binding EGF
Hepatocellular carcinoma
Human epidermal growth factor receptor 2
Hepatocyte growth factor receptor
Insulin-like growth factor 1 receptor
IGF binding proteins
Insulin-like growth factors
International NB risk group staging system
International NB staging system
Janus kinase/signal transducers and activators of
transcription
xii
mABs
MAPK
Mcl-1
MDM-2
mTOR
NB
NSCLC
ODC
PDGFR
PH
PI3K
PIP2
PIP3
PLK1
PNET
PTEN
PTPN11
RKIP
RTK
RTKIs
Shc
Sos
TGFα
TrkA
TrkB
VEGFR
WHO
Monoclonal antibodies
Mitogen-activated protein kinase
Myeloid cell leukemia 1
Mouse double minute 2 homolog
Mammalian target of rapamycin
Neuroblastoma
Non-small cell lung cancer
Ornithine decarboxylase
Platelet-derived growth factor receptor
Pleckstrin homology
Phosphoinositide 3-kinases
phosphatidylinositol 4,5 biphosphate
phosphatidylinositol-3,4,5 trisphosphate
Polo-like kinase 1
Primitive neuroectodermal Tumor
Phosphatase and tensin homolog deleted on
chromosome ten
Protein tyrosine phosphatase non-receptor 11
Raf kinase inhibitory protein
Receptor tyrosine kinase
Receptor tyrosine kinase inhibitors
Src-homology collagen protein
Son of sevenless
Transforming growth factor-α
Tropomyosin receptor kinase A
Tropomyosin receptor kinase B
Vascular endothelial growth factor receptor
World health organization
Material & Methods
BCA
BSA
CI
DMSO
ECL
EDTA
FBS
PBS
RIPA
ROI
SDS
Bicinchoninic Acid
Bovine serum albumin
Combination indices
Dimethyl sulfoxide
Enhanced chemiluminescence
Ethylenediaminetetraacetic acid
Fetal bovine serum
Phosphate buffered saline
Radioimmunoprecipitation assay
Region of interest
Sodium dodecyl sulfate
xiii
Units of Measure
%
°C
g
mg
µg
M
mM
µM
mL
µL
ng
V
v/v
Percent
Degrees Celsius
Gram
Milligram
Microgram
Molar (mol / L)
Millimolar
Micromolar
Millilitre
Microlitre
Nanogram
volt
mL solute per 100 mL solution
xiv
Chapter One: Introduction
1.1 Paediatric Cancer and Incidence
All cancers begin at a cellular level. In the cell, hundreds of genes intricately control the
process of cell division, proliferation, growth suppression and cell death to maintain normal
development. However, in cancer cells, these processes are deregulated mostly due to genetic
alterations (Lengauer, Kinzler and Vogelstein 1998). A tumor is caused by the abnormal
proliferation of cells and could be benign or malignant. A benign tumor remains confined to its
original location without invading adjacent tissues. A malignant tumor, however, is capable of
both invasion and metastasis (Cooper 2000).
Cancer cell growth is different from normal growth and it involves a multistep process in
which cells gradually become malignant through genetic alterations, which affect the tightly
controlled systems for growth. The development of a cancer is thought to occur in three stages:
initiation, promotion and progression (Figure 1). Genomic alterations occur during all three
stages and this enable cells to acquire the “hallmarks of cancer”, including (1) evasion of growth
suppressors, (2) sustaining proliferation signaling, (3) replicative immortality, (4) resisting cell
death, (5) inducing angiogenesis, and (6) acquiring the ability to invade and metastasize
(Hanahan and Weinberg 2000).
Cancer is one of the leading causes of death worldwide. An estimated 14.1 million new
cancer cases and 8.2 million cancer-related deaths occurr worldwide every year (Torre et al.
2015). Whereas most adult cancers are carcinomas, childhood cancers are histologically diverse.
Adult tumors are generally described according to the location in the body where they develop,
such as the breast, lung, colon or prostate. However, malignancies in children are generally
embryonal or sarcomatous in origin and named according to the type of cells in which they
1
develop, such as neuroblastoma (nervous system cells), retinoblastoma (retinal cells of the eye)
and nephroblastoma (kidney cells). The biology of childhood cancer differs from that in adults.
In adults, cancer generally occurs when normal cells accumulate genetic mutations and begin to
grow abnormally. However, children are usually diagnosed when the cancer cells have
accumulated fewer genetic abnormalities and metastasized to other organs. Childhood cancers
are often the result of alterations to the DNA that take place very early in life, sometimes even
before birth. The most common types of cancer diagnosed in children are leukemia, lymphomas,
brain and central nervous system (CNS) tumors, NB, retinoblastoma, Wilms tumor,
hepatoblastoma or hepatocellular carcinoma (HCC), osteosarcoma or Ewing's sarcoma (ES),
rhabdomyosarcoma, and germ-cell tumors (Stiller 2004).
The overall incidence for cancer in children aged 14 years and younger has increased at
an annual rate of 0.6% since 1975 (Siegel et al. 2012). It is estimated that 1 in 285 children will
be diagnosed with cancer before 20 years of age (MacDonald 2010). Incidence for childhood
cancer are highest among young children aged 0–4 years. Childhood cancer represents a
collection of cancer types with varying survival. Children with acute lymphocytic leukemia
(ALL), as well as for lymphomas and kidney cancer, are most likely to benefit from current
treatment protocols. Consistently curative treatment of other types of cancers such as brain
malignancies or CNS tumors remains elusive. Mortality rates for these types of tumors are
unchanged from 1996 to 2006 (Childhood cancer incidence and mortality in Canada 2015).
Cancer is the second leading cause of death in Canadian children (after accidents) and the
leading cause of death by disease past infancy (Childhood cancer incidence and mortality in
Canada 2015). Despite dramatic increases in five-year survival rates for pediatric malignancies
2
in general, the outcome for patients who relapse are poor. New therapies and treatment protocols
are urgently needed to improve the cure rate in these children.
3
Figure 1-1. The multistep process involved in cancer development
The first step, initiation, involves genetic alteration of a single cell leading to abnormal
proliferation. The promotion stage involves the selective clonal expansion of the initiated cell
through an increase in cell growth or a decrease in apoptosis. The third step, progression,
involves more genetic changes, which results in a change from the pre-neoplastic state to the
neoplastic state.
4
1.2 Embryonal tumors of childhood
Embryonal tumors are generally characterized by the malignant proliferation of cells of the
developing embryo. Such malignancies are encountered in children only and are often diagnosed
in the first few years of life. Embryonal tumors can be divided into two sub-groups: the CNS
embryonal tumors and non–CNS embryonal tumors (Tulla et al. 2015). The non–CNS
embryonal tumors include NB, ganglioneuroblastoma, Wilms tumor, retinoblastoma,
hepatoblastoma, pulmonary blastoma, and pleuropulmonary blastoma (Gatta et al. 2012). The
CNS embryonal tumor group, which accounts for approximately 20% of childhood CNS tumors,
includeS
medulloblastoma,
Atypical
teratoid/rhabdoid
tumor
(AT/RT),
primitive
neuroectodermal tumors [PNETs] and medulloepithelioma (Louis et al. 2007).
Wilms tumor, NB and retinoblastoma are the three common types of embryonal tumors
in children (Ward et al. 2014). NB alone accounts for approximately 22% of all cancers
diagnosed in the first year of life (Heck et al. 2009). AT/RT is a newly recognized tumor that
was previously misclassified with medulloblastoma and has a very high mortality rate in infants
and young children (Rorke, Packer and Biegel 1996). Details regarding the origin of these
embryonal neoplasms, and how their molecular details might relate to therapeutic response and
clinical outcome are still being debated.
The incidence and survival rates of embryonal tumors vary greatly between the
subgroups. The survival probability for retinoblastoma is greater than other tumors whereas
AT/RT has the worst survival rate (Tulla et al. 2015). In the case of NB, the survival probability
declines steeply for children under the age of one at diagnosis (Tulla et al. 2015). The prognosis
of advanced stage NB and AT/RT are still poor and more effective treatments are needed to
improve the overall survival.
5
1.2.1 Neuroblastoma
Neuroblastoma was originally described by Virchow in 1863 (Lonergan et al. 2002). This
is a malignant embryonal tumor of the sympathetic nervous system and is derived from primitive
neural crest cells. The neural crest forms in the third to fourth week of embryonal development
and some of these cells differentiate and migrate to create the sympathetic nervous system.
Nearly all childhood cancers of the sympathetic nervous system are NB. Although epidemiologic
studies have investigated environmental factors associated with NB, no strong or consistent risk
factors have been identified (Ferrís i Tortajada et al. 2005). A family history of NB is present in
1% to 2% of cases (Maris 2010).
NB is the third most common childhood cancer, accounting for 7% to 8% of cancer cases
among children younger than 15 years of age (Ward et al. 2014). This incidence is fairly uniform
throughout the world. NB is the most common cancer diagnosed during the first year of life and
the incidence is slightly higher in boys than girls (ratio 1.2:1) (Hallett and Traunecker 2012).
NB belongs to the malignant small round blue cell tumors, which is a descriptive
category of a number of childhood tumors that tend to occur in children with a similar
histological appearance of uniformly sized round cells, with an immense dark nuclei and scant
cytoplasm (Rajwanshi, Srinivas and Upasana 2009). Such tumors may arise anywhere along the
sympathetic nervous system, but most frequently occur in the adrenal gland (50% of tumors)
followed by abdomen (20%), or the sympathetic ganglia in the neck, thorax and pelvis (30%)
(Bjørge et al. 2008). Neuroblastoma commonly metastasizes to cortical bone, bone marrow, skin,
lymph nodes and the liver (Papaioannou and McHugh 2005). Stage IV NB carries an extremely
poor prognosis despite intensive multi-modality therapy and may acquire a substantially drug
resistant phenotype following exposure to chemotherapeutic agents (Keshelava, Seeger and
6
Reynolds 1997). The resistance progressively increases with the intensity of the therapy
delivered especially in stage IV (London et al. 2011).
Despite recent advances in treatment, including bone marrow transplantation, NB
remains a highly difficult to cure tumor, accounting for approximately 15% of cancer deaths in
children. As such, ongoing research and innovative clinical studies are being conducted to reduce
the morbidity and mortality in these children.
1.2.2 Atypical teratoid/rhabdoid tumor
The CNS AT/RT is an extremely rare and aggressive tumor of early childhood. Beckwith
and Palmer first coined the term ‘rhabdoid tumor’ to describe an aggressive variant of Wilms
tumors with rhabdomyosarcomatous features that was associated with an extremely poor
prognosis (Beckwith and Palmer 1978). The name ‘atypical teratoid/rhabdoid tumor’ exemplifies
the disparate mixtures of rhabdoid, primitive neuroepithelial, mesenchymally and/or epithelially
differentiated tumor cells. AT/RT was first defined as an entity by Rorke et al in 1995 (Rorke,
Packer and Biegel 1995). It is classified as a grade IV brain tumor by the World Health
Organization (WHO) as of the year 2000 and is estimated to account for approximately 10% of
all CNS tumors in children (Robson 2001).
AT/RT is the most common malignant CNS tumor in children below 1 year of age and
accounts for approximately 40%–50% of all embryonal CNS tumors in the first year of life
(Frühwald et al. 2016). AT/RT often feature several histopathological patterns similar to other
CNS embryonal tumors (Rorke, Packer and Biegel 1996). Apart from their histologically typical
appearance with sheets of rhabdoid cells, AT/RT can also display histological features of typical
PNETs. AT/RT has been misdiagnosed in the past as PNET because of such overlapping
7
histologic characteristics. AT/RT can be distinguished from PNET and other embryonal tumors
by the presence of rhabdoid cells and by specific immunohistochemistry (Ho et al. 2000).
Typical rhabdoid cells have an enlarged, vesicular, eccentric nucleus containing a prominent
nucleolus (Yang et al. 2014). Morphological features of these rhabdoid cells, however, are
variable; some may be small and spindle shaped with an ovoid nucleus, whereas others are
relatively large and have a nucleus with a wrinkled border and two nucleoli (Strother 2005).
These large nuclei often have an empty appearance. The cell body may be large and plump, or
spindle-shaped, with homogeneous appearance or prominent hyaline cytoplasmic inclusions.
More than 50% of AT/RT are found in the cerebellum or brain stem; however, they may appear
anywhere in the CNS (Strother 2005).
Like rhabdoid tumors of the kidney, CNS AT/RT are also associated with poor survival
when compared with other embryonal tumors. Children with AT/RT rarely respond to treatment
despite of the use of aggressive chemotherapy and/or radiotherapy, and median survival is
reported to be less than 10 months (Rorke, Packer and Biegel 1995; Tekautz et al. 2005). AT/RT
remains a therapeutically challenging tumor with a poor prognosis, and to date there is no
established standard treatment for children with AT/RT. Therefore, intensive research is required
to comprehend the biology of this polymorphic tumor so that more targeted therapies can be
developed.
1.3 Clinical aspects of NB and AT/RT
The most important clinical aspects that predict outcome are the stage of disease and the
age of the patient at diagnosis. Accurate staging is essential for prognostic stratification,
treatment planning and comparative analysis of clinical outcomes.
8
1.3.1 Age at diagnosis
The peak age for presentation for NB is 1–2 years with the median age at diagnosis of 22
months; most deaths occur within two years of diagnosis (Papaioannou and McHugh 2005).
About 81.5% of cases are diagnosed by the age of 4 years and another 15% by the age of 9 years
(Kembhavi et al. 2015). The outcome of infants who are less than 1 year of age at diagnosis is
considerably better than older patients. Patients older than 1 year of age typically have an
aggressive disease at the time of diagnosis, and their overall prognosis is poor (Park, Eggert and
Caron 2010).
The onset of AT/RT occurs from birth to adulthood, with the highest incidence in the first
two years of life (mean age at diagnosis, 17 months) (Burger et al. 1998). Although it occurs
primarily in young children, approximately 45 cases of AT/RT in adults have been reported so
far (Souki et al. 2014). Males appear to be affected slightly more frequently than females, with a
reported ratio of 3:2 to 2:1 (Burger et al. 1998). Children with AT/RT have a median survival of
9–17 months (Ginn and Gajjar 2012).
1.3.2 Tumor stage
The most important clinical variable in predicting a patient outcome is the stage of the
disease at diagnosis. Based on the localization of the primary tumor, lymph node involvement
and the pattern of metastasis, NB can be classified into different stages. The first staging system
was proposed in 1971 by Evans and colleagues and evolved into the International NB Staging
System (INSS) in the late 1980s with revisions in 1993 (Brodeur et al. 1993) (Table 1). In the
INSS classification system, stage 1 NB are localized tumors that can be completely removed by
surgery. Stage 2A and 2B are localized tumors with incomplete gross excision. In stage 2B,
9
tumors have begun to spread into nearby lymph nodes. Stage 3 tumors are unresectable and have
begun to spread into surrounding organs and structures, but not to distant areas of the body.
Stage 4 tumors show remote disease involving the skeleton, liver and other organs, and distant
lymph nodes. Stage 4S tumors have metastases restricted to liver, skin and/or bone marrow and
is limited to infants less than 1 year old.
A new staging system known as the International NB Risk Group Staging System
(INRGSS) was introduced in 2008 (Monclair et al. 2009). The INRGSS is based on imaging and
includes two stages of localized (L1 and L2) and two stages of metastatic (M and MS) disease
(Table 1-2). The INSS is a post-surgical staging system whereas INRGSS is a pre-treatment
staging system. The INRGSS is not intended to substitute for the INSS but is to be used in
parallel for pre-surgical risk stratification. The use of INRGSS system will result in significant
changes in the initial evaluation and subsequent treatment of NB patients and in a more
consistent approach to NB risk stratification.
In the case of AT/RT, the tumor is classified as newly diagnosed or recurrent (Ginn and
Gajjar 2012). At the time of diagnosis, the tumor may be localized, occurring in one location in
the brain, or disseminated and spread to distant areas of the body. In addition, for AT/RT, the
Chang staging system for CNS tumors (mainly for medulloblastoma) has been applied for
staging purpose (Chi et al. 2009; von Hoff et al. 2011). According to this system, an increase in
tumor size and invasion defines the aggressive behavior of the tumor. These are classified as T1
to T4. The letter T stands for primary tumor, and subdivided according to tumor size and the
extent of involvement. In addition to "T" staging, Chang staging has been modified by the
inclusion of "M" staging, where the "M" stands for metastasis. The M staging consists of 5
possible groups: M0 (no metastasis), M1 (presence of tumor cells in the cerebral spinal fluid),
10
M2 (nodular seeding in the cerebellar or cerebral subarachnoid space or in the third or lateral
ventricle), M3 (nodular seeding in spinal subarachnoid space) and M4 (metastases outside the
cerebrospinal axis). Each patient is assigned a combination of one T stage and one M stage.
11
Stage
Description
1
Localized tumor with complete gross excision
2A
Localized tumor with incomplete gross excision
2B
Localized tumor with or without complete gross excision, with
ipsilateral lymph nodes positive for tumor
3
,
Unresectable unilateral tumor infiltrating across the midline , with or
without lymph node involvement
4
Any primary tumor with dissemination to distant lymph nodes, bone,
bone marrow, liver, skin, and/or other organs (except as defined for
stage 4S)
4S
Localized primary tumor (as defined for stage 1, 2A, or 2B), with
dissemination limited to skin, liver and/or bone marrow (limited to
infants <1 year of age)
Table 1-1. International NB staging system (INSS)
12
Stage
Description
L1
Localized disease without image-defined risk factors
L2
Localized disease with one or two image-defined risk factors
M
Metastatic disease
MS
MS is equivalent to stage 4S
Table 1-2. International NB Risk Group Staging System (INRGSS)
13
1.3.3 Biological aspects
The genetic and molecular alterations have a fundamental role in the development of
cancer, both in children and adults. Genetic studies and molecular biology have improved our
understanding of NB and AT/RT and contributed to current diagnostic, risk stratification and
treatment protocols for these children.
1.3.3.1 Neuroblastoma
The biological hallmark of NB is the complexity of the genetic abnormalities that
characterize the tumor cells, and some of these abnormalities have been found to be powerful
prognostic markers independent of the clinical features. Many genetic features of NB, such as
oncogene amplification or allelic loss and the ploidy status have now been identified and shown
to correlate with clinical outcome (Brodeur et al. 2014). NB can be characterized by either a
near-diploid or hyperdiploid karyotype. Near-triploid NB is characterized by whole chromosome
gains and losses without structural genetic aberrations and are associated with favorable outcome
(Gisselsson et al. 2007). Near-diploid NB is characterized by the presence of genetic aberrations,
such as MYCN amplification, 17q gain and allelic loss at sites such as chromosome 1p, and are
linked to more aggressive tumors and poor prognosis (Brodeur 2003; Maris 2005; Park, Eggert
and Caron 2010)
The complete or partial gain of the chromosome 17 occurs in greater than 60% of NB
cases (Park, Eggert and Caron 2010). Chromosome 1p loss occurs more frequently in older
children who have stage 3 and 4 NB and is a strong predictor of outcome (Maris 2005). Other
genetic factors such as allelic gain or amplification of 4q, 6p, 7q, 11q and 18q, and other sites
14
have been identified but their prevalence and clinical significance are currently unclear (Brodeur
2003).
Amplification of MYCN is predominantly associated with advanced stages of the disease
and the overall prevalence of MYCN amplification in NB is about 22% (Brodeur 2003). MYCN
was first identified as an MYC-related oncogene amplified in NB (Brodeur et al. 2014). MYCNamplified NB is characterized by highly aggressive behavior that is coupled with an unfavorable
outcome (Gisselsson et al. 2007). Tropomyosin receptor kinase A and B (TrkA and TrkB) have
also been implicated in the clinical and biological behavior of NB. High levels of TrkA
expression correlates with younger age, lower stage and absence of MYCN amplification (Light
et al. 2012). Furthermore, TrkA expression have shown correlation with favorable outcome in
NB patients (Brodeur et al. 2009). Tropomyosin receptor kinase B is co-expressed with its
ligand, brain-derived neurotrophic factor (BDNF), in approximately 36% of NB and is correlated
with unfavorable one (Brodeur et al. 2014).
Approximately 6% to 10% of sporadic NB carry somatic anaplastic lymphoma kinase
(ALK) activating mutations (Zage, Louis and Cohn 2012). ALK encodes a receptor tyrosine
kinase (RTK) that is prevalently expressed in the nervous system. The other gene alterations
identified in NB include ornithine decarboxylase (ODC), forkhead box R1 (FOXR1) and protein
tyrosine phosphatase non-receptor 11 (PTPN11) (Brodeur et al. 2014b). In NB, PTPN11
regulates the phosphorylation status of intracellular signaling proteins, and ODC modulates the
polyamine status (Brodeur et al. 2014a). Polyamines are organic cations that enhance
transcription, translation, replication and polyamines are sometimes co-amplified with MYCN in
high-risk NB (Hogarty et al. 2008). Despite major improvements in our understanding of the
15
mechanisms involved in NB progression, only a few potential molecular targets for novel
therapeutic strategies have been identified.
1.3.3.2 Atypical teratoid/rhabdoid tumor
The biological characteristics and some of the histological features of AT/RT are similar
to malignant rhabdoid tumors of the kidney. Molecular genetic investigations of most AT/RT
specimens have demonstrated that the vast majority of AT/RT demonstrates monosomy 22 or
deletions of chromosome band 22q11 (Biegel et al. 2000). Inactivating deletions or mutations of
the tumor suppressor gene hSNF5/INI-1, which is subsequently located in the chromosomal
region 22q11.2, are now regarded as a critical step in the molecular pathogenesis of most AT/RT
(Strother 2005). The INI1 gene encodes a core member of the adenosine triphosphate (ATP)dependent SWI/SNF complex involved in chromatin remodeling, an essential process in the cell
nucleus designed to regulate gene expression (Roberts and Orkin 2004). The gene is inactivated
by truncating mutations or by partial or total deletion of the gene. Germ-line and acquired
mutations of the hSNF5/INI1 tumor suppressor gene have been reported in approximately 85%
of the cases (Biegel et al., 2002). Targeted deletion of the INI1 gene causes rhabdoid tumors in a
subgroup of mice, which clearly demonstrates its crucial role in the pathogenesis of rhabdoid
tumors (Roberts et al. 2000). The loss of INI1 not only distinguishes AT/RT from other true
rhabdoid tumors but also allows distinction of AT/RT from histologically similar CNS entities.
While treatment of relapsed and refractory AT/RT remains a challenge, novel treatments can be
developed once the tumor biology is thoroughly investigated. The summary of clinical and
biological aspects of NB and AT/RT is provided in table 1-3.
16
Feature
NB
AT/RT
22 months
17 months
Male > Female
Male > Female
Median patient age
at diagnosis
Sex ratio
Neuroblastic (N), substrateRhabdoid cells, which are
adherent (S) and intermediate
Morphologic features
either large, pale, or bland
phenotype (I) cell subtypes are
cells are present
present
Gain of chromosome 17q, loss of
Loss of SMARCB1
Biologic and Molecular
chromosome 1p, MYCN
(hSNF5/INI1) protein
features
amplification, High Trk receptors
expression
expression
Table 1-3. Clinical and biological aspects of NB and AT/RT
17
1.3.4 Therapeutic aspects
The considerable contribution to better outcomes comes from advances in our
understanding of the biological aspects of any disease. Recent therapeutic approaches for the
treatment of childhood cancers rely on biologic and targeted therapies. The increasing
recognition of genetic and molecular causes of cancer favors the continued development of novel
therapeutic targets.
1.3.4.1 Neuroblastoma
NB treatment requires a multimodal treatment approach including surgical debulking,
chemotherapy, radiotherapy, immunotherapy and more often, hematopoietic stem cell
transplantation (Brignole et al. 2003). At diagnosis, most of the cases with advanced stage NB
require chemotherapy as the primary approach for treatment. The most common
chemotherapeutic regimens used in the treatment of NB include cyclophosphamide, cisplatin,
vincristine, doxorubicin and etoposide (Maris 2010). Despite recent advances, 50–60% of
patients with high-risk NB will develop recurrent disease, and, to date, there are no wellestablished, curative treatment regimens for these patients (Cole and Maris 2012).
Tumor specific therapy is aimed at eradicating, inducing maturation and growth arrest of
NB cells. In children with high-risk NB, treatment with a differentiating agent, such as 13-cisretinoic acid and all-trans-retinoic acid (ATRA), reduces the risk of recurrence after high-dose
chemotherapy (Reynolds et al. 2003). In the last decade, anti- disialoganglioside (GD2) therapies
represent the latest major therapeutic advance for high-risk NB, and the addition of GD2
immunotherapy to retinoid maintenance therapy has constituted a significant advance in NB
treatment (Parsons et al. 2013).
18
Several drugs synthesized to specifically inhibit tyrosine and serine/threonine kinases
have also been tested in clinical trials for NB. Recent in vivo studies reported the antiproliferative ability of phosphoinositide 3-kinases (PI3K)/ protein kinase B (AKT)/ mammalian
target of rapamycin (mTOR) inhibitors in NB (Reynolds et al. 2013; Seitz et al. 2013). Polo-like
1 (PLK1) is another kinase recently recognized as a potential therapeutic target in NB and PLK1
inhibitors have shown to inhibit tumor growth, both as single agents and in combination with
irinotecan in a xenograft model (Grinshtein et al. 2011a). Other tyrosine kinase inhibitors
targeted against janus kinase/signal transducers and activators of transcription (JAK/STAT) axis
and aurora kinases have been shown to have anti-tumor activity in NB xenografts (Maris et al.
2010; Yan, Li and Thiele 2013). As mentioned, despite all efforts the prognosis of children with
advanced-staged NB still remains poor. As conventional chemotherapy and radiotherapy are not
always favorable because of their excessive side effects, the need for novel therapies that
produce fewer side effects is critical.
1.3.4.2 Atypical teratoid/rhabdoid tumor
AT/RT tumors remain a significant challenge in pediatric oncology and to date, no
standard therapeutic approach has been established. The current treatment for AT/RT includes
aggressive resection followed by radiation and chemotherapy. Although an optimal
chemotherapy regimen has not been defined for children with AT/RT, several chemotherapeutic
protocols have been used based on the evidence collected from infant brain tumor therapeutic
trials. The chemotherapeutic regimens used in AT/RT treatment include cisplatin,
cyclophosphamide, vincristine, and etoposide (Ginn and Gajjar 2012).
19
Multiple therapeutic approaches have been attempted over the last two decades as we
have learned more about the biological mechanisms that drive tumor formation and proliferation
in AT/RT. More often, the genomic aberration encountered in AT/RT is monosomy 22 or the
deletion or translocation of 22q11.2. Inactivating mutations of the hSNF5/INI-1 at 22q11.2 is
thought to be a crucial step in AT/RT tumorigenesis (Biegel 2006). Recent studies have shown
the association of INI1 and cyclin D1, and an altered expression of cyclin D1 in many primary
AT/RT tumor samples (Tsikitis et al. 2005; Venneti et al. 2011). These results provide insight as
to how the deregulation of the cell cycle contributes to the pathogenesis of AT/RT and may lead
to the discovery of new potential therapies to treat AT/RT.
The other genetic abnormality in CNS tumors is the activation of RTKs, of which the
aberrant expression of EGFR/ human epidermal growth factor receptor 2 (HER-2) is the most
frequently seen abnormality (Zhu et al. 2009; Saletta et al. 2014). A recent study has shown
increased HER-2 expression in a considerable number of AT/RT samples and this suggests that
HER-2 may be implicated in the pathogenesis of AT/RT, thus representing a potential target for
novel therapies (Patereli et al. 2010). Recent studies have also suggested the role for insulin as an
autocrine growth factor for AT/RT cell lines (D’cunja et al. 2007; Narendran et al. 2008). A
number of other tyrosine kinases, such as insulin-like growth factor 1 receptor (IGF-1R),
vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor
(PDGFR) and MET can also represent potential therapeutic targets for AT/RT, due to their
aberrant activation in pediatric CNS malignancies (Sikkema et al. 2009). More laboratory studies
are needed to identify effective targets for novel therapeutics to formulate the next generation
clinical trials for AT/RT.
20
1.4 Role of Receptor Tyrosine Kinases in cancer
Tight control of cell proliferation and differentiation in conjunction with programmed cell
death (apoptosis) is required to maintain normal tissue homeostasis. Genetic and molecular
alterations that cause an imbalance of the cellular signals that control these events may suppress
apoptosis, promote unregulated cell growth, and metastasis and result in tumorigenesis. Many of
these signals are regulated by role of RTKs. All RTKs are membrane-spanning proteins that are
comprised of an extracellular domain that contains a ligand-binding site. Furthermore, RTKs
include a single transmembrane α-helix domain and a cytoplasmic region that contains the
protein tyrosine kinase domain. The activity of RTKs is typically tightly regulated. The ATP
binding intracellular catalytic domain shows the highest level of conservation between the RTKs
and catalyzes phosphorylation of selective tyrosine residues in target proteins (Paul and
Mukhopadhyay 2004). In the absence of ligand, RTKs reside in the plasma membrane as
inactive enzymes. Binding of ligand to receptor promotes receptor dimerization and a change in
conformation that leads to activation of the kinase, which subsequently stimulates downstream
signal-transduction cascades. The activated normal cell has mechanisms in place to
downregulate the receptor through receptor internalization. This involves ligand-stimulated
endocytosis and subsequent intracellular degradation of both ligand and occupied receptor
(Lemmon and Schlessinger 2010). However, when deregulated through genetic alterations, many
RTKs can become potent oncogenes causing cellular transformation. Aberrant RTK activation in
human cancers is mediated by four principal mechanisms: chromosomal translocations, autocrine
activation, RTK overexpression or gain-of-function mutations (Hanahan and Weinberg 2000).
Mutations that affect RTK signaling or components of downstream pathways such as mitogen-
21
activated protein kinase (MAPK) and PI3K often lead to cell transformation; this is observed in a
wide variety of malignancies (Regad 2015).
The sequencing effort of the Human Genome Project has revealed that approximately
20% of the human coding genes encode proteins involved in signal transduction and to date, 58
genes encoding RTKs have been identified in the human genome (Blume-Jensen and Hunter
2001). Out of 58 genes, approximately 30 tumor-suppressor genes have been found to be
dysregulated in human cancers (Blume-Jensen and Hunter 2001). Of these deregulated
subfamilies, epidermal growth factor receptor (EGFR/ErbB), insulin receptor (IR)/ IGF-1R,
PDGFR, VEGFR and hepatocyte growth factor receptor (HGFR) are strongly associated with
tumorigenesis (Ségaliny et al. 2015).
EGFR and IGF-1R are two of the most important tyrosine kinase receptors that play a
central role in the pathogenesis and the progression of different carcinoma types. The EGF
ligand/receptor and IGF ligand/receptor systems are involved in early embryonic development
and in the stem cell renewal of the skin, liver and gut (Raggi et al. 2015; Ziegler, Levison and
Wood 2015). These two receptors are frequently overexpressed in a variety of childhood cancers,
and therefore, could be used as potential candidates for targeted cancer therapy (Wang et al.
2013).
1.4.1 EGFR and its role in cancer
EGFR belongs to the ErbB family of RTK and is also one of the most extensively studied
receptors for its role in human cancer. The ErbB family comprises four distinct receptors: EGFR
(also known as ErbB-1/HER-1), ErbB-2 (neu, HER-2), ErbB-3 (HER-3) and ErbB-4 (HER-4)
(Yarden 2001). The ErbB family of receptors are composed of an extracellular ligand-binding
22
domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase-containing
domain (Normanno et al. 2006). The extracellular domains are less conserved (about 40% to
60%) among the four receptors and have different specificity in ligand binding, whereas the
intracellular tyrosine kinase domain is highly conserved (about 60% to 80%) (Downward et al.
1984). Upon ligand binding, the ErbB receptors form homo- or hetero-dimers, which are
essential for the activation of the intracellular tyrosine kinase domain and phosphorylation of the
C-terminal tail (Wieduwilt and Moasser 2008). Six mammalian ligands that bind to ErbB family
receptors have been characterized, including epidermal growth factor (EGF), transforming
growth factor-α (TGFα), heparin-binding EGF (HB-EGF), amphiregulin (AR), epiregulin and
betacellulin (Bodey et al.2005). Human epidermal growth factor receptor 2 is unique within the
ErbB family receptors, as none of the known ErbB family ligands activates the HER-2
homodimers (Normanno et al. 2006). In contrast to the other family members, HER-2 is only
activated following hetero-dimerization with other ErbB family receptors (Klapper et al. 1999).
Upon ligand binding, receptor activation then leads to phosphorylation of specific tyrosine
residues within the cytoplasmic tail and further stimulates multiple signal pathways including
PI3K/AKT, signal transducer, RAS/mitogen-activated protein kinase (MAPK), and activator of
transcription (STAT) pathways and others (Douziech et al. 1999; Nicholson and Anderson
2002).
ErbB receptors are usually expressed in various tissues of epithelial, mesenchymal, and
neural origin. The crucial roles of ErbB receptors and EGF-like peptides in the development of
organs and tissues have been demonstrated by using different mouse models. Mice deficient in
ErbB family members die from preimplantation to 3 weeks postnatally, depending on their
genetic background (Miettinen et al. 1995). Mice lacking HER-2, HER-3, or HER-4 are
23
embryonic lethal and have defects in cardiac and neuronal development (Gassmann et al. 1995;
Erickson et al. 1997). The phenotypes of mice deficient in ErbB members show the critical role
these receptors play in the normal development, however; the aberrant activity of members of
ErbB family has been shown to play a key role in the development and growth of tumor cells
(Wieduwilt and Moasser 2008). The expression of ErbB receptors and their ligands have been
reported to be associated with a worse prognosis (Lo et al. 2005). It has been reported that
approximately 50% to 70% of lung, colon and breast carcinomas have overexpression of EGFR
or HER-3 (Normanno et al. 2003). The overexpression of the EGFR family receptors and their
ligands is correlated with poor prognosis in colorectal, head and neck, ovarian, cervical, bladder
and esophageal cancer (Nicholson, Gee and Harper 2001).
The role of EGFR and HER-2 in human carcinomas is demonstrated by a number of
studies that have shown overexpression of these receptors in the majority of cancers. Several
studies in transgenic mice have demonstrated the transforming properties of EGFR and HER-2
(Pattengale et al. 1989; Guy, Cardiff and Muller 1996). About 40 % of primary glioblastoma
multiforme (GBM) and 22% to 32% of patients with non-small cell lung cancer (NSCLC) have
gene amplification of EGFR (Hirsch et al. 2003; Nicholas et al. 2006). Overexpression of the
ErbB2 receptor has been reported in 20 – 30 % of breast cancers and is associated with decreased
survival and increased relapse rate (Slamon et al. 1987). EGFR expression is detected in a broad
range of childhood cancers including osteosarcoma, Wilms tumor, hepatoblastoma, NB,
pediatric high-grade gliomas and ependymoma (Fouladi et al. 2010). HER-2 expression has been
studied in many pediatric solid tumors, including medulloblastoma, ependymoma, osteosarcoma
Ewing’s tumor, and Wilms’ tumor (Gilbertson 2005). Studies of HER-2 expression in
24
medulloblastoma have shown a significant association between expression and clinical outcome
(Gilbertson et al. 1997).
EGFR appears to contribute to the growth and survival of neoplastically-transformed
cells, as well maintain the normal cellular function. Therefore, ErbB receptors and their ligands
represent suitable targets for novel therapeutic approaches in human carcinomas.
25
Figure 1-2. EGFR pathway and its role in cancer
Schematic diagram representing EGFR/HER-2 mediated phosphorylation and activation steps of
downstream pathways. Binding of EGF to individual EGFR subunits causes receptor
dimerization and autophosphorylation of tyrosine residues in the cytoplasmic domain. These
phosphorylated tyrosine residues then recruit docking proteins, most notably IRS, which in turn
recruits the p85 and p110 subunit to the receptor. Phosphatidylinositol 4,5 biphosphate (PIP2) is
then phosphorylated by the p110 subunit to form phosphatidylinositol-3,4,5 trisphosphate (PIP3).
The conversion of PIP3 to PIP2, and the subsequent inactivation of PI3K downstream signaling,
is facilitated by the phosphatase and tensin homolog deleted on chromosome ten (PTEN)
phosphatase. PIP3 can activate a large number of proteins, including phosphoinositol-dependent
kinase-1 (PDK1) and protein kinase C (PKC). PKC activates mTOR, and mTOR associated with
rictor forms mTOR complex 2 (mTORC2), which can phosphorylate Ser473 on AKT. PDK1
26
phosphorylates Thr308 on AKT, which then activate mTOR complex 1 (mTORC1)
(mTOR/Rictor) via phosphorylation. Active AKT is then able to promote cell survival, growth,
and proliferation by phosphorylation of key substrates such as Bad and GSK3β. In parallel, the
alternative RAS/RAF/MEK/ERK pathway can also be stimulated by receptor tyrosine kinases.
Black arrows represent positive regulation and red bars represent negative regulation. Red bars
leading to phosphorylation represent repression via phosphorylation.
27
1.4.1.1 Targeting EGFR/HER-2
The EGFR signal transduction pathways play a key role in the development of
malignancies through various processes, such as effects on cell cycle progression, angiogenesis,
tumor cell motility and metastases. Members of the EGFR family, especially EGFR and the
HER-2 receptor, have frequently been implicated as prognostic indicators in a variety of human
malignancies (Normanno et al. 2003). Therefore, many strategies to block or down-regulate
EGFR and HER-2 have been developed to inhibit tumor proliferation and improve overall
clinical outcome. Several anti-receptor therapeutic strategies have been pursued, with two of the
anti-receptor therapeutics now being developed clinically. One strategy is the generation of small
molecules that competitively bind the ATP-binding region of the kinase domain of ErbB
receptors, thus blocking receptor activation and the transduction of downstream signals. Several
anti-cancer agents targeting EGFR are currently in clinical trials, such as lapatinib, gefitinib and
erlotinib, which have been also approved for clinical use. Gefitinib and erlotinib are mostly
active against EGFR and lapatinib is equally active against EGFR and HER-2 (Stamos,
Sliwkowski and Eigenbrot 2002; Wood et al. 2004).
The second approach utilizes humanized monoclonal antibodies (mAbs) generated
against the receptor's ligand binding extracellular domain. These antibodies block binding of
receptor-activating ligands and possibly induce endocytosis and degradation (Mosesson et al.
2003). Three humanized mAbs targeting ErbB receptors have been approved for clinical use
including trastuzumab, cetuximab and panitumumab (Wieduwilt and Moasser 2008).
Trastuzumab is a HER-2-targeting mAb and is widely used in the treatment of HER-2-amplified
breast cancer.
28
The important role of aberrant EGFR signaling in the progression of malignant gliomas
makes EGFR-targeted therapies of particular interest for brain tumors (Kuan, Wikstrand and
Bigner 2000). The expression of EGFR has been reported in malignant rhabdoid tumor cell lines
and EGFR targeted treatment has been effective in inhibiting the proliferation of cancer cells in
vivo as well as in clinical trials (Kuwahara et al. 2004). In another study, 70% of children, with
HER-2 positive MEDs/PNETs, succumbed to cancer suggesting that HER-2 is probably a
prognostic marker for limited survival (Bodey et al. 2005). Since AT/RT was previously
confused with PNET, a defined targeted therapy is still unclear.
However based on the
overlapping characteristics of AT/RT with PNET, the use of anti-EGFR therapies can represent a
promising new approach for the treatment of AT/RT in children. Clinical studies already suggest
that targeting EGFR, either alone or in combination with standard chemotherapies, can induce
favorable clinical responses and tumor disease stabilization in a variety of common solid
neoplasms (Wieduwilt and Moasser 2008).
1.4.2 IGF-1R and its role in Cancer
IGF-1R is a RTK that belongs to the IR family, which includes the Insulin receptor (IR)
(a homodimer), IGF-1R (a homodimer), IGF-1R/IR (heterodimer), and the IGF-2R (also known
as mannose 6-phosphate receptor) (Larsson, Girnita and Girnita 2005a). IGF-1R is a tetrameric
receptor complex compromised of two α and two β subunits, which are joined by disulfide
bridges. The α-subunits are exclusively extracellular and bind the ligands, and the β-subunits
comprise a transmembrane region and an intracellular tyrosine kinase domain (Kim et al. 2009).
The IGF-1R can be activated by the ligands insulin-like growth factor-1 (IGF-1) or insulin-like
growth factor-2 (IGF-2) (Chen and Sharon 2013). IGF-2R does not seem to have a signaling
29
function and may act as a sink to modulate IGF2 ligand bioavailability (Farabaugh, Boone and
Lee 2015). IGF-1 is secreted primarily by the liver upon stimulation by pituitary-derived growth
hormone (GH), whereas IGF-2 is not dependent on pituitary-derived GH and is expressed in a
variety of tissues (HOLT 2002). The bioavailability of IGF-1 and IGF-2 is modulated by IGF
binding proteins (IGFBPs), which have a high affinity for these ligands and limit ligand access to
IGF receptors (Pollak 2008). Binding of IGF1 or IGF2 to IGF-1R results in autophosphorylation
of the IGF-1R kinase domain and activation of intracellular signaling cascades. Intracellular
signaling of IGF-1R is mediated through IR substrates (IRS-1 through -4) and Src-homology
collagen protein (Shc) (Baserga 1999a). The IRS and Shc substrates link the activated receptors
to numerous intracellular adaptor proteins and activate downstream signaling cascades such as
PI3K/AKT and RAS/MAPK pathways (Chen and Sharon 2013). A detailed description of IGF1R and its downstream signaling pathways are provided in Figure 1.3.
IGF-1R is critical in cell survival and proliferation, and cells lacking this receptor cannot
be transformed by most oncogenes (Baserga 1999b). It has been shown that fibroblasts cultured
from IGF-1R knockout mice grow more slowly than fibroblasts from wild-type mice and they
are unable to proliferate under anchorage-independent conditions (Sell et al. 1994). The
importance of the IGF-1R in normal mammalian development has been demonstrated by a study,
which reported that IGF-1R knockout mice were minimally viable and exhibited severe growth
retardation (Liu et al. 1993). The IGF-1R system is required for early and as well as late
development and exerts a pivotal role in cell growth and homeostasis.
A number of publications implicate a crucial role for IGF-1R signaling in the
development and progression of cancer. IGF-1R appears to be an important growth factor
receptor in cancer cells and plays an essential role in the establishment and maintenance of the
30
transformed phenotype (Riedemann and Macaulay 2006). Altered IGF-1R signaling is a
common event in several malignancies and is associated with over-expression of IGF-1R and
over-activation or production of IGF-1 and IGF-2 (Camirand and Pollak 2004). Studies have
demonstrated that overexpression of the human IGF-1R in fibroblasts causes the receptor to
function as a ligand-dependent oncogenic protein and subsequently promotes tumor development
in vivo (Kaleko, Rutter and Miller 1990). Similar findings were observed by another group that
reported that overexpression of IGF-1R accelerated the development of tumors in a mouse model
(Lopez and Hanahan 2002).
The significance of IGF-1R expression in breast cancer has been reported where IGF-1R
signaling induced by IGF-1 contributes to breast cancer growth by promoting cell proliferation
and chemotherapy resistance (Gooch, Van Den Berg and Yee 1999). IGF-1R also has an antiapoptotic role and the activation of IGF-1R protects breast cancer cells from apoptosis induced
by a number of anti-cancer drugs (Dunn et al. 1997). Preclinical studies have shown the
sensitization of cancer cell lines to chemotherapy agents by IGF-1R (Tang et al. 2013). More
specifically, IGF-1R blockade improved the response to paclitaxel and doxorubicin in breast
cancer cells (Beech, Parekh and Pang 2001). Similar results were obtained when NSCLC,
AT/RT cells and gastric cancer cells were used, yet again the aforementioned cancer cells
became sensitized to chemotherapy agents after blocking IGF-1R (Min et al. 2005;
Warshamana-Greene et al. 2005; D’cunja et al. 2007).
IGF-1R blockade improves cancer cell response to chemotherapy, and presently IGF-1R
inhibition is being actively explored. Increased IGF-1R signaling has been linked to an increase
in cancer risk or to a more aggressive cancer phenotype (Denduluri et al. 2015). The IGF-1R has
clear biological implications in ES and the inhibition of IGF-1R with a small molecule kinase
31
inhibitor reduces the growth of ES cells in vitro and in vivo (Scotlandi et al. 2005). In a phase I
trial with different IGF-1R blocking antibodies, durable responses have been achieved in ES
patients (Tolcher et al. 2009). IGF-1R has been linked to GBM, as perivascular tumor cells
express higher levels of IGF-1R and induce glioblastoma tumor invasion (Schlenska-Lange et
al.). Previous studies have shown that the treatment of glioblastoma cells with IGF-1 increases
cellular migration and growth inhibition occurs when IGF-1R is down regulated or inhibited
(Schlenska-Lange et al.; Resnicoff et al. 1994).
1.4.2.1 Targeting IGF-1R
The insulin-like growth factors (IGFs) and their receptors play a pivotal role in cellular
signaling.
More
specifically,
IGFs
regulate
cell
growth,
differentiation,
apoptosis,
transformation, angiogenesis and other important physiological progresses. Inhibition of IGF-1R
action has been studied intensively in cultured cells and in tumor xenografts. Currently, the
feasibility and efficacy of growth factor receptor targeting with monoclonal antibodies (mAbs)
or small molecule receptor tyrosine kinase inhibitors (RTKIs) have been established and some of
these treatments are already used in the clinic while others are being investigated in clinical trials
(Palazzo, Iacovelli and Cortesi 2010). Most of the available mAbs are selective and a large
number of pre-clinical studies are ongoing to evaluate the effect of IGF-1R mAbs as single
agents or in combination with standard treatments for various cancers (Heidegger et al. 2011).
As mentioned above, IGF-1R is a RTK and requires kinase activity for signal
transduction. Thus, one direct strategy would be to interfere with IGF-1R activity with selective
RTK inhibitors. The RTKIs are small molecules that bind to the intracellular TK catalytic
domain of the receptor, blocking activation and preventing downstream signaling. Since the first
32
IGF-1R RTKIs was introduced in 2004, several different RTKIs have been developed and tested
in preclinical experiments (Heidegger et al. 2011). One major advantage of this approach is that
small molecules have the considerably higher bioavailability compared to antibodies (Larsson,
Girnita and Girnita 2005b).
For approximately two decades, IGFs have been implicated in the pathogenesis of
childhood cancers and there are several IGF-1R targeted therapies currently being tested in
clinical trials (Kim et al. 2009). Previous studies have shown that IGF-2 expression and signaling
through the IGF-1R are important for NB proliferation and survival (El-Badry et al. 1991a). In
addition, increased IGF-1R expression increases the protection of NB cells from apoptosis
induced by chemotherapeutic agents (Singleton, Randolph and Feldman 1996). Neuroblastoma
preferentially metastasizes to bone and patients with bone metastases have a very high mortality
rate (Philip 1992). One feasible explanation for this phenomenon is that bone tissue expresses
large amounts of IGF, which support NB metastasis formation in the bone (Middleton et al.
1995; van Golen et al. 2006). According to the “seed and soil” hypothesis of metastasis, NB cells
expressing high IGF-1R (seed) and the bone secretion of IGF-1 (soil) lead to NB migration
through endothelium toward IGF-1 in bone (Fidler 2003; van Golen et al. 2006a). NB is a
heterogeneous tumor and a previous study has shown that N-type (neuronal) NB cells (e.g. SKN-BE(2)) have increased expression of the IGF-1R compared to the glial like S (stromal) cells
(e.g. SK-N-AS) (Kim, van Golen and Feldman 2004a).
Unfortunately, the development of these highly promising agents has been thwarted by
issues with toxicity (Riedemann and Macaulay 2006). However, new compounds with increased
selectivity have been developed and are currently being evaluated in clinical trials.
33
Figure 1-3. IGF-1R and its role in cancer
The IGF/IGF-1R system is composed of ligands (IGF-1 and IGF-2), receptor (IGF-1R), and
ligand binding proteins (IGFBPs). IGFBPs bind with IGF-1 and IGF-2 and limit the
bioavailability of these ligands to the receptor. The IGF-1R contains two α (cysteine rich
domain) and two β (tyrosine kinase domain) subunits, which are joined by disulfide bridges to
form a heterotetrameric receptor complex. The IGF/IGF-1R interaction results in
phosphorylation of tyrosine residues in the tyrosine kinase domain. The activated receptors in
turn activate downstream effectors mainly the PI3K and MAPK pathways. The phosphorylation
34
of the kinase domain of the receptor then recruits a plethora of adaptor proteins (e.g, Shc,
GRB2), which form a complex with guanine nucleotide exchange factors (e.g, SOS) that convert
inactive, GDP-bound RAS to its active GTP-bound form. Activated RAS induces the
phosphorylation of the RAF kinases (A, B, and C). Consequently, RAF activates MEK, which,
in turn, phosphorylates ERK. Activated ERK enters the nucleus and acts as a transcription factor,
initiating the transcription of several genes that lead to cellular proliferation and cell survival.
IGF-2R is structurally distinct from IGF-1R and is not a receptor tyrosine kinase. Once IGF-2
binds, IGF-2R targets IGF-2 to endocytosis-mediated lysosomal degradation.
35
1.4.3 Role of serine/threonine kinases in cancer
Cellular signaling includes phosphorylation events that occur through interactions of
kinases that are localized in the cell membrane, cytosol and nucleus. Perturbation of kinase
signaling, by mutations or other genetic alterations, results in their deregulated activity, and it is
often associated with malignant transformation (Baselga 2006). Protein kinases catalyze the
following reaction:
MgATP1- + Protein-OH = Protein–OPO32- + MgADP + H+
Based on the nature of the phosphorylated –OH group, protein kinases are classified as proteinserine/threonine kinases and protein-tyrosine kinases. The aforementioned kinases activate
numerous signaling pathways and play an important role in cell proliferation, cell cycle
regulation, differentiation, apoptosis and migration (Cohen 2001). The protein kinase family
includes 385 serine/threonine kinases, 90 tyrosine kinases, and 43 tyrosine-kinase like proteins.
Of the 90 protein-tyrosine kinases, 58 are receptor and 32 are non-receptor kinases (Roskoski
2004). A small group of dual-specificity kinases, which include MEK1 and MEK2, catalyzes the
phosphorylation of both threonine and tyrosine on target proteins (Blume-Jensen and Hunter
2001).
Serine/threonine kinases play a paramount role in cellular homeostasis and signaling
through their ability to phosphorylate transcription factors, cell cycle regulators, and a plethora
of cytoplasmic and nuclear effectors. In particular, the deregulation of the MAPK, PI3K, Aurora
kinase and PLK families have been associated with increased tumor growth, metastasis and poor
clinical outcome (Tsatsanis and Spandidos 2004). Tyrosine phosphorylation of RTKs creates
binding sites for downstream substrates such as IRS and Shc, which subsequently activate the
AKT/PI3K and RAS/RAF/MAPK pathways (Batzer et al. 1994; Riedemann and Macaulay
36
2006). Activation of the MAPK and the PI3K cascades are critical events during cell activation
and proliferation.
1.4.3.1 The PI3K pathway
A family of lipid kinases, termed PI3Ks, has been shown to play a key regulatory role in
a variety of important cellular processes including cell survival, proliferation, differentiation,
apoptosis, migration, and vesicular trafficking (Vivanco and Sawyers 2002). The PI3K isoforms
function as major effectors downstream of the RTKs and G-protein-coupled receptors (GPCRs)
pathways (Ghigo and Li 2015).
Upon stimulation, the receptors can activate PI3K by two mechanisms. In the first
mechanism, the regulatory subunit, p85, directly binds to phosphotyrosine residues on RTKs
and/or adaptors. This binding recruits the catalytic subunit of PI3K, p110, to this complex. In the
second mechanism, activated RAS stimulates PI3K. After receptor activation, Shc protein binds
to the receptor to enable the son of sevenless (Sos) and growth factor receptor-bound protein 2
(Grb2) proteins to form a complex, which results in the activation of RAS. The activated RAS
then directly binds to the p110 subunit of PI3K. Activated PI3K converts PIP2 into PIP3, which
results in the membrane localization of PDK1 via its pleckstrin homology (PH) domain.
Subsequently, AKT is recruited to the plasma membrane by its PH domain and is phosphorylated
at residues T308 and S473 by PDK1 and mTOR complex 2 (mTORC2), respectively (Martini et
al. 2014; Regad 2015). AKT is the primary mediator of PI3K-initiated signaling and it
phosphorylates several target proteins such as the glycogen synthase kinase 3α (GSK3α),
mammalian target of rapamycin (mTOR), the forkhead family of transcription factors (FOXO3),
mouse double minute 2 homolog (MDM-2), B-cell lymphoma 2-interacting mediator of cell
37
death (BCL-2-BIM) and BCL2-associated agonist of cell death (BAD) and others (Hemmings
and Restuccia 2012) (Figure 1.2). The PI3K pathway is often activated in various cancers and
represents an attractive target for therapies based on small molecule inhibitors (Regad 2015).
The recent awareness that PI3K signaling is altered at multiple levels has motivated researchers
to develop targeted therapies against individual enzymes involved in this signaling cascade.
1.4.3.2 The MAPK pathway
The MAPK pathway signals from cell surface receptors to transcription factors, which
regulate cell cycle progression, apoptosis and migration (McCubrey et al. 2007) (Figure 1.3).
This pathway is often activated in a number of tumors through chromosomal translocations,
mutations in the cytokine receptors or through the overexpression of wild type or mutated
receptors (McCubrey et al. 2007). The MAPK pathway also plays a key role in the regulation of
apoptosis by the post-translational phosphorylation of apoptotic regulatory molecules including
Bad, Bim, myeloid cell leukemia 1 (Mcl-1), caspase 9 and Bcl-2. Additional signal transduction
pathways interact with the MAPK pathway to positively or negatively regulate its activity.
Following binding of ligands to their appropriate receptors, activation of the coupling
complex Shc/Grb2/SOS occurs. Upon stimulation by Shc/Grb2/SOS, the inactive RAS
exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and undergoes a
conformational change and becomes active. RAS is a small GTP-binding protein, which is
upstream molecule of RAF/MEK/ERK pathway (Peyssonnaux et al. 2000). Three RAS proteins
have been identified so far, namely HRAS, NRAS, KRAS (Fernández-Medarde and Santos
2011). The GTP-bound active RAS can then recruit RAF to the cell membrane. RAF is a
serine/threonine kinase that consists of A-RAF, B-RAF and RAF-1 (C-RAF). RAF can become
38
activated by many events, which include the recruitment to the plasma membrane, which is
mediated
by
an
interaction
with
RAS,
the
dimerization
of
RAF
proteins,
phosphorylation/dephosphorylation on different domains of RAF and disassociation of RAF
from RAF kinase inhibitory protein (RKIP) (McCubrey et al. 2007). Activated RAF in turn
activates MEK, which is a tyrosine and serine/threonine dual specificity protein kinase (Alessi et
al. 1994). Activity of MEK1 and MEK2 is positively regulated by RAF phosphorylation and
interestingly, all three RAF family members are able to phosphorylate and activate MEK1/2.
RAF activates MEK1/2 by phosphorylating Ser218 and Ser222 in the activation loop (Dhillon et
al. 2007).
Activated MEK1/2 then activates ERK1/2, the final kinases in the RAS-RAF-MEK-ERK
signaling and the only known substrates for MEK1/2. Active ERKs can directly phosphorylate
numerous cytoplasmic and nuclear targets, including kinases, phosphatases and transcription
factors and around 160 substrates have already been discovered for ERKs. (Yoon and Seger
2006). Thus, the signaling pathway appears to be a linear one:
Activated RTK → RAS → RAF→ MEK1/2 → ERK1/2.
The MAPK pathways are highly conserved and finely regulated by multiple mechanisms,
including the scaffolding of proteins, and the phosphorylation/dephosphorylation of MAPKs
(McCubrey et al. 2007). Mutations affecting the RTK/MAPK signaling cause many human
diseases, including cancer. Since the cloning of HRAS, the first human oncogene, the MAPK
pathway has been a preferential subject of cancer research and for developing effective drugs
that could have a significant impact on cancer treatment (Malumbres and Barbacid 2003).
39
1.4.3.2.1 Targeting MAPK pathway in cancer
Target-based therapies are widely investigated for the cancer treatment and much
consideration has been focused on developing inhibitors of the MAPK pathway. In cancer,
constitutive and aberrant activation of MAPK pathway results in increased proliferation, survival
and metastasis. Abnormal activation of this pathway occurs in some human cancers due to
mutations at upstream membrane receptors or to the downstream components such as RAS, B
RAF and MEK1/2 (Roberts and Der 2007). All three RAS proteins (HRAS, KRAS, and NRAS),
are frequently mutated in human cancers; however, NRAS mutations are more commonly
encountered in melanoma (15%–20%) (Daud and Bastian 2012). BRAF is one of three RAF
proteins identified in 2002 and has high rate of mutations in a plethora of cancer (Davies et al.
2002). Over 30 mutations of the BRAF gene associated with human cancers have been
identified, mostly in two regions of the kinase domain (Wan et al. 2004). For the reasons stated
above, the RAS/RAF/MEK/ERK pathway is an important pathway to target for therapeutic
intervention. Inhibitors of MAPK pathways have been developed and many are currently in
clinical trials.
1.4.3.2.2 Targeting MAPK pathway through MEK1/2 Inhibition
Because of the importance of MAPK in cancer, this pathway has been in focus for drug
discovery for almost 15 years with RAS, RAF and MEK as the main targets. The fact that
ERK1/2 is the only known catalytic substrate of MEK1/2 has fueled the development of
pharmacological inhibitors of MEK as a means to block ERK1/2 activation. The small molecule
inhibitors of MEK1/2 are highly specific protein kinase inhibitors. The first MEK inhibitor to
40
enter clinical trials was PD184352, an orally active, highly potent and selective inhibitor of
MEK1/2 (Sebolt-Leopold et al. 1999). Clinical trial evaluation found that the activity of
phosphorylated ERK1/2 was significantly reduced in patients treated with PD184352, indicating
that the target was indeed inhibited (Wang et al. 2007). After the success of PD184352 as
MEK1/2 inhibitor, two-second generation MEK1/2-specific inhibitors (PD325901 and
ADZ6244) have been developed and are currently in clinical trials. Several MEK1/2 inhibitors
are also in later phase of clinical trials, including selumetinib (AZD6244) and cobimetinib
(GDC-0973).
In contrast to the majority of protein kinase inhibitors, MEK inhibitors are non-ATP
competitive inhibitors and bind to a unique pocket adjacent to the ATP-binding site, which may
account for their highly selective properties (Ohren et al. 2004). The only known downstream
target of MEK is ERK and downstream ERK has multiple targets which have roles in cell cycle
regulation, proliferation, apoptosis and others. Thus, therapeutic targeting of MEK1/2 is
relatively specific and holds much promise in cancer treatment.
1.4.4 Lapatinib as an EGFR/HER-2 inhibitor
Lapatinib (GW572016) is an orally bioavailable dual tyrosine kinase inhibitor that targets
EGFR and HER-2. Lapatinib is an orally effective FDA-approved drug designed for the
treatment of HER2-positive metastatic breast cancer (Roskoski 2014). Lapatinib, when used as a
single agent or in combination with trastuzumab, exhibited activity against HER-2 positive
advanced or metastatic breast cancer that had progressed after trastuzumab therapy (Konecny et
al. 2006). This study also presented significant correlation between lapatinib activity and HER-2
expression. Although some preclinical work suggested that both EGFR and HER-2 were targets
41
of lapatinib, there are no pre-clinical data available that show whether EGFR alone is an
effective target of lapatinib (Oakman et al. 2010). However, It has been shown that lapatinib
sensitivity is independent of EGFR expression in HER-2 overexpressing breast cancer cells
(Zhang et al. 2008) .
The frequency of brain metastases among women with metastatic breast cancer is
estimated to be 10–15 % (Lin et al. 2013). For patients with established CNS metastases,
trastuzumab therapy has poor outcome due to its limited ability to penetrate the blood–brain
barrier (BBB) (Pestalozzi and Brignoli 2000). Lapatinib is a small molecule with the theoretical
capability to cross BBB and there is pre-clinical evidence that supports the activity of lapatinib
against CNS disease (Gril et al. 2008a). Due to its ability to cross BBB, lapatinib was tested in
children with recurrent brain tumors, and there is evidence that shows that lapatinib prolonged
disease stabilization with minimal side effects (Fouladi et al. 2010). Although the activity of
lapatinib and other inhibitors of ErbB signaling are well established in breast cancer, lapatinib's
efficacy in other malignancies that overexpress EGFR and/or HER-2 is under evaluation.
1.4.5 Ponatinib as an IGF-1R inhibitor
Ponatinib (AP24534) is an orally bioavailable, small molecule, pan-BCR-ABL inhibitor
that has been recently approved for chronic myeloid leukemia (CML) patients (Jabbour and
Kantarjian 2016). Despite the success with RTKIs in most CML patients, some patients still
experience resistance or intolerance to current RTKIs such as imatinib. Ponatinib has been
designed as an agent in the management of imatinib-resistant disease. Ponatinib is a third
generation clinical candidate for CML patients and it inhibits the growth, proliferation, and
42
signaling mediated by native BCR-ABL leukemia, including patients with the T315I mutation
(Zhou et al. 2011).
Ponatinib also targets the fibroblast growth factor receptor (FGFR) family in myeloid and
lymphoid malignancies. A recent report has demonstrated that ponatinib has in vitro inhibitory
activity against a specific subset of protein RTKs, including fms-related tyrosine kinase 3
(FLT3), FGFR-1 and PDGFR-α (Gozgit et al. 2011). The fact that FLT3, FGFR, and PDGFR
family kinases are potential targets in a variety of other malignancies, support the potential
testing of ponatinib in a wider range of cancers. However, the effect of ponatinib in solid tumors
is still unknown and a detailed study is required to determine the targets of ponatinib in solid
malignancies.
1.4.6 Cobimetinib as an MEK1/2 inhibitor
Cobimetinib (GDC-0973, XL-518, and RG7421) is a potent, orally bioavailable, smallmolecule inhibitor of MEK 1 (Akinleye et al. 2013a). Currently, thirteen MEK inhibitors have
been clinically tested but only trametinib (GSK1120212) and cobimetinib have emerged as the
first MEK inhibitors to show efficacy in a phase III clinical trial (Akinleye et al. 2013b; Richman
et al. 2015). MEK inhibitors are sub-divided into two major classes; ATP non-competitive
(allosteric) inhibitor and ATP competitive inhibitors. Most of the known MEK inhibitors
including cobimetinib are noncompetitive and show high specificity compared to ATP
competitive inhibitors (Akinleye et al. 2013). Inhibition of MEK1/2 with MEK inhibitors blocks
ERK1/2 activation and consequently relieves the negative feedback loop. Therefore, it facilitates
the activation of upstream pathway components, including RAS and RAF. Thus, the majority of
MEK inhibitors including cobimetinib, do not disrupt the phosphorylation of the MEK1/2
43
activation loop sites, and treatment with these inhibitors cause relief of negative feedback loop,
which results in the accumulation of phosphorylated MEK1/2 (Caunt et al. 2015). Differences in
the mechanism of action may identify distinct interactions between the MEK inhibitors and the
MEK1/2 activation loop residues (Hatzivassiliou et al. 2013a). Since, MEK1 and MEK2 are the
only activators of ERK1 and ERK2, and serve an entirely unique role as critical 'ERK1 and
ERK2 gatekeeper' kinases, MEK1 and MEK2 as therapeutic targets can have a promising role in
cancer treatment.
1.4.7 Cancer cell migration and metastasis
Cell migration and metastasis are key features of aggressive tumors. Cell migration is a
multistep process where tumor cells propagate from the primary tumor to a distant organ. The
commonly proposed five-step model of cell migration involves the following steps: (1)
detachment of cancer cells from the primary tumor site, (2) invasion into surrounding tissue, (3)
intravasation into blood or lymphatic vessels, (4) circulation in the blood stream or the lymphatic
system and, finally, (5) extravasation and outgrowth at a secondary site (Bozzuto, Ruggieri and
Molinari 2010). The complexity of tumor cell migration suggests that multiple signals control
the process and that the tumor cell may respond to these signals in additive or synergistic manner
(Yamaguchi, Wyckoff and Condeelis 2005). Tumor cells have a motile response to many agents
including extracellular matrix components, host-derived motility and growth factors, and tumorsecreted factors (Liotta and Kohn 2003). Many growth factors that stimulate tumor cell motility
include the IGFs, EGF, HGF, FGF, and TGF-β, among others (Kohn et al. 1990; Bacac and
Stamenkovic 2008). Cell migration is a complex and heterogeneous process, and depending on
the cell type and tissue environment, cells can migrate in two major pathways: individually
44
(when cell-cell junctions are absent), or collectively as multicellular groups (when cell-cell
adhesions are maintained) (Friedl and Wolf 2010).
The ability to metastasize is a hallmark of malignant tumors, and is often the frequent
cause of death in patients with cancer. At the time of cancer diagnosis, the majority of the
patients already present with clinically detectable metastatic lesions (Martin et al. 2000).
Metastasis is the process where cancer cells spread to tissues and organs beyond where the tumor
originated and subsequently forms new tumors. The metastatic process is composed of a number
of sequential events, all of which must be successfully completed to give rise to a metastatic
tumor. Tumor metastases occurs via three major routes: lymphatic vessels, blood vessels and
serosal surfaces (Bacac and Stamenkovic 2008). During the metastatic process, a cancer cell
from a primary tumor performs the following sequence of steps: cancer cells invade the
surrounding tissue and enter the blood circulatory system (intravasation), survive in the
circulation system and translocate to the distant tissues through the bloodstream, exits from the
bloodstream (extravasation), survives in the distant tissues and finally adjust to the foreign
microenvironment of these tissues and forms a macroscopic secondary tumor (colonization)
(Chaffer and Weinberg 2011).
Pediatric patients with solid tumors mostly have better outcomes than adults, but the
overall survival for patients with metastatic or relapsed disease is poor (Pui et al. 2011). Similar
to other cancers, metastasis is associated with a poor prognosis in patients with NB and AT/RT
(Sohara, Shimada and DeClerck 2005; Ginn and Gajjar 2012). About 40% of NB patients have
metastatic lesions at the time of diagnosis and in the case of children over one year of age,
approximately 75% of cases present with disseminated metastases (Kiyonari and Kadomatsu
45
2014). Bone marrow metastasis is the most commonly reported one in patients with NB, and this
is related with poor prognosis.
AT/RT often presents with metastatic disease and approximately one third of patients
have widespread metastasis at presentation (Tekautz et al. 2005). The usual AT/RT metastasis
sites are the lungs, liver and other locations of the CNS (mainly cerebrospinal pathway). Of all
childhood cancers, embryonal tumors have the worst survival rate, and would greatly benefit
from more targeted therapeutic approaches with less long-term toxicity (Saletta et al. 2014). In
summary, our understanding of altered signaling pathways responsible for childhood
malignancies is potentially improving the treatment approaches and this information will be
beneficial when improving the outcomes of pediatric patients in future.
1.5 Hypothesis
Given the potential use of targeted therapy in cancer treatment and role of RTK signaling
in childhood cancer, this study was designed to help in developing novel targeted therapeutic
targets for the treatment of NB and AT/RT. We hypothesize that selective RTKs provide an
effective target for therapeutics in refractory NB and AT/RT.
1.5.1 Specific Aims
To test the hypothesis, the research work described in this thesis was focused on three
specific aims:
1. To identify potential therapeutic targets for the treatment of AT/RT
2. To identify potential therapeutic targets for the treatment of NB
3. To define the role of targeted inhibition of MEK1 by cobimetinib in NB
46
Chapter Two: Material and Methods
2.1 Cell Culture
Cell lines used in this study were cultured in Opti-MEM medium (Gibco, Life
Technologies Corporation, Burlington, Ontario) supplemented with 5% heat inactivated fetal
bovine serum (FBS) (Gibco), 100 units/ml penicillin and 100 units/ml streptomycin (Gibco) in
T25 flasks (Nalgene Nunc, Rochester, New York). Confluent cells were trypsinized with 0.25%
Trypsin-EDTA in Ca2+ and Mg2+ free balanced salt solution (Gibco) every three to five days.
All cell cultures were maintained in incubators at 37 °C in a humidified atmosphere with 5%
CO2.
Characteristics of NB cell lines used in this study are summarized in Table 2.1.
2.1.1 Freezing and thawing of cells
Cells to be frozen were harvested by trypsin- ethylenediaminetetraacetic acid (EDTA)
(Gibco) treatment, centrifuged at 1200 rpm, and resuspended in a freezing medium containing
FBS and 10% sterile dimethylsulfoxide (DMSO, Sigma, Oakville, ON) to yield a concentration
at approximately 1×106 cells/ml. One ml aliquots of cell suspension were transferred to cryo
vials (Nalgene) and kept at -80°C over night. Frozen cells were then stored in liquid nitrogen
for long-term storage. To thaw cells, a vial of frozen cells was removed from liquid nitrogen,
placed in a 37°C water bath for 2-3 minutes and suspended in 11ml of culture medium and
centrifuged at 1200 rpm for five minutes. The pellet containing cells was resuspended and
transferred to a fresh flask containing culture medium with 10% FBS and was incubated at 37ºC
in a CO2 incubator.
47
2.1.2 Passaging of cells
Cells upon attaining ~80-90% confluence were washed with phosphate buffer saline
(PBS) and dislodged from the culture flask using trypsin-EDTA. An appropriate volume of
media containing 10% FBS was then added to stop the trypsin activity and cells were
centrifuged at 1200 rpm for five minutes. The cells were then resuspended in fresh medium and
plated in a fresh tissue culture flask with uniform spreading. All cell lines were split at 1:4 to
1:8 ratios depending upon the experimental setup.
48
Cell Line
Patient
(yr.mo/sex)
IMR-32
1.1/M
IMR-5
1.1/M
SK-N-AS
8/F
SK-N-SH
4/F
SHEP
4/F
SK-NBE(2)
2.2/M
Description
Deletion of the short arm of chromosome 1 and
amplification of the MYCN gene. Two cell types
are present; predominant is a small neuroblast-like
cell and other is a large hyaline fibroblast. IMR-32
is more tumorigenic N (Neuronal) type.
Genetically modified cell line (A clone of IMR32). Deletion of the short arm of chromosome 1
and amplification of the MYCN gene. IMR-5 is I
(intermediate) type.
Deletion of the short arm of chromosome 1 and
lack MYCN amplification. SK-N-AS is less
tumorigenic S (Schwannian or stromal) type.
Lack 1p alteration and MYCN amplification. SKN-SH is N type.
Genetically modified cell line (A clone of SK-NSH). Lack 1p alteration and MYCN amplification.
SHEP is S type.
Deletion of the short arm of chromosome 1 and
amplification of the MYCN gene. Chromosome 17
alteration is present. SK-N-BE(2) is N type.
References/Sources
ATCC
(Miller, Evans and
Cohn 1993)
ATCC
ATCC
(Reddy et al. 1991)
(Barnes et al. 1981a)
Table 2-1. Characteristics of NB cell lines investigated in this study
49
2.2 Small molecule kinase inhibitors and chemotherapeutic agents
A small molecule kinase inhibitor library was tested in this study. This library included
151 inhibitors synthesized, purity checked and provided by Chemietek (Indianapolis, Indiana).
All of the inhibitors were prepared in DMSO to a stock concentration of 10 mM and stored at 20°C. For subsequent experiments, the inhibitors were diluted in culture media to the
appropriate concentrations. In addition, cis-RA used in this study was reconstituted in DMSO
and stored at -20°C in 10 mM aliquots. Stock solutions of cis-RA were obtained from the
Alberta Children’s Hospital Pharmacy (Calgary, Alberta) and stored at room temperature.
2.3 Cell viability assays
2.3.1 Single drug cell cytotoxicity assay
For single drug cell cytotoxicity assay, individual inhibitor or vehicle control (DMSO)
was diluted in duplicate in 100 µl of Opti-MEM per well in 96 well plates (Grenier Bio-One,
Monroe, North Carolina) at four concentrations (0.01, 0.1, 1 and 10 µM). NB and AT/RT cells
were trypsinized at a concentration of 5 × 103 cells per well for a final volume of 200 µl per
well and plates were incubated at 37ºC in a CO2 incubator. After four days in culture, cell
viability was quantified by automated microscope (Cyntellect Inc, San Diego, CA) and percent
cell survival was determined by comparison to cells treated with vehicle control.
2.3.2 Drug combination studies
For combination studies, inhibitor A was diluted in 100µl of Opti-MEM per well in 96
well plates in triplicate at final concentrations ranging from 1 X 10-6 to 10 µM. In next step,
IC25 concentration (concentration inducing 25% growth inhibition) of inhibitor B was added at a
50
volume of 10 µl per well to already diluted concentrations of inhibitor A in triplicate. The
control wells included inhibitor A (concentrations ranging from 1 X 10-6 to 10 µM), the IC25
concentration of inhibitor B and vehicle control in duplicate. After drug dilution, 1 X 103 cells
per well were seeded for a final volume of 200 µl and plates were incubated for four days. For
both single agent and combination studies, cell survival was measured by automated inverted
microscope (Cyntellect Inc, San Diego, CA). In order to determine combination indices (CI),
calculations described by Zhao and colleagues were employed (Zhao, Wientjes and Au 2004).
A ratio of the concentration of inhibitor A required in combination with inhibitor B to achieve
50% survival (Inhibitor A CI50) compared to the half maximal inhibitory concentration of
inhibitor A alone (Inhibitor A IC50) was added to the ratio of the concentration of inhibitor B
used in combination (Inhibitor B IC25) to the half maximal inhibiting concentration of inhibitor
B alone (Inhibitor B IC50), as indicated in the following formula:
Based on these calculations, CI of less than, equal to, and more than 1 indicates synergy,
additivity, and antagonism, respectively(Zhao, Wientjes and Au 2004) .
2.3.3 Automated inverted microscopy
After incubation for four days, cell viability was quantified by automated inverted
microscope using the Celigo Cell Cytometer (Cyntellect, San Diego, California). The number
of cells in each well was determined using the direct cell counting application. This method
uses bright field imaging combined with image analysis software to yield cell count and
51
viability measurements. The percent survival was then calculated as a ratio of the number of
cells in treated wells to the number of cells in control wells multiplied by 100.
2.4 Preparation of cellular extracts
Exponentially growing cells were seeded into 6 well plates (~70-80% confluence), 24
hours prior to drug treatment. Following drug treatment conditions, cells were harvested and
washed with 1X PBS, centrifuging at 1200 rpm at 4°C for five minutes. After first wash,
supernatant was removed and the pellet was resuspended in radioimmunoprecipitation assay
(RIPA) buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate;
0.1% sodium dodecyl sulphate (SDS)) supplemented with 1% phosphatase inhibitor (SigmaAldrich), 1% protease inhibitor (Sigma-Aldrich) and 1% sodium orthovanadate (Alfa Aesar,
Ward Hill, Massachusetts). Cell suspensions were transferred to microcentrifuge tubes,
incubated on ice for 10 minutes, vortexed and then centrifuged at 10,000 rpm for 10 minutes.
Supernatants were collected in a clean microcentrifuge tube prior to protein quantification. For
future experiments, samples were stored at -20°C.
2.5 Immunoblotting
Protein quantification of the lysates was measured using the Bicinchoninic Acid (BCA)
Protein Assay Kit (Pierce, Rockford, Illinois), as per manufacturer’s instructions using bovine
serum albumin (BSA) as a standard. Absorbance readings were measured at 562nm using a
SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, California). Appropriate
volumes of samples and loading buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1%
β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue) were mixed, and heated for 5
52
minutes at 95°C prior to loading. For the majority of experiments in this study, 25-30 µg of
protein per well was loaded. Samples were resolved by SDS-PAGE (polyacrylamide gel
electrophoresis) using various percentages of acrylamide gels (8% - 12%), depending on the
mass of the proteins of interest. SDS-PAGE was electrophoresed in 1X running buffer (25 mM
Tris, 192 mM glycine and 0.1% SDS) and then transferred to nitrocellulose membranes in
transfer buffer (48 mM Tris, 39 mM glycine and 20% (v/v) methanol) at either 100 volts for
one hour or 30V overnight at 4°C. Immunoblots were then blocked in 5% skim milk in trisbuffered saline with 0.1% Tween-20 (TBS-T; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1%
(v/v) Tween-20 (Sigma-Aldrich)) for one hour. Immunoblots were then incubated with selected
primary antibodies diluted in TBS-T with 0.1% gelatin (Bio-Rad, Mississauga, Ontario) and
0.05% sodium azide (Sigma) over night. The specifications of the primary antibodies used in
these experiments are summarized in Table 2.2. Following washes with 1X TBS-T three times
for 10 minutes, immunoblots were incubated with appropriate secondary antibodies conjugated
to horseradish peroxidise (Sigma) diluted in 1X TBS-T plus 5% skim milk at a ratio of 1:5000
for one hour at room temperature. Immunoblots were subsequently washed three times in 1X
TBS-T, and were exposed to combined enhanced chemiluminescence (ECL) reagents (Reagent
1: 100 mM Tris-HCl (pH 8.5), 5.4 mM hydrogen peroxide; Reagent 2: 100 mM Tris-HCl (pH
8.5), 2.5 mM luminol, 0.4 mM p-coumaric acid) for one minute and developed by exposure to
X-ray film (Christie InnoMed, Montreal, Quebec).
53
Antigen
Species
Working
dilution
Β-Actin
Rabbit
1:5000
AKT1
Mouse
P-AKT1/2/3
Company
Phosphorytion
site
Catalog
Sigma-Aldrich
-
A2066
1:1000
Santa Cruz
-
SC-5298
Rabbit
1:1000
Santa Cruz
S473
ERK1/2
Rabbit
1:2000
Cell Signaling
-
9102
P-ERK1/2
Rabbit
1:2000
Cell Signaling
Thr185/Tyr187
9101
IGF-1R
Mouse
1:1000
R&D Systems
-
P-IGF-1R
Rabbit
1:1000
Santa Cruz
Caspase-9
Rabbit
1:1000
Cell Signaling
-
9502
PARP
Rabbit
1:2000
Cell Signaling
-
9542
MEK1/2
Rabbit
1:1000
Cell Signaling
-
9122
P-MEK1/2
Rabbit
1:1000
Cell Signaling
S217/221
9121
Stat3
Rabbit
1:1000
Santa Cruz
P-Stat3
Rabbit
1:1000
Cell Signaling
Tyr705
9131
c-RAF
Rabbit
1:1000
Cell Signaling
-
9422
P-c-RAF
Rabbit
1:1000
Cell Signaling
S296
9423
EGFR
Mouse
1:1000
Santa Cruz
-
sc-377229
P-EGFR
Rabbit
1:1000
Santa Cruz
Tyr1173
SC-101668
mTor
Rabbit
1:1000
Cell Signaling
-
2972
P-mTor
Rabbit
1:1000
Cell Signaling
S2448
2448
Src Antibody kit
Rabbit
1:1000
Cell Signaling
Tyr416
9935
Tyr1165/1166
-
SC-7985-R
MAB391
SC-101704
SC-482
Table 2-2. Summary of primary antibodies used in immunoblotting studies
54
2.6 Antibody Array
In this study, we used a human phospho-RTK Array Kit (R&D Systems, Inc., Minneapolis,
Minnesota) to simultaneously detect the relative phosphorylation of 49 different RTKs. Second
human phospho-kinase array (R&D Systems) used in this study can detect the relative sitespecific phosphorylation of 43 kinases simultaneously. Cells were seeded in six well culture
plates (Nunc) at 1 x 106 cells/ml and incubated overnight. Fresh culture medium containing drug
or vehicle control was added and after a two-hour incubation, cells were washed with ice cold
1X PBS and treated with lysis buffer (50 mM Tris, 5 mM EDTA, 0.1 % SDS, 1 % Triton X-100,
0.5 % sodium deoxycholate) containing phosphatase and protease inhibitors (Sigma). Protein
quantification of the samples was carried out and lysates were used immediately. Protein
concentration used in this assay was 100-300 µg for each sample. In one experiment, two arrays
were used for treatment and control condition each. Each array was incubated for one hour at
room temperature in array buffer provided with kit. After blocking, prepared lysate samples were
added to each array and incubated overnight at 2-8 °C. After overnight incubation, each array
was washed with 1X wash buffer for 10 minutes for a total of three washes. Each array was then
incubated with anti-phospho-tyrosine-HRP detection antibody for two hours at room
temperature. Arrays were subsequently washed three times with 1X wash buffer, and were
exposed to combined ECL reagents for one minute and developed by exposure to X-ray film.
The
arrays
were
scanned
and
the
spot
(http://rsb.info.nih.gov/ij/ version 1.4.3.67).
55
densities
were
quantified
with
ImageJ
2.7 Cell Migration Assay
A scratch assay was used to analyze the cell migration effect. NB and AT/RT cells were
plated in six well culture plates (Nunc). Cells were grown to ~70% confluence in serum-free
medium prior to scratch. On the day of the assay, the cell monolayer was scraped in a straight
line with a 10 µl pipette tip and washed with 1X PBS to remove any floating cell. New culture
medium containing different treatment conditions was added to each well. The plate was kept in
a tissue culture incubator at 37 °C for 8–24h. The photographs of each well were taken using
the Celigo Cell Cytometer (Cyntellect, San Diego, California) at 0h, 8h and 24h. The images
acquired for each sample were further analyzed quantitatively by using Image J software. For
each image, distances between one side of scratch and the other was quantified to analyze the
cell migration.
2.8 Immunocytochemical Assay
Before adding cells, 24 x 30mm coverslips (Nunc) treated with 5N NAOH (Sigma) and
Poly-L-Lysine (Sigma) were placed in six well plate. Cells were treated with cobimetinib (1 µM)
and cis-RA (10 µM) alone or in combination along with vehicle control for 24 hours. Briefly, the
cells were fixed with 4 % paraformaldehyde (Sigma) for 15 min and permeabilized with 0.05 %
Triton X-100 (Sigma). The cells were washed three times with 1X PBS. The cells were
incubated with antibodies to Nestin (R&D Systems, 1:1000), GFAP (Sigma, 1:1000) and MAP-2
(Sigma, 1:800) for two hours at 37 °C. The cells were then washed with 1X PBS and incubated
with fluorescently labeled secondary antibody (Invitrogen, 1:500) at room temperature for one
hour. After incubation, cells were washed with 1X PBS and in the last wash, cells were stained
56
with DAPI solution (3ng/ml or 1in 2ml 1X PBS) for 5-10 min and coverslip were then
transferred on a slide.
Staining of treated and untreated cells was then visualized by fluorescence microscopy for
detection of differentiation markers.
2.9 Tumor Xenograft Study
2.9.1 Generation of tumor cells stably expressing firefly luciferase and eGFP
BT16 cells expressing enhanced firefly luciferase (effLuc) and eGFP were generated
using a self-inactivating lentiviral vector encoding the internal U3 region from mscv, effLuc, the
IRES element from emcv, and eGFP (Bai et al. 2011). The virus was packaged in 293-FT cells
using pMD2.G (VSV.G env) and pCMV-deltaR8.91 and concentrated 50 times using Amicon
Ultra-15 100,000 NMWL centrifugal concentration units (Millipore, Billerica, MA).
Concentrated viral supernatants were used to transduce BT16 cell lines via spinfection for two
hours at 2200 rpm at 30°C (approximate multiplicity of infection [MOI] = 10). After 72 h, eGFP
expression was observed via fluorescence microscopy (Zeiss inverted microscope, Axiovert
200 M) and used to calculate transduction efficiency by flow cytometry on a FACS Calibur
instrument (BD Biosciences, San Jose, CA). The effLuc based bioluminescent activity was
calculated using an IVIS 200 (Caliper Life Sciences, Alameda, CA).
2.9.2 In vivo real-time monitoring of tumor growth (bioluminescence imaging)
Six to eight week-old female CD-1 mice from Charles River Laboratories were used in
this study. All protocols were reviewed and approved by the Animal Care Committee of the
University of Calgary. All animal work procedures were in accordance with the Guide to the
57
Care and Use of Experimental Animals published by the Canadian Council on Animal Care and
the Guide for the Care and Use of Laboratory Animals issued by national institutes of health
NIH.
The AT/RT intracranial animal model established with BT16GFPFluc cells and the
stereotactic techniques used to implant BT16GFPFluc cells in the right putamen have been
described previously (Studebaker et al. 2010). Two groups of randomly assigned CD-1 Nude
mice (n = 5 per group, total 10 animals) were implanted in the flank with 5 × 106 BT16GFPLuc
cells. After allowing approximately two weeks for the tumors to establish, tumor-bearing animals
were randomly divided into two groups for control vehicle treatment and for lapatinib treatment,
which was given as a twice-daily oral administration for 3 weeks (5-days on, 2-days off) at a
dose of 160 mg/kg (320 mg/kg/day). The imaging process was carried out by the Xenogen IVIS
200 (Xenogen Corporation, Alameda, California) system to record the bioluminescent signal
emitted from the tumors. Every week after tumor implantation, all mice were imaged to record
bioluminescent signal emitted from tumors. Anesthesia was given in an induction chamber with
2.5% isoflurane in 100% oxygen at a flow rate of 1 L/min and maintained in the IVIS with a
1.5% mixture at 0.5 L/min. The mice were then injected with d-luciferin (126 mg/kg, Xenogen
Corp.) dissolved in PBS (15 mg/ml) by intraperitoneal administration. Subsequently, mice were
placed in a prone position in the IVIS instrument and bioluminescent acquisitions were collected
until the maximum signal was reached. Data were analyzed based on total photon flux emission
(photons/s) in the region of interest (ROI) over the tumor. After 17 days of treatment, the animals
were sacrificed and the tumors were removed and photographed under a dissection microscope
with a fluorescent filter or white light, where a scale was included for size comparison.
58
Chapter Three: Profiling pathway-specific novel therapeutics in preclinical assessment for
central nervous system atypical teratoid rhabdoid tumors (CNS AT/RT): Favorable
activity of targeting EGFR- ErbB2 signaling with lapatinib
Many childhood cancers show the overexpression of various RTKs and the autocrine
production of their ligands. It has been shown that the modulation of these receptors decreases
the growth or otherwise hinders the malignant potential of the transformed cell. A direct strategy
to interfere with RTK activity is to induce selective inhibition of its tyrosine kinase activity by
selective small-molecule inhibitors. We proposed to achieve this by first identifying effective
agents in a drug discovery screening assay. Further, we proposed that inhibition of RTK
signaling will inhibit the growth of cancer cells and will provide avenues for the development of
novel therapeutics. The following report1 summarizes these studies and presents a preclinical
evaluation pathway to identify novel therapeutic regimens for rare pediatric malignancies such as
CNS AT/RT.
Author’s contribution: Anjali Singh performed and analyzed all in vitro experiments with the
support of her supervisor, Dr. Aru Narendran. Xueqing Lun contributed to in vivo section
(figure 3.8). AJ and HO facilitated the maintenance of some of the cell lines used and assisted
with some of the experimental studies. DS, PF, SC, and AS provided reagents and cells. All
authors have reviewed and approved the final manuscript. The original publication has been
reproduced as Chapter Three in this thesis. Figures and references have been reformatted
according to the guidelines outlined by the University of Calgary Faculty of Graduate Studies.
1
Anjali Singh, Xueqing Lun, Aarthi Jayanthan, Halah Obaid, Douglas Strother, Peter Forsyth,
Susan N Chi, Amy Smith and Aru Narendran. Profiling Pathway Specific Novel Therapeutics in
Preclinical Assessment for Central Nervous System Atypical Teratoid Rhabdoid Tumors (CNS
AT/RT). Favorable Activity of targeting EGFR- ErbB2 signaling with lapatinib. Molecular
Oncology 2013 Jun;7(3):497-512.
59
3.1 Abstract
Despite intensifying multimodal treatments, children with CNS AT/RT continue to endure
unacceptably high mortality rates. At present, concerted efforts are focusing on understanding
the characteristic INI1 mutation and its implications for the growth and survival of these tumors.
Additionally, pharmaceutical pipeline libraries constitute a significant source of potential agents
that can be taken to clinical trials in a timely manner. However, this process requires efficient
target validation and relevant preclinical studies. As an initial screening approach, a panel of 129
small molecule inhibitors from multiple pharmaceutical pipeline libraries was tested against
three AT/RT cell lines by in vitro cytotoxicity assays. Based on these data, agents that have
strong activity and corresponding susceptible cellular pathways were identified. Target
modulation, antibody array analysis, drug combination and in vivo xenograft studies were
performed on one of the pathway inhibitors found in this screening. Approximately 20% of
agents in the library showed activity with IC50 values of 1 µM or less and many showed IC50
values less than 0.05 µM. Intra cell line variability was also noted among some of the drugs.
However, it was determined that agents capable of affecting pathways constituting ErbB2,
mTOR, proteasomes, Hsp90, Polo like kinases and Aurora kinases were universally effective
against the three AT/RT cell lines. The first target selected for further analysis, the inhibition of
ErbB2-EGFR pathway by the small molecule inhibitor lapatinib, indicated inhibition of cell
migration properties and the initiation of apoptosis. Synergy between lapatinib and IGF-IR
inhibition was also demonstrated by CI values. Xenograft studies showed an effective antitumor
activity of lapatinib in vivo. We present an experimental approach to identifying agents and drug
combinations for future clinical trials and provide evidence for the potential of lapatinib as an
effective agent in the context of the biology and heterogeneity of its targets in AT/RT.
60
3.2 Introduction
Primary CNS AT/RT is a malignant embryonal tumor that commonly affects infants and
very young children (Rorke, Packer and Biegel 1996). There are infrequent cases of long-term
survivors described in the literature following treatment with intensive multimodal therapy
(Reddy 2005). However, presently no standard or generally effective treatment protocols exist
for the treatment of these children. AT/RT cells are distinguished by alterations of the INI1
tumor-suppressor gene located on chromosome band 22q11.2 (Bikowska, Grajkowska and
Jóźwiak 2011). Mechanistically, INI1/hSNF5 is a component of the ATP-dependent chromatin
remodeling SWI/SNF complex and shown to mediate cell cycle arrest due to the direct
recruitment of HDAC activity to the cyclin D1 promoter, leading to its repression and
subsequent G0-G1 arrest (Zhang et al. 2002; Fujisawa et al. 2005). Currently, however, the
pathways by which this molecular abnormality leads to the aggressive growth phenotype are not
completely understood. Recent literature suggests that INI1 is capable of interacting with key
signaling molecules and modifying processes such as cell cycle progression and growth factor
response. For example, the interaction between the key signal transducer AKT and members of
the hSWI/SNF chromatin remodeling complex leading to AKT activation has been demonstrated
(Foster et al. 2006). A number of studies have also investigated specific cytokine driven growth
regulatory pathways in AT/RT cells. These include the growth dependency on IGF-I and IGF-II
and the inhibition of these cytokines by small molecule inhibitors or antisense oligonucleotides
(D’cunja et al. 2007; Narendran et al. 2008; Ogino et al. 1999; Ogino et al. 2001). Data from
Foster and colleagues have shown the dependency of these cells on AKT activation, which may
occur through aberrant stimulation of the IGF-IR pathway (Foster et al. 2009). Similarly,
61
autocrine signaling by insulin, via the PI3K/AKT pathway, leading to increased growth and
survival of AT/RT cell lines has also been demonstrated (Arcaro et al. 2007). These studies
indicate that mechanistic associations exist between the distinctive genetic abnormalities of
AT/RT and altered sensitivity to specific growth factor mediated signaling processes. Hence,
directed interference of these pathways provides unique opportunities to discover effective
targets for future therapeutics.
In the recent past, efforts have intensified to identify molecular mechanisms that regulate
AT/RT cell growth and to detect targets for novel therapeutics. For example, supported by the
previous finding that cyclin D1 is a key target of INI1, Smith and colleagues have shown that the
derivatives of fenretinide have the ability to down-modulate Cyclin D1, inducing cytotoxicty in
rhabdoid cell lines (Smith, Das and Kalpana 2011). Similarly, Knipstein and co-workers
demonstrated the utility of histone deacetylase inhibitors (HDI) to induce radiosensitization and
apoptosis in AT/RT cells (Knipstein et al. 2012). Our previous studies have provided evidence
for an effective drug combination consisting of multi-tyrosine kinase inhibitors with irinotecan
(Jayanthan et al. 2011). Recently, the generation of genetically engineered INI1+/− mice that
spontaneously develop tumors, including CNS lesions, has provided valuable means to test new
therapeutic agents and drug resistance mechanisms in AT/RT (Guidi et al. 2001). The Pediatric
Oncology Experimental Therapeutics Investigators' Consortium (POETIC) is currently
evaluating libraries of pipeline agents to identify drugs that hold promise in future clinical trials
for currently difficult to treat pediatric malignancies. This report describes the initial screening of
such a drug panel against cell lines established from AT/RT patients and the identification of
agents that are able to induce cytotoxicity at sub-micromolar concentrations. More detailed
analysis of one of these agents, lapatinib, provides information on target modulation and
62
evidence for effective drug synergy with IGF-IR inhibition. Lapatinib also showed in vivo
activity in a xenograft model of AT/RT, validating an approach to develop future clinical studies
in the treatment for AT/RT.
3.3 Material and Methods
3.3.1 Cell lines and cell culture
BT12 and BT16 cell lines were established from infants with CNS AT/RT and
generously provided by Drs. Peter Houghton and Jaclyn Biegel (Nationwide Children's Hospital,
Columbus, Ohio and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
respectively). These cell lines have been used extensively in preclinical studies in AT/RT. The
cell line KCCF1 was established in our laboratory from the CSF cells of a two-month-old male
infant with AT/RT. Characterization of this cell line has been described previously (Jayanthan et
al. 2011). The Hs68 primary skin fibroblast cells were provided by the Sung-Woo Kim
laboratory (University of Calgary) and the EGFR over-expressing glioblastoma multiforme
(GBM) cell line T98G was a gift from the laboratory of Dr. Greg Cairncross (University of
Calgary). These cell lines were cultured in Opti-MEM medium (Gibco, Invitrogen Corporation,
Burlington, Ontario) containing 5% FBS (Fetal Bovine Serum), 100 units/ml penicillin and
100 units/ml streptomycin (Gibco). Cells were trypsinized with 0.25% Trypsin-EDTA in Ca2+
and Mg2+ free balanced salt solution (Gibco) every three to five days. All cell cultures were
maintained in incubators at 37 °C in a humidified atmosphere with 5% CO2.
63
3.3.2 Antineoplasic agents
All targeted therapeutic agents used in the screening analysis were synthesized, purity checked
and provided by Chemi-etek (Minneapolis, MN). Lapatinib was kindly provided by
GlaxoSmithKline (Collegeville, PA). These agents were dissolved in DMSO to a final
concentration of 10 mM and stored frozen at −20 °C and diluted appropriately in culture medium
at the time of study.
3.3.3 Cell growth inhibition assays
Atypical teratoid/rhabdoid tumor cells were trypsinized and placed in 96 well plates
(Grenier Bio One, Monroe, NC) at a concentration of 5 × 103 cells per well. Increasing
concentrations of study agents were added to these wells to a final volume of 200 µl per well.
Corresponding dilutions of the vehicle DMSO was used as control. After four days in culture,
cell survival was quantified by automated cytometer (Celigo, Cyntellect Inc., San Diego, CA,
USA), according to the manufacturer's protocol (Nabzdyk et al. 2011). The half maximal
inhibitory concentration (IC50) values were calculated for each agent based on individual
cytotoxicity plots. For drug combination studies involving lapatinib, IC25 concentration of
lapatinib (i.e., the amount that induced 25% cell death by itself) was added to cultures containing
increasing concentrations of the second agent. The new IC50 values corresponding to the
combination were then calculated and used to derive combination index (CI) values as described
previously (Chou 2010). A CI of less than 1 indicates synergy between the two agents under the
experimental conditions used.
64
3.3.4 Intracellular signaling studies
Atypical teratoid/rhabdoid tumor cells were grown to approximately two-third confluence
in six well culture plates (Nunc, Rochester, NY) and the culture medium was replaced with fresh
medium containing lapatinib or drug combinations as indicated in individual experiments. After
incubation for 12 h, the cells were washed with ice cold PBS and lysed in buffer containing
50 mM Tris, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate with
phosphatase and protease inhibitors (Sigma, Oakville, ON). Protein concentrations of the lysates
were quantified by BCA Protein Assay (Pierce, Rockford, IL, USA). Proteins were then
separated on a 10% polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC)
membranes (Bio-Rad, Mississauga, ON). The membranes were blocked for 2 h at 4 °C with 5%
skim milk powder in PBS containing 0.1% Tween-20 (Sigma). The blots were incubated with
primary antibodies (Cell Signaling Technology, Danvers, MA) overnight at 4 °C, washed and
probed with appropriate secondary antibodies conjugated to horseradish peroxidase (HRPO)
(Sigma), followed by a luminol based substrate (Mandel, Guelph, ON) and developed by
exposure to x-ray film (Christie InnoMed, Montreal, QC).
3.3.5 In vitro cell migration assay ("scratch" test)
The scratch test to quantify inhibition in cell migration was performed as described
previously (Liang, Park and Guan 2007a). Atypical teratoid/rhabdoid tumor cells were grown to
confluence in six well culture plates (Nunc). On the day of the assay, the cell monolayer was
scraped in a straight line with a 10 µl pipette tip and the culture medium was replaced with 3 ml
of new medium or medium containing varying concentrations of lapatinib (0.001–1 µM).
65
Pictures of the scratch at the same spot of all plates were taken at various time points (at 0 h, 8 h
and 24 h) using an inverted microscope.
3.3.6 Tumor xenograft studies
3.3.6.1 Generation of BT16 cells stably expressing firefly luciferase and eGFP
BT16 cell line expressing enhanced firefly luciferase (effLuc) (Rabinovich et al. 2008)
and eGFP were generated (BT16GFPFluc) using a self-inactivating lentiviral vector encoding the
internal U3 region from mscv, effLuc, the IRES element from emcv, and eGFP (Bai et al. 2011).
Virus was packaged in 293-FT cells using pMD2.G (VSV.G env) and pCMV-deltaR8.91 and
concentrated 50 times using Amicon Ultra-15 100,000 NMWL centrifugal concentration units
(Millipore, Billerica, MA). Concentrated viral supernatants were used for transduction in the
BT16 cell line. After 72 h, eGFP expression was observed via fluorescent microscopy (Zeiss
inverted microscope, Axiovert 200 M) and used to calculate transduction efficiency by flow
cytometry on a FACS Calibur instrument (BD Biosciences, San Jose, CA). The effLuc based
bioluminescent activity was calculated using an IVIS 200 (Caliper Life Sciences, Alameda, CA).
3.3.6.2 In vivo real-time monitoring of tumor growth (bioluminescence imaging)
Six to eight week-old female CD-1 mice from Charles River Laboratories were used in
this study. All protocols were reviewed and approved by the Animal Care Committee of the
University of Calgary. All animal work procedures were in accordance with the Guide to the
Care and Use of Experimental Animals published by the Canadian Council on Animal Care and
the Guide for the Care and Use of Laboratory Animals issued by NIH.
66
Two groups of randomly assigned CD-1 Nude mice (n = 5 per group, total 10 animals)
were implanted in the flank with 5 × 106 BT16GFPLuc cells. After allowing approximately 2
weeks for the tumors to establish, tumor-bearing animals were randomly divided into two groups
for control vehicle treatment and for lapatinib treatment, which was given as a twice-daily oral
administration for 3 weeks (5-days on, 2-days off) at a dose of 160 mg/kg (320 mg/kg/day).
Xenogen IVIS 200 system (Xenogen Corporation, Alameda, CA) was used to monitor the animal
tumor growth in vivo. Every week after tumor implantation all mice were imaged to record
bioluminescent signal emitted from tumors. Anesthesia was given in an induction chamber with
2.5% isoflurane in 100% oxygen at a flow rate of 1 L/min and maintained in the IVIS with a
1.5% mixture at 0.5 L/min. The mice were then injected with d-luciferin (126 mg/kg, Xenogen
Corp.) dissolved in PBS (15 mg/ml) by intraperitoneal administration. Subsequently, mice were
placed in prone position in the IVIS instrument and bioluminescent acquisitions were collected
until the maximum signal was reached. Data were analyzed based on total photon flux emission
(photons/s) in the region of interest (ROI) over the tumor (Lun et al. 2010).
3.4 Results
3.4.1 Cytotoxicity profiling of a panel of novel therapeutic agents against AT/RT cells
A panel of targeted small molecular weight inhibitors (n = 129) was evaluated against
three AT/RT cell lines using in vitro cytotoxicity assays. These agents were selected based on
their known activities in other tumor cell systems and their potential to be used in human clinical
trials. Table 3.1 provides the IC50 values obtained in cytotoxicity studies as described in
Methods. Data provided in this table show a wide range of drug sensitivity values across the
three cell lines. There were 16 agents that showed IC50 values of 0.1 µM or less in at least two
67
out of the three cell lines. Of these, eight showed such activity in all three cell lines. Specifically,
seven agents, INK-128, carfilzomib, NVP-AUY922, AV951, BI6727, AZD1152 and YM155,
showed significant activity with IC50 values less than 0.05 µM in at least two out of the three cell
lines, however carfilzomib, NVP-AUY922 and BI6727 showed this activity in all three cell lines.
Figure 3.1A is a diagrammatic representation of these data showing the distribution of IC50
values across all 129 agents for individual cell lines. To categorize the agents with relatively high
cytotoxicity values, those with a median IC50 values less than the arbitrarily defined value of
1 µM, were selected and analyzed for cytotoxicity in an expanded range of drug dilutions (10–
10−5 µM). Data obtained from these studies are summarized in Figure 3.1B. In this figure, for
each drug treatment the vertical lines represent the lowest, median and highest values and the
horizontal lines represent the range of the IC50 values. The identities of these agents are given
along the Y axis of the graph presented. Agents inhibiting a multitude of targets are represented
in this group with the proteasome inhibitor carfilzomib showing the lowest IC50 values. This
figure also shows that certain agents such as bortezomib and NVP-AUY922 are equally effective
against all three cell lines, while others such as AZD-4547 and BIBW 2992 carry differential
sensitivity among the cell lines tested. These data have provided us with a short list of agents for
further evaluation and more extensive pre-clinical studies. Among the agents presented in
Figure 3.1A, two of the molecules, lapatinib and CUDC-101 are targeted inhibitors of HER2 and
EGFR kinases. Based on currently available data demonstrating efficacy, tolerability and the
potential for CNS penetration by lapatinib in adult clinical trials, we have selected this agent as
the first in a series of additional studies for preclinical development.
68
69
70
Table 3-1. Cytotoxicity profiling of a panel of targeted agents against three AT/RT cell lines.
Exponentially growing tumor cells were treated with four different concentrations of individual
drugs (10 µM, 1 µM, 0.1 µM and 0.01 µM) in duplicate cultures. After four days in culture, cell
growth inhibition was quantified by automated cytometer and percentage growth inhibition was
calculated in comparison to vehicle-only untreated control cells. The IC50 values are from a
single complete study and trends observed are representative of three separate experiments.
71
A.
72
B.
Figure 3-1. Differential sensitivity of AT/RT cell lines against the drug panel.
A: A schematic representation of the range of IC50 values presented in Table 1 to show that
sensitivities differ greatly among the various agents tested. The top horizontal line indicates
agents with IC50 values of 10 µM or higher (low sensitivity) and the lowest line indicates agents
that showed IC50 of 0.01 µM or less (high sensitivity). B: Expanded studies of agents with
relatively high cytotoxicity against AT/RT cells in the screening assay. Drugs that showed IC50
values of 1 µM or lower in the screening assays were further evaluated in more expanded
concentration range of 10–10−5 µM of each drug. Data obtained are summarized schematically.
For each agent the vertical lines represent the lowest, median and highest values, and the
horizontal line represents the range of IC50 values for the three cell lines tested. Names of the
agents used in this analysis are given along the Y axis. Data presented above is representative of
three separate experiments.
73
3.4.2 Activity of lapatinib against AT/RT cell lines
Atypical teratoid/rhabdoid tumor cells and the (control) non-malignant fibroblast cell line
Hs68 were treated with increasing concentrations of lapatinib and cell viability was evaluated
after four days in culture. Results presented in Figure 3.2 show that cell lines BT16 and KCCF1
are highly sensitive to lapatinib with IC50 of 0.1 µM and BT12 was sensitive for lapatinib activity
at IC50 of 5 µM. The non-malignant Hs68 cells showed no significant inhibitory effects by
lapatinib at concentrations as high as 10 µM.
74
Figure 3-2. In vitro cytotoxicity of lapatinib against AT/RT cell lines.
A: Effect of Lapatinib alone in AT/RT cell lines. Exponentially growing AT/RT and control
non-malignant fibroblast cells were incubated with increasing concentrations of lapatinib or
corresponding DMSO control in triplicate. After four days in culture, cell viability in each
condition was measured by automated cytometer and percentages of cell survival were calculated
with respect to DMSO control wells. Data presented above is representative of three separate
experiments.
75
3.4.3 Target validation of lapatinib in AT/RT cells
Next we aimed to look at the presence of the targets for lapatinib in AT/RT cells that are
highly and moderately sensitivity to its effects. We also wanted to identify the off-target effects
by lapatinib. The antibody array technique provides an effective tool to screen for inactivation of
receptor tyrosine kinases by targeted therapeutic agents. BT16 (high sensitivity) and BT12
(moderate sensitivity) cells were treated with lapatinib and the resulting dephosphorylation of 42
separate receptor tyrosine kinases (RTKs) were analyzed. Results presented in Figure 3.3 show
the loss of phosphorylation of both EGFR and other ErbB family of receptors in BT16 cells and
the loss of activity of EGFR in BT12 cells, which did not show measurable ErbB activity at
baseline. Both cell lines, however, expressed active IGF-IR that was not affected by lapatinib.
Figure 3.4 shows further analysis of the effect of lapatinib on EGFR with respect to
phosphorylated tyrosine 1173 (Y1173), which has previously been shown to be a critical
activation site. Interestingly, BT16 and KCCF1 cells expressed lower amounts of absolute EGFR
than BT12 cells, but had highly phosphorylated Y1173, suggestive of an apparent correlation
between receptor activity and lapatinib sensitivity.
76
Figure 3-3. Identification of potential targets of lapatinib activity on AT/RT cells.
Antibody arrays to a panel of receptor tyrosine kinases were used to screen for
dephosphorylation effects by lapatinib. Proteins were extracted from BT12 and BT16 cells
treated with 20 µM of lapatinib and cell lysates were made with lysis buffer containing protease
and phosphatase inhibitors. DMSO treated cells were used as control. Antibody arrays were
incubated with 100 µg of proteins overnight at 4 °C with gentle mixing and probed with HRPO
77
conjugated anti-phosphotyrosine antibodies as per manufacturer's protocol. A: Antibody array
showing changes in phosphorylation as seen by a decrease or loss of signal as indicated by
arrows. B: A diagrammatic representation of the array map showing the positions of capture
antibodies to different RTKs. The arrows are numbered in sequence to indicate EGFR in BT12
cells and EGFR, ErbB2, ErbB3, ErbB4 and PDGFRα in BT16 cells. Data presented above is
representative of two separate experiments.
78
Figure 3-4. Expression and activity of EGFR in AT/RT cells.
Exponentially growing cells were treated with lapatinib (20 µM) or DMSO control for 4 h,
washed and lysed in the presence of protease and phosphatase inhibitors and total cellular
proteins were probed for total EGFR and pEGFR by Western blot analysis. Data presented above
is representative of three separate experiments.
79
3.4.4 Target modulation effects of lapatinib on AT/RT cells
Given that antibody array studies demonstrated that the primary targets of lapatinib are
affected in treated AT/RT cells, we then investigated the downstream consequences of this effect
by looking at the modulation of critical signaling cascades linked to RTK mediated pathways. In
this study, the activity modulation of three critical signaling nodes, AKT1/2/3, ERK1/2 and Stat3, were evaluated upon exposure to lapatinib. Proteins from untreated and lapatinib treated cells
were extracted in the presence of protease and phosphatase inhibitors and the phosphorylation
status of AKT1/2/3, ERK1/2 and Stat-3 were probed with phospho-specific antibodies by
Western blot analysis. Data given in Figure 3.5 show that ERK1/2 is phosphorylated in all three
cell lines but demonstrate measurable dephosphorylation by lapatinib only in BT16 and KCCF1
cells. Secondly, measurable phosphorylation of AKT1/2/3 is seen in only BT16 and KCCF1 cells
and in both cases becomes dephosphorylated in the presence of lapatinib. These data also
categorize lapatinib activity in different AT/RT cell lines with respect to modulations in distinct
signaling pathways with corresponding drug sensitivity profile.
80
Figure 3-5. Intracellular signaling and lapatinib activity.
Atypical teratoid/rhabdoid tumor cells were treated with lapatinib (20 µM) or DMSO control for
4 h, washed and lysed in the presence of protease and phosphatase inhibitors and total cellular
proteins were probed for total and phosphorylated forms of ERK, AKT and Stat-3. Findings
presented above are representative of two experiments.
81
3.4.5 Synergistic activity of lapatinib with IGF-IR inhibitors
Previous studies have demonstrated that IGF-IR activity contributes to the growth and
survival of AT/RT cells. The antibody array analysis presented in figure 3.3 also showed the
activated status of IGF-IR in both BT12 and BT16 cells. These data provided a mechanistic
rationale to investigate the hypothesis that a combined inhibition of lapatinib with IGF-IR
inhibitors would show synergy against these cells. In the next set of experiments, we investigated
lapatinib and the targeted IGF-IR inhibitor AEW541 (Novartis Pharma AG, Basel, Switzerland)
in drug combination studies. Studies of lapatinib in combination with the targeted IGF-IR
inhibitor AEW541 were carried out as described in Materials and Methods. A graphic
representation of cell survival when treated with drug combination is given in Figure 3.6. The
combination indices (CI) (Chou 2010) calculated from these experiments are given in Table 3.2.
In this analysis, a CI value equals to 1, less than 1 and more than 1 indicates additive, synergistic
and antagonistic effects, respectively, between the two agents. Values presented in Table 3.2
show synergy between lapatinib and AEW541. Lower CI values seen in BT16 and KCCF1 cells
also indicate that these cell lines are most susceptible to the combined effect of lapatinib and
IGF-IR inhibition.
82
A.
Figure 3-6. Drug combination study of Lapatinib with AEW-541 in AT/RT cell lines.
A: In vitro synergy of lapatinib-induced cytotoxicity with the IGF-IR inhibitory agent AEW541.
AT/RT cells were incubated with increasing concentrations of AEW541 alone or increasing
concentrations of AEW541 (X axis) plus a constant IC25 concentration of lapatinib. Cell growth
inhibition was measured after four days in culture as describe above. The IC25 values of lapatinib
used were 2 µM, 0.01 µM and 0.01 µM for BT12, BT16 and KCCF1 respectively that were
calculated from Figure 3.2. Data presented above is representative of three separate experiments.
83
B.
Cell lines
AEW-541+ Lapatinib
(CI)
BT12
0.70
BT16
0.3
KCCF1
0.22
Table 3-2. Activity of combined IGF-1R inhibition and lapatinib against AT/RT cells.
IC50 values for single agent AEW541 and in combination with lapatinib were calculated from
data presented in Figure 3.6 and used to calculate combination indices according to the method
of Chou and Talalay (Chou 2010). A CI value less than 1 indicates drug synergy under the
specific experimental conditions used.
84
3.4.6 Lapatinib inhibits BT16 cell migration in vitro
In addition to the cytotoxicity effects observed after four days of exposure to lapatinib,
we evaluated the effects of lapatinib on the migration of AT/RT cells during a shorter time
period, presumably before the initiation of apoptosis. In the scratch assay, the movement of cells
across a scratch line is evaluated as an indication of the capability of an agent to inhibit cell
migration. Photographs presented in Figure 3.7A show a concentration-dependent loss of cell
migration over the scratch line when treated with lapatinib, demonstrating its potential ability to
prevent BT16 cell migration. The similar trend was observed in KCCF1 cell line. Cell migration
into the detection zone was quantified by counting cell number using ImageJ software
(http://rsb.info.nih.gov/ij/) (version 1.4.3.67). To differentiate cell proliferation versus cell
migration to account for the additional cells in the scratch zone, viable cell count was measured
by Alamar blue assay and a graphic representation for cell migration with corresponding cell
counts are given in Figure 3.7B.
85
A.
86
B.
Figure 3-7. In vitro cell migration assay.
A: BT16 cells were plated in 6-well plate and a scratch was introduced when cells were 80%
confluent. Images were acquired at 0 h, 8 h and 24 h following the in vitro scratch assay. The
dotted lines define the areas lacking cells. B: The rate of migration was measured by quantifying
the total distance that cells moved from the edge of the scratch toward the center of the scratch
(marked by imaginary dotted lines). After 24 h in culture, the quantity of viable cells in each
condition was measured by Alamar blue assay. Data presented above are representative of three
separate experiments.
87
3.4.7 In vivo activity of lapatinib
It is likely that cells that express highly active ErbB2 will be more susceptible to
treatment with lapatinib or similar agents. The BT16 cell line represents such a tumor phenotype.
Hence, we selected this cell line for the next proof-of-concept in vivo studies. BT16 cells were
labeled with luciferase and GFP (BT16GFPLuc) and used to generate xenografts in CD-1 Nude
mice. After allowing sufficient time for tumors to establish, randomly assigned groups received
either lapatinib or vehicle control. Lapatinib was given as a twice-daily oral administration for 3
weeks (5 days on, 2 days off) at a dose of 160 mg/kg (320 mg/kg/day). The tumor growth
differences between the two groups were measured at weekly intervals by total flux emission
photon/second as described in methods. Images presented in Figure 3.8A show luminescence
imaging of the two groups of mice. The mice that received lapatinib show measurably less signal
intensity indicating the inhibition of tumor growth. Quantitative analysis of this data presented in
Figure 3.8B show growth inhibition of the tumors compared to the continuous increase seen in
control animals. On day seventeen the animals were sacrificed and the tumors were removed.
Figures 3.8C and D show the fluorescent and gross images of the tumors respectively, illustrating
the antitumor effect of lapatinib on tumor xenografts.
88
Figure 3-8. Lapatinib inhibits the tumor activity in vivo.
Tumor cells labeled with luciferase and GFP (BT16GFPLuc) were implanted in the flank
(5 × 106 cells per animal) of mice to generate xenografts. After allowing 10–14 days for tumors
to establish, two randomly assigned groups received either lapatinib or vehicle control. The drug
was given as a twice-daily oral administration for 3 weeks (5 days on, 2 days off) at a dose of
160 mg/kg (320 mg/kg/day). Xenogen IVIS 200 system (Xenogen Corporation, Alameda, CA)
was used to monitor and quantify tumor growth in vivo (A, B). Day 0 = first day of treatment, i.e.
2 weeks after implantation. After 17 days of treatment the animals were sacrificed and the
tumors were removed and photographed under a dissection microscope with a fluorescent filter
or (C) or white light, where a scale was included for size comparison (D).
89
3.5 Discussion
Atypical teratoid/rhabdoid tumor is currently considered to be one of the most malignant
and difficult to cure tumors in the pediatric population. Although defects in the chromatin
remodeling apparatus by the SWI/SNF complex is likely to be the key molecular feature in
AT/RT, the pathways and nodes that constitute deregulated growth regulatory mechanisms are
critical for the identification of effective targets for future therapeutics. We report the initial
screening of a comprehensive library of targeted therapeutic agents using in vitro cytotoxicity
assays. Our data show that among these, there are individual agents, as well as the inhibition of
specific growth regulatory pathways, that can significantly interfere with the growth and survival
of these cells. Such an approach also provides essential initial data that can be further developed
by different research groups with expertise in diverse experimental systems and new agents to
complete additional preclinical studies without delay.
Among the agents that have been found to be effective, we selected lapatinib for further
studies for a number of reasons. Recent clinical trials in adults have shown tolerability of this
agent (de Souza et al. 2012) and it has been suggested to possess the ability to cross the blood–
brain barrier, which supports its use in patients with CNS tumors (Mukohara 2011). However,
drug distribution during treatment for brain metastases of breast cancer appears to be partially
restricted by blood-tumor barrier permeability (Taskar et al. 2012). Importantly, in one of the
earlier studies, the targets of lapatinib, EGFR and ErbB2, have been found in 3/7 and 6/7 of
AT/RT primary tumor specimens, respectively (Patereli et al. 2010). The contribution of offtarget effects notwithstanding, three independent agents that target ErbB2 family, lapatinib,
CUDC101 and canertinib, showed significant cytotoxicity in our screening, suggesting the utility
of targeting these receptors in AT/RT (Table 3.1; Figure 3.1). The IC50 values of lapatinib in the
90
AT/RT cells were similar for two of the three cell lines, BT16 and KCCF1, but higher for the
BT12 cell line (Table 3.1, Figure 3.2). These values are within the range reported for susceptible
Her2-positive breast cancer cell lines (O’Donovan et al. 2011). The higher IC50 of BT12 cells is
closer to the values described for lapatinib hypo-responsive breast cancer cell lines MDA-MB468 and T47D, which express low basal levels of ErbB2 and the IC50 seen for BT16 and KCCF1
cells are similar to the lapatinib-responsive lines, BT474 and SKBr3, that constitutively overexpress ErbB2 (Hegde et al. 2007). These findings are in line with the molecular and phenotypic
heterogeneity of AT/RT and underscore the importance of target validation studies in the
stratification of patients for Her2 EGFR-based therapies in the future.
Data from the antibody array studies showed loss of activation signals of the previously
described lapatinib targets EGFR, ErbB2 and ErbB4 (Figure 3.3). We have also noted loss of
signals with respect to ErbB3 and PDGFRα. However, the exact mechanisms for the additional
activity against ErbB3 and PDGFα are currently unclear. It is possible that these effects may be
due to the ability of lapatinib to interfere with dimerization of Her family of receptors and to
disrupt previously formed receptor dimers (Sánchez-Martín and Pandiella 2012), or due to
previously not described off-target effects of this agent.
Autophosphorylation at the tyrosine residue 1173 (Tyr 1173) has been shown to be a
significant event and important for signal transduction following ligand binding and receptor
dimerization of EGFR (Chattopadhyay et al. 1999). Data presented in Figure 3.4 show effective
dephosphorylation of Y1173 by lapatinib in the two highly sensitive lines BT12 and KCCF1,
although there appears to be EGFR phosphorylation in all three cell lines, as determined by panphospho antibodies in Figure 3.3. This prompted us to further evaluate the effect of lapatinib on
intracellular signaling pathways in relation to EGFR and ErbB2 activities (Figure 3.5). In these
91
studies, a loss of ERK and AKT activities was seen in the highly sensitive and ErbB active cells
(BT16 and KCCF1). However, although all three cell lines expressed Stat-3 with detectable
phosphorylated bands, no significant changes in response to lapatinib was seen in Stat-3. The
major signaling cascades that are initiated as a consequence of EGFR and ErbB activation are
thought to be mediated by PI3, RAS-RAF (MAPK), JNK and PLCγ kinases (Eccles 2011).
Consequently, these activities lead to a multitude of cellular functions necessary for the growth
and survival of tumor cells. It is known that AKT and ERK functions are critical for the flow of
many of these pathways. Our findings are also in agreement with the microarray and
phosphorylated protein findings of Hedge and colleagues, in which phosphorylation and gene
expression changes in breast cancer cell lines in response to lapatinib were explored (Hegde et
al. 2007). This study showed that the cells highly responsive to lapatinib significantly downregulated a number of transcripts, including AKT1, whereas the non-responsive lines only weakly
down-regulated the AKT pathway. Phosphorylated AKT also decreased in response to lapatinib.
Furthermore, gene expression profiling showed that lapatinib modulated many of the genes
involved in cell cycle control and in the regulation of metabolic pathways such as glycolysis and
fatty acid metabolism (Hegde et al. 2007). Metabolomic studies are currently in progress in our
laboratory to evaluate such an effect in AT/RT cells, especially the ways in which such changes
can be monitored in the CSF of patients who may receive ErbB2-directed treatments in the
future.
Findings from the cell motility inhibition studies (scratch tests) demonstrated that lapatinib
inhibits the movement of BT16 cells in a concentration-dependent manner within hours
(Figure 3.7). Previous reports have provided evidence that, in addition to their positive
contribution to cell proliferation, activation of EGFR-ErbB2 receptors also promote cell adhesion
92
and motility (Freudenberg et al. 2009). In the breast cancer model, a multitude of studies have
shown that HER2 in metastatic cells promote cell motility (Eccles 2011). A recent investigation
by Siedel and colleagues has shown that breast cancer cells that express HER2 neu+ and not the
HER2-neu
(+/−)
phenotype show decreased invasion and loss of anchorage when treated with
lapatinib. This appears to be independent of the surface receptor CUB domain-containing protein
1 (CDCP1) activity (Seidel et al. 2011). In the glioma tumor model, it has been found that
lapatinib interferes with cellular migration through the interruption of EGFR-integrin β(1)
complex formation (Dimitropoulos et al. 2010). Frequently AT/RT presents with significant
infiltration into brainstem, making tumor resection a difficult task. Recently, a case report by
Beschorner and colleagues described a child who received incomplete resection, followed by
multi-modal therapy, but presented with a second tumor, possibly a late metastasis of the original
AT/RT. This lesion appearing along the right trigeminal nerve was not in continuity with the
primary tumor (Beschorner et al. 2006). Although the rare possibility of a radiation-induced
second AT/RT cannot be ruled out, this case reveals the potential of aggressive AT/RT cells for
invasion and migration.
Lapatinib has shown promising result in trastuzumab-refractory metastatic breast cancer in
Phase I, II and III studies and a number of trials are currently in progress to further understand its
utility in CNS metastasis. Further studies are needed to define the contribution of inhibition of
tumor migration in the context of surgical and overall management of AT/RT in the future.
Generation of resistance to RTK-targeted therapeutics has been a major obstacle in the
utility of this family of agents. In addition, tolerability concerns have also limited the
effectiveness of single agent RTK-targeted therapies in the past. Our current results (Figure 3.6,
Table 3.2) as well as previously published studies have alluded to the critical role of IGF-IR
93
activity in AT/RT cells (D’cunja et al. 2007). As lapatinib has shown strong growth inhibition in
only two of the three cell lines studied, we wanted to investigate the effect of combining IGF-IR
inhibition with lapatinib, especially for tumors that may have lower Erb expression. Our in vitro
studies show enhanced activity in all three cell lines with combination indices less than 1,
suggesting drug synergy under the experimental conditions used ( Table 3.2). In the recent past,
effective drug combinations with lapatinib have been explored in a number of tumor models. For
example, combining lapatinib with the notch inhibitor (γ-secretase inhibitor) MRK-003 GSI
showed significant reduction of tumor growth in ErbB-2-positive breast cancer xenografts
(Pandya et al. 2011). Treatment of animals carrying orthotopic CNS tumor isolates with lapatinib
and Bcl-2 homology domain-3 (BH3) mimetic obatoclax (GX15-070) prolonged survival of
these animals (Cruickshanks et al. 2012). The ability of lapatinib to synergize with HDAC
inhibitors has been shown in previous studies (LaBonte et al. 2011). Interestingly, in breast
cancer cells, the failure of the EGFR inhibitor transtuzumab appears to be mediated by the
upregulation of IGF-IR and lapatinib may actively block Erb2 and IGF-IR cross talk in
transtuzumab-resistant cells (Nahta et al. 2007). It is conceivable that lapatinib also blocks IGFIR and ErbB2 cross talk in AT/RT cells, thus providing an additional mechanism to enhance the
effect of a combined inhibition of these two pathways. Additional studies are needed to
experimentally demonstrate this interesting activity. Such information including in vivo studies
using combined lapatinib and AEW-541, is crucial for the development of future drug
combination therapies to optimize cell killing and reducing toxicity and the potential for drug
resistance.
In the following set of experiments, in vitro cytotoxic activity of lapatinib was further
evaluated using in vivo xenograft experiments (Figure 3.8). Our initial studies used the cell line
94
that expressed all targets as we envision that such tumors will be most suitable for future clinical
studies with lapatinib. We used a regimen of twice-daily oral administration for 3 weeks (5 days
on, 2 days off) at a dose of 160 mg/kg (320 mg/kg/day). This dose was based on previous studies
including a report by Gorlick and co-workers who evaluated lapatinib for activity in pediatric
tumor xenografts (Gorlick et al. 2009). Our findings are in agreement with previous xenograft
studies of other ErbB2 and EGFR over-expressing tumor models (Rusnak et al. 2001a; Konecny
et al. 2006). Future xenograft studies are needed to evaluate drug combinations that would
benefit the complete spectrum of EGFR ErbB-expressing AT/RT tumors. In our studies,
lapatinib alone gave significant tumor kill at low and non-toxic concentrations, making data from
in vivo drug combination studies difficult to interpret. It has been suggested that the utility of a
IGF-IR inhibitors will be of significance for patients who have generated treatment resistance
(Jin and Esteva 2008; Abraham et al. 2011). We are currently in the process of generating
variants of AT/RT cell lines to test this hypothesis in future studies.
Abnormal expression and activity of ErbB family of proteins have been described in a
number of tumors and are central in the development, metastasis and treatment of breast cancer.
Our report provides evidence for the first time that these molecules present an effective target for
therapeutics in at least a sub group of CNS AT/RT. Although there are reports showing the
existence of ErbB family of proteins in rhabdoid tumors, additional studies are needed in an
expanded cohort of specimens to precisely define the incidence of ErbB expression and
activation in AT/RT. Studies are currently in progress in our laboratory using
immunohistochemical analysis of tissue microarrays (TMA) of CNS AT/RT specimens. In
addition to the effects on ErbB family of proteins, potential off-target effects of lapatinib need to
be evaluated. For example, a recent report by Dolloff and colleagues has shown the effect of
95
lapatinib on TRAIL death receptor expression and signaling that is independent of EGFR and
HER2 inhibition (Dolloff et al. 2011). Importantly, information is also needed on the pathways
that may link the loss of INI1 to the activity of these molecules leading to an aggressive tumor
physiology. For instance, recent reports indicate that EGFR may act as a transcriptional regulator
of cyclin D1 (Burness et al. 2010; Eccles 2011), and the loss of INI1 has been known to lead to
derepression of cyclin D1 in rhabdoid tumors (Smith, Das and Kalpana 2011). Targeting ErbB
should be further evaluated in the context of patients who are receiving other treatment
modalities such as surgical debulking. This is important as it has been postulated that ErbB2
positive breast cancers show proliferative responses to growth factors found in postsurgical
wound fluids and this can be blocked by anti-HER2 antibodies (Tagliabue et al. 2003). Recent
studies using high-resolution genome-wide analysis have failed to show recurrent genomic
alterations other than SMARCB1 in AT/RT (Hasselblatt et al. 2013). In addition Kieran and
colleagues have examined tumor specimens with INI1 alterations for changes in oncogenes and
tumor suppressor genes and found no evidence of mutations in canonical pathways critical for
adult cancers (Kieran et al. 2012). Although these findings may limit the possibility of
identifying single effective agents based on highly prevalent molecular abnormalities, an
extension of the approach of screening diverse drug libraries can provide an avenue to identify
effective agents based on operational growth regulatory pathways and individually tailored,
tumor-defined therapeutic interventions. Approaches to target epigenetic modifiers with small
molecule inhibitors have led to the availability of novel candidate epidrugs which can also be
used in similar studies (Andreoli et al. 2013). Interestingly, a recent study demonstrating the
suppression of HER-2 receptor in subgroups of breast cancer after treatment with 5-aza-2′-
96
deoxycytidine (DAC) (Radpour et al. 2011) indicates the feasibility of effective evaluation such
agents in novel therapeutic opportunities.
In summary, as a proof-of-concept, we present a preclinical study pathway to identify
novel therapeutic regimens for rare pediatric malignancies such as CNS AT/RT. The selection of
agents to screen comes from a library of drugs that have been validated in a range of more
common adult tumors. The safety and dosing data that are becoming available on these drugs
provides the feasibility of the successful candidates to be considered for early phase clinical trials
in a timely manner. Most often these agents already have key information such as CNS
penetration data, potential toxicities and pharmacokinetic profiles. Secondly, initial in vitro
studies in representative cell lines help to gain preliminary data on potential drug combination
and treatment schedules that can be further validated in additional xenograft studies. Intracellular
target modulation experiments provide information that can be incorporated into selecting the
most targetable patient population based on the analysis of pre-treatment biopsy specimens.
Importantly, the identification of multiple active agents and survival pathways in early
preclinical studies is practical and expedient for “pick the winner” trial designs that are needed
for the treatment of rare cancers such as AT/RT.
3.6 Acknowledgment
This research was supported by grants from the Kids Cancer Care Foundation of Alberta
(KCCFA) Alberta Children's Hospital Foundation (ACHF) and the Brain Tumor Foundation of
Canada. AJ holds a Canadian Institutes for Health Research (CIHR) Training Program in
Genetics, Child Development and Health Graduate Studentship awarded by Alberta Children's
97
Hospital for Child and Maternal Health (ACHRI) and a McCarthy Tetrault Graduate Studentship
awarded by ACHF.
3.7 References
References are added to the end in this thesis.
98
Chapter Four: In vitro sensitivity profiling of neuroblastoma cells against a comprehensive
small molecule kinase inhibitor library to identify agents for future therapeutic studies
In the previous Chapter, we described the drug screen approach to identify potential
therapeutic agents for AT/RT treatment. In this report, we focused on the distinct biological
characteristics of NB and identified selective therapeutic agents using a comprehensive panel of
clinically feasible drugs. Results summarized in this report1 present a preclinical evaluation
pathway to identify novel therapeutic agents for refractory NB. Our data provide initial proof-ofconcept information on the potential utility of ponatinib as an effective targeted therapeutic agent
against refractory NB.
Author’s contribution: Anjali Singh performed and analyzed all the experiments with the
support of her supervisor, Dr. Aru Narendran. Aarthi Jayanthan participated in manuscript
preparation and assisted with the preparation of cell culture. All authors have reviewed and
approved the final manuscript. The original publication has been reproduced as a Chapter Four in
this thesis. Figures and references have been reformatted according to the guidelines outlined by
the University of Calgary Faculty of Graduate Studies.
1
Anjali Singh, Aarthi Jayanthan and Aru Narendran. In vitro sensitivity profiling of
neuroblastoma cells against a comprehensive small molecule kinase inhibitor library to identify
agents for future therapeutic studies. Current Cancer Drug Targets, submitted.
99
4.1 Abstract
Solid tumors represent one of the most widespread causes of death in children across the
world. Neuroblastoma constitutes about 8% of all childhood tumors, yet accounts for more than
15% of death and an unacceptable overall survival rate of only one in five patients. Despite the
current multimodal therapeutic approaches involving surgery, radiation, chemotherapy with
myeloablative therapy and hematopoietic stem cell rescue, there is growing realization of the
limitations of conventional agents to improve the outcome in high risk metastatic disease. Hence,
efforts have intensified to identify new targets and novel therapeutic approaches for the treatment
of these children. Among the significant number of new therapeutics that are being evaluated for
cancer each year, the agents that have been developed for common adult malignancies have the
added advantage of having usable toxicity data already available for consideration. To identify
potential therapeutic targets, we screened a small molecule library of 151 small kinase inhibitors
against NB cell lines. Based on our initial screening data, we further examined the potential of
Bcr-Abl targeting small molecule inhibitors to affect the growth and survival of NB cells. Our
findings confirm the diversity in activity among the currently available Bcr-Abl inhibitors,
possibly reflecting the molecular heterogeneity and off-target activity in each combination. In
depth analyses of ponatinib, an orally bioavailable multi-target kinase inhibitor and an effective
agent in the treatment of refractory Philadelphia chromosome (Ph) positive leukemia, show
growth inhibition at sub-micromolar concentrations. In addition, we also identified the potential
of this agent to interfere with insulin-like growth factor-1 receptor (IGF-1R) mediated growth
regulatory pathways and Src activity. Ponatinib also induced apoptosis, indicated by activation of
caspase 9 and PARP cleavage. Furthermore, at sub lethal conditions ponatinib significantly
inhibited the ability of these cells to migrate. Our findings provide initial data on the potential of
100
ponatinib to target key growth regulatory pathways and provide the rationale for further studies
and its evaluation in future early phase clinical trials for the treatment of refractory NB.
4.2 Introduction
Neuroblastoma is one of the most frequent and difficult to treat cancers in pediatrics and
ranks high among the diseases with an unacceptable fatality rate. Although cure rates have
improved greatly over the past two decades, event-free survival still remains below 50% for
patients with high-risk metastatic disease (Matthay, George and Yu 2012). Hence, novel research
strategies are urgently needed to identify therapeutic targets to advance the timely development
of innovative treatment approaches for these children. Known molecular aberrations in NB
include N-Myc oncogene amplification or allelic loss, near triploid karyotype, deletion of short
arm of chromosome 1, chromosomal rearrangements involving chromosome 11q and abnormal
expression of neurotrophin receptors (Davidoff 2012). Currently, the management of NB
requires a multimodal treatment approach including surgical debulking, chemotherapy,
radiotherapy, as well as biological and immunological therapies (Gains et al. 2012). Advancedstage tumors and those with MYCN gene amplification typically show the emergence of
treatment resistance and are often associated with aggressive disease progression. Continuing
investigations are focusing on uncovering informative genetic features and growth regulatory
pathways associated with poor outcome.
Constitutive activation of receptor tyrosine kinases (RTKs) caused by gain-of-function
mutation, fusion with a partner protein or by autocrine or paracrine means, leads to increased
tumor growth and metastasis. The existence of many such aberrant RTK-mediated growth
regulatory pathways has been reported in NB including the stimulatory loop promoting the
101
activity of IGF-1R (El-Badry et al. 1989). A functional implication of this mechanism in NB was
confirmed by antisense RNA and blocking antibody studies (Liu et al. 1998; Maloney et al.
2003). Such findings have prompted the evaluation of IGF-1R mediated pathway components as
targets for therapeutics in NB (Tanno et al. 2006).
The Bcr-Abl chimeric protein plays a predominant role in Philadelphia (Ph) chromosomepositive leukemia and has led to the development of a number of effective targeted therapeutic
agents in the recent past (Mughal et al. 2013). The tyrosine kinase Src is a cytoplasmic/ nonreceptor tyrosine kinase (TK) that is over-expressed or hyper-activated in a variety of solid
tumors(Brunton and Frame 2008). Specifically, c-Src has been reported to contribute to the
differentiation, cell adhesion and survival of NB cells and considered to be a prospective target
for therapeutics (Navarra et al. 2010; Radi et al. 2011). As Bcr-Abl and Src share significant
sequence homology and hold significant structural resemblance, the compounds originally
designed as potent inhibitors of Bcl-Abl also carry the exciting possibility to inhibit Src mediated
pathways in cancer cells (Musumeci et al. 2012). For example, the Bcr-Abl inhibitors imatinib
and dasatinib have shown to have anti-NB activity both in vitro and in vivo pre-clinical studies
(Vitali et al. 2003; Beppu et al. 2004; Meco et al. 2005). Ponatinib (AP24534, Ariad
Pharmacutical) is an orally bioavailable multi-tyrosine kinase inhibitor that has shown effective
activity against primary and mutated forms of Bcr-Abl (Cortes et al. 2012). An in vitro studies
have demonstrated the additional capability of ponatinib to inhibit a wide range of kinases
involved in the growth, survival and metastasis of cancer cells. For instance, studies by Gozgit
and colleagues have found that ponatinib blocks in vitro kinase activity of FGFR (Gozgit et al.
2012). In addition, in the BaF3 cell model, ponatinib has been shown to inhibit the
phosphorylation and activity of downstream effectors, such as PLCγ, Stat5 and Src (Ren et al.
102
2013b). In this study, we used an in vitro drug screening approach to examine the effect of 151
small kinase inhibitors on NB cells and finally evaluated the cytotoxicity profile and target
modulatory activities of ponatinib against a panel of NB cell lines. The purpose of this
investigation is to provide initial proof-of-concept data and biological correlates of activity to
support further studies leading to the formulation of early phase clinical trials in the near future.
4.3 Material and Methods
4.3.1 Cell lines and cell culture
The following NB cell lines were used: SK-N-AS (ATCC-CRL-2137), SK-N-SH (ATCC
HTB-11), SK-N-BE(2) (ATCC CRL- 2271), IMR-32 (ATCC CCL-127), SHEP, IMR-5. SHEP
and IMR-5 cell lines were a gift from Dr. Herman Yeger (The Hospital for Sick Children,
Toronto, ON, Canada). These cells were maintained in Opti-MEM media (Gibco, Invitrogen
Corporation, Burlington, ON, Canada) supplemented with 5% fetal bovine serum and 100
units/ml penicillin and 100 units/ml streptomycin (Gibco). Confluent cells were trypsinised with
0.25% Trypsin-EDTA in Ca2+ and Mg2+ free balanced salt solution (Gibco) every three to five
days. All cell cultures were maintained in incubators at 37 °C in a humidified atmosphere with
5% CO2.
4.3.2 Small molecule kinase inhibitors
A library of small molecule kinase inhibitors consisting of 151 drugs active against at
least 65 individual kinases was synthesized, purity checked and provided by ChemieTek
(Indianapolis, IN, USA). These agents were dissolved in DMSO to a final concentration of 10
103
mM and stored frozen at -20oC and diluted appropriately in culture medium at the time of the
study.
4.3.3 In vitro cytotoxicity assays
NB cells were trypsinized and placed in 96 well plates (Grenier Bio One, Monroe, NC,
USA) at a concentration of 5 X 103 cells per well. Increasing concentrations of study agents and
a corresponding dilution of DMSO were added to a final volume of 200 µl per well. After four
days in culture, cell survival was quantified by an inverted microscope (Cyntellect Inc, San
Diego,
CA,
USA;
http://www.nexcelom.com/Celigo/direct-cell-counting-assays-for-
immunotherapy.php#feature6). The half maximal inhibitory concentration (IC50) values were
calculated for each agent based on individual cytotoxicity plots.
4.3.4 Human antibody array
NB cells were seeded in six well culture plates (Nunc, Waltham, MA, USA) at 1 X 106
cells/ml and incubated overnight. Fresh culture medium containing ponatinib or vehicle control
was added and after a four-hour incubation, cells were washed with ice cold PBS and treated
with lysis buffer (50 mM Tris, 5mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium
deoxycholate) containing phosphatase and protease inhibitors (Sigma, Oakville, ON, Canada).
Human phopsho-RTK array (R&D Systems, Inc., Minneapolis, MN, USA) were incubated with
ponatinib-treated and control cell lysates (150 µg) overnight, washed and probed with HRPO
labeled anti-pTyr antibodies according to manufacturer’s protocol. The arrays were scanned and
the
spot
densities
were
quantified
with
http://rsb.info.nih.gov/ij/ (version 1.4.3.67)).
104
ImageJ
(National
Institutes
of
Health,
4.3.5 Western blot analysis for protein and phosphorylated protein detection
Each NB cell line was grown to 70 to 80% confluence in six well culture plates (Nunc)
and incubated overnight to allow for cell adherence. The cells were then incubated with fresh
culture medium containing ponatinib or vehicle control as indicated in individual experiments.
After a four-hour incubation, cells were washed with ice cold PBS and treated with lysis buffer
containing phosphatase and protease inhibitors. Protein concentrations of the lysates were
quantified by BCA Protein Assay (Pierce, Rockford, IL, USA). Proteins were then separated on
an 8% polyacrylamide gel by electrophoresis and transferred onto nitrocellulose (NC)
membranes (Bio-Rad, Mississauga, ON, Canada). The membranes were blocked for two hours at
4°C with 5% skim milk powder in PBS containing 0.1% Tween-20 (Sigma). The blots were
incubated with appropriate primary antibodies overnight at 4oC, washed and probed with
relevant secondary antibodies conjugated to horseradish peroxidase (HRPO) (Sigma) followed
by luminol based substrate (Mandel, Guelph, ON, USA) and developed by exposure to x-ray
film (Christie InnoMed, Montreal, QC, Canada).
4.3.6 In vitro cell migration assay (“scratch” test)
Previous studies have shown that the NB cell line SK-N-AS is highly relevant for cell
migration and metastasis analysis (Khanna et al. 2002; Yoon et al. 2008; Feduska et al. 2013).
For instance, using comparative analysis of subcutaneous and adrenal orthotopic models, Khanna
and colleagues have demonstrated appropriate cellular biology of SK-N-AS initiated tumors
including angiogenic properties and the ability for distance metastasis (Khanna et al. 2002). The
test to quantify inhibition in cell migration was performed as described previously (Liang, Park
and Guan 2007b). Briefly, NB cells were grown to 80-90% confluence in six well culture plates
105
(Nunc) and on the day of the assay, the cell monolayer was scraped in a straight line with a 10 µl
pipette tip and the culture medium was replaced with 3 ml of new medium containing varying
concentrations of ponatinib (0.01 - 1 µM) or vehicle control. Pictures of the scratch at the same
spot were taken at various time points (0 hr, 8 hr and 24 hr) using an inverted microscope. Cell
migration into the detection zone under each condition was quantified by using ImageJ software
(http://rsb.info.nih.gov/ij/) (version 1.4.3.67).
4.4 Results
4.4.1 cytotoxicity profiles of small molecule kinase inhibitors against NB cell lines
Neuroblastoma cell lines were tested against a panel of targeted small molecular weight
inhibitors (n = 151) in a four-­‐point dilution series (0.01, 0.1, 1 and 10 µM). These inhibitors were
selected based on their known activities in other types of malignancies and their potential to be
used in clinical trials. After four days in culture, cell viability was quantified by automated
microscopy and percent growth inhibition was determined by comparison to cells treated with
DMSO. Table 4.1 summarizes the IC50 values determined from cytotoxicity study. Those agents
exhibiting IC50 values less than 1 µmol/L in at least 4 cell lines were considered effective for
inhibiting NB cell growth and proliferation.
Among the 151-kinase inhibitors tested, 45 inhibitors demonstrated IC50 values less than
1 µM in the majority of the cell lines tested. These inhibitors target several kinases including
Aurora kinases (VX-680, AZD1152), HER2/EGF-R (CUDC-101), mTOR (Pp242, AZD8055,
INK128, PF-4691502, GDC-0980), IGF-1R (BMS-754807), HDACs (LBH-589, MS-275,
MGCD0103, vorinostat, belinostat), c-Met (PF-2341066, ARQ197, foretinib), topoisomerases
(SN-38, topotecan, etoposide), nucleic acid synthesis (gemcitabine, doxorubicin), proteasomes
106
(bortezomib, carfilzomib), HSP90 (17-DMAG, 17-AAG, NVP-AUY922), JAK (AZD1480),
tubulin stabilization (docetaxel, paclitaxel), PI3K (PIK-75, NVP-BKM120), MEK1/2
(PD0325901, RDEA119, GSK 1120212, ARRY-162, Cobimetinib), Bcr-Abl (ponatinib), VEGFR/PDGF-R (Sunitinib), Src (AZD05030, Dasatinib), PLK (BI 2536, BI 6727, GSK461364),
checkpoint kinases (AZD7762, SCH900776) CDKs (PD-0332991, Dinaciclib), BET (JQ1, IBET151), NPM-ALK (NVP-TAE684), survivin (YM155), ERK2 (VX-11e) and NEDD8activating enzyme (MLN4924).
This data is graphically represented in Figures 4.1A, B and C with each graph showing
the calculated IC50 values across all 151 inhibitors tested against each cell line. The results from
this study identified several potential novel therapeutics targets for NB and we focused on BcrAbl inhibitor for further studies because of the limited information on its role as a potential
therapeutic target for NB.
107
Number
Inhibitor
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
ZM 447439
VX-680
AZD1152
Lapatinib
Canertinib (CI-1033)
BIBW 2992 (Tovok)
BMS-599626
Gefitinib (Iressa)
Erlotinib, Hydrochloride
CUDC-101
WZ4002
Dacomitinib
Pp242
Rapamycin (Sirolimus)
AZD8055
INK128
OSI-027
PF-04691502
GDC-0980
Deforolimus
OSI-906
BMS-754807
LBH-589 (Panobinostat)
MS-275
MGCD0103
Vorinostat (SAHA)
Belinostat (PXD101)
Tubacin
Tubastatin A
MK-2206
GSK690693
Ramatroban (Bay u3405)
TM30089
PF-2341066
ARQ 197 (Tivantinib)
BMS-777607
EMD1214063
PF-04217903
Foretinib
SN-38
Topotecan (Hycamtin)
Etoposide
Capecitabine (Xeloda)
Pemetrexed Disodium (Alimta)
Gemcitabine, HCl (Gemzar)
Doxorubicin (Adriamycin)
CVT-6883
CGS 21680
Bortezomib (Velcade)
Carfilzomib (PR-171)
Activity
Aurora Kinase
Inhibition
HER2/EGF-R
Inhibition
mTOR Inhibition
IGF-IR Inhibition
HDAC Inhibition
Akt Inhibition
CRTH2-R Antagonist
c-MET Inhibition
Topoisomerase
Inhibition
NA Synthesis
Inhibition
Adenosine Receptor
Antagonist
Proteasome
Inhibition
Cell line (IC 50 (µmol/L)
IMR-5
1
0.01
0.01
5
4
2
10
6
10
0.08
10
5
1
7
0.09
0.05
1
0.7
6
10
8
0.7
0.01
0.4
0.01
0.6
0.1
1
6
6
10
10
10
0.6
0.05
7
7
10
0.7
0.01
0.01
0.3
10
10
0.01
0.01
10
10
0.01
0.01
108
IMR-32
8
0.5
0.1
6
0.5
7.5
10
6
10
0.7
10
5
1
0.01
0.03
0.04
0.5
1
0.3
8
5
1
0.01
0.8
0.7
1
0.3
10
10
3
10
10
10
0.8
0.08
6
5.5
10
0.35
0.01
0.06
0.3
10
10
0.01
0.01
10
10
0.01
0.01
SK-N-BE(2) SK-N-AS SK-N-SH
10
10
10
0.01
0.01
0.05
0.01
0.02
0.1
8
6.5
10
5
7
1
8
0.1
9
10
10
10
10
10
10
10
10
10
0.9
0.5
0.6
10
10
10
5
7
0.8
9
1
0.5
10
0.1
10
0.05
0.04
0.1
0.07
0.07
0.03
7
5
4
0.78
1
0.075
0.7
0.7
1
10
10
10
0.1
10
10
0.01
5
0.1
0.01
0.01
0.01
0.9
0.8
0.5
0.7
0.5
0.5
7
0.7
0.6
0.7
0.6
0.65
8
7
2
10
10
6
10
2
7.5
10
10
0.1
10
10
10
10
10
10
0.8
0.6
0.7
0.6
0.1
0.7
10
8
9
10
6.5
7
10
10
10
0.5
0.7
0.7
0.01
0.01
0.01
0.01
0.08
0.01
0.5
4
0.5
10
10
10
10
10
10
0.01
0.01
0.01
0.01
0.09
0.01
10
10
10
10
10
10
0.01
0.01
0.01
0.01
0.01
0.01
SHEP
0.6
0.01
0.06
6
7
7.5
10
6
6
0.5
10
3
5
0.01
0.01
0.01
1
10
0.7
0.01
8
0.6
0.1
0.7
0.2
1
0.5
10
10
7
10
10
10
0.5
0.1
0.6
1
10
1
0.01
0.01
0.1
10
0.06
0.01
0.01
10
10
0.01
0.01
Number
Inhibitor
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
17#DMAG
17#AAG
NVP#AUY922/(VER)52296)
CP#690550
AZD1480
CYT#387
INCB018424/(Ruxolitinib)
TG101348
PLX4032 /(RG7204)
PLX4720
Dabrafenib
Docetaxel
Paclitaxel/ (Taxol)
TGX#221
PIK#75,/Hydrochloride
GDC#0941
CAL#101
LY294002
TG100#115
XL#147
NVP#BKM120
PD/0325901
RDEA119
AZD/6244/(ARRY)142886)
ARRY#162
GSK1120212
AS703026/(MSC1936369B)
Cobimetinib/
ABT#263
ABT#737
AT101
Axitinib /(AG)013736)
Vandetanib /(Zactima)
Vatalanib/Dihydrochloride
Motesanib /(AMG)706)
Sorafenib
Sunitinib
Tandutinib
AV#951/(Tivozanib)
Pazopanib /(Votrient)
XL#184/(Cabozantinib)
AZD05030/(Saracatinib)
Dasatinib
Bosutinib /(SKI)606)
Nilotinib
Imatinib
Ponatinib /(AP24534)
FTY720,/Hydrochloride
FK#506
Activity
IMR-5
0.01
0.01
0.01
10
0.6
7
10
1
10
8
HSP/90/Inhibition
JAK/Inhibition
Raf/Inhibition
Tubulin/Stabilization
PI3K//Inhibition
MEK//Inhibition
Bcl)2/Family//Inhibition
VEGF)R/PDGF)R
/Inhibition
Src//Inhibition
Bcr)Abl//Inhibition
Immunosuppression
109
IMR-32
0.01
0.01
0.01
10
0.1
0.8
10
1
10
7
Cell line (IC50 (µmol/L)
SK-N-BE(2) SK-N-AS SK-N-SH
0.01
0.01
0.01
10
2
10
10
0.8
10
7
0.01
0.09
0.01
10
5.5
7.5
10
6
10
6
0.5
0.1
0.01
10
0.07
5
10
0.9
8.5
6
10
5
10
10
10
0.01
0.01
10
0.01
10
10
6
10
10
0.01
0.01
10
0.01
10
10
9
10
10
0.01
0.01
10
0.01
0.1
10
7.5
10
10
0.01
0.01
10
0.01
10
10
10
10
10
0.01
0.01
10
0.05
0.2
9
5
10
10
0.8
2
0.9
0.72
0.8
10
10
10
10
7
10
8
5
1
6
4.5
8
10
10
5.5
0.1
10
5.5
10
6
1
7
3
10
10
1
6
10
0.1
0.8
3.5
10
0.1
0.5
0.05
2
5
5
1
7
10
10
5
0.01
0.9
1
10
8
0.2
0.01
1
10
8
0.1
7
10
0.01
0.1
7
1
0.01
0.01
0.1
9
5
5
3.5
10
10
10
3
5
10
10
10
5
1
10
9
10
10
1.5
6
10
0.01
0.01
0.1
0.02
0.01
0.01
5
8
10
10
9
10
10
10
7
2
10
9
10
7.5
10
0.01
6
10
10
0.7
1
10
0.01
1
7
0.1
0.01
10
1
4
0.1
6
7.5
9
10
10
5
1
10
0.01
10
6
1
0.01
8.5
10
10
1
9
10
SHEP
0.01
0.01
0.01
10
0.7
6.5
10
10
10
10
10
0.01
0.01
6.5
0.01
10
1
10
10
10
0.6
0.5
0.8
5
1
0.01
0.01
1
10
8
3
0.6
7
10
10
10
1
10
5
10
10
5
0.1
5
10
10
0.6
5
10
Number
Inhibitor
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
ABT$888& (Veliparib)
AZD&2281-(Olaparib)
AG014699 -(PF201367338)
BSI$201 -(Iniparib)
MK$4827
BI&2536
BI&6727 -(Volasertib)
GSK461364
AC220 -(Quizartinib)
Crenolanib-(CP2868596)AZD4547
NVP$BGJ398
PD$0332991
Dinaciclib -(SCH-727965)
AZD7762
SCH900776
SGI$1776&
AZD1208
JQ1PF$1
I$BET151&(GSK1210151A)GDC$0449
Bicalutamide-(Casodex)
Laropiprant
Raltegravir
Odanacatib& (MK20822)
NVP$TAE684
VX702
Maraviroc-(UK2427857)
AN2728
Bexarotene -(Targretin)
Hypothemycin
Dimebolin&Hydrochloride
Montelukast&Sodium-(Singulair)
Rofecoxib -(Vioxx)
BI$D1870
A$769662&
Atorvastatin&Calcium-(Lipitor)
Dutasteride& (Avodart)
Eprosartan&Mesylate& (Teveten)
Lenalidomide -(CC25013)
NVP$LDE225
Regorafenib-(BAY-7324506)
SR1
Varespladib -(LY315920)
VX$765
VX$950-(Telaprevir)
YM155
PCI$32765 -(Ibrutinib)
VX$11e
PTC124 -(Ataluren)
MLN4924
Activity
IMR-5
PARP--Inhibition
PLK--Inhibition
FLT3-Inhibition
FGFR-inhibition
CDK-Inhibition
Checkpoint-kinase-inhibition
Pim-inhibition
BET-inhibition
Hedgehog-pathway-Inhibition
Androgen-receptor-Inhibition
PGD22R-Antagonist
HIV-integrase-Inhibition
Cathepsin-Inhibition
NPM2ALK-Inhibition
p38-MAPK-Inhibition
CCR5-Antagonist
PDE4-Inhibition
RXR-Activation
T-cell-activation-Inhibition
Antihistamine
LTR-Antagonist
COX22-Inhibition
p90-RSK-Inhibition
AMPK-Activation
HMG2CoA-reductase-inhibition
5α2reductase-inhibitor
Angiotensin-receptor-Inhibition
Immunomodulation
SMO-Inhibition
Multi2kinase-Inhibition
AHR-Antagonist
Phospholipase-A2-Inhibition
ICE/Caspase21-Inhibition
Protease-Inhibition
Survivin-Suppression
Bruton's-TK-inhibition
ERK2-inhibition
Nonsense-mutation-suppressant
NEDD82Activating-Enzyme-inhibitor
IMR-32
10
8
5.5
9
5
0.01
0.01
0.6
8.5
6.5
5
10
10
7
9
6
0.03
0.01
0.7
10
5
1
Cell line (IC50 (µmol/L)
SK-N-BE(2) SK-N-AS
10
9
7
10
0.8
0.01
0.01
0.6
10
7
0.01
SK-N-SH
10
10
7.5
10
5
0.01
0.01
0.9
10
10
6
10
10
7.5
10
8.5
0.01
0.01
0.5
10
10
0.01
9
0.01
7
6
10
0.1
0.01
10
0.01
0.6
0.01
0.7
10
1
0.01
0.8
1
6
10
0.1
3.5
0.7
0.07
0.6
4
5
0.3
1
0.5
0.6
0.85
7
10
0.8
8
10
0.5
1
10
10
10
10
10
0.4
1
5
10
1
5
0.8
10
10
10
10
10
0.7
10
10
10
10
3
10
10
10
10
10
6
10
10
10
10
6.5
7.5
10
10
10
0.01
10
10
10
10
10
0.5
10
10
10
10
0.8
10
10
10
10
10
5.5
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
0.05
10
10
10
10
6.5
10
10
10
10
10
10
10
10
10
10
5
6
10
10
10
0.01
10
10
10
10
10
1
10
10
10
10
1
10
10
10
10
10
10
10
10
10
10
6.5
9
10
10
10
0.01
10
10
10
10
10
0.6
10
10
10
10
1
10
10
10
10
10
1
10
0.8
8.5
6.5
1
6
10
10
10
0.01
10
1
2.8
0.6
10
0.9
10
0.7
7.5
0.85
10
0.06
10
0.5
10
0.7
10
10
10
0.8
SHEP
0
8
3
10
10
10
0.01
10
10
1
3.5
1
0.01
0.01
1
0.7
5
10
0.2
0.8
0.6
10
10
10
10
10
0.8
10
10
10
10
1
10
10
10
5
10
5
10
10
10
10
6
9
10
10
10
1
8.5
0.5
10
0.01
Table 4-1. Summary of cytotoxicity profiles of small molecule kinase inhibitors against six
NB cell lines.
Neuroblastoma cell lines were tested in a 4-­‐point dilution series (0.01, 0.1, 1 and 10 µM). After
four days in culture, cell viability was quantified by automated microscopy and percent growth
inhibition was determined by comparison to cells treated with DMSO. The calculated IC50 values
are from one complete study.
110
1A
111
1B
112
1C
Figure 4-1. Graphical representation of the IC50 values of small molecule kinase inhibitors
against IMR-5, IMR-32 and SHEP NB cell lines presented in Table 4.1.
The diverse sensitivity of these inhibitors against NB cell lines SK-N-SH, SHEP, IMR-5, IMR32, SK-N-AS and SK-N-BE(2) is shown in graphs A, B and C. Each graph indicates IC50 values
in a tested range from 1 X 10-2 to 10 µM. Points at 1 X 10-2 µM represent IC50 values of 1 X 10-2
µM or less and points at 10 µM represent IC50 values of 10 µM or more. Values are calculated
averages from three separate experiments.
113
4.4.2 Cytotoxicity profiling of small molecule Bcr-Abl kinase inhibitors against NB cells
A panel of six Bcr-Abl targeted small molecular weight inhibitors was evaluated against
five NB cell lines using in vitro cytotoxicity assays. These agents were selected based on their
known activities in leukemia and their activity in human clinical trials. Table 4.2 provides a
summary of the IC50 values obtained in cytotoxicity studies. Data provided in this table show a
wide range of drug sensitivity values across the five cell lines. Among the agents studied,
ponatinib showed consistent activity in all NB cell lines with IC50 values ranging from of 0.1 µM
to 1.5 µM.
4.4.3 Inhibition of NB cell proliferation by ponatinib
Cells from NB cell lines were treated with increasing concentrations of ponatinib and cell
viability was evaluated after four days in culture. Results presented in Figure 4.2 show that
ponatinib effectively inhibited NB cell growth and IC50 values were approximately 1.5 µM or
less. Under our experimental conditions, ponatinib showed no significant cell death in normal
lymphocytes (data not shown).
114
Table 4-2. Cytotoxicity profiling of Bcr-Abl targeting agents against five neuroblastoma
cell lines.
Exponentially growing tumor cells were treated with four different concentrations of individual
drugs (10 µM, 1µM, 0.1 µM and 0.01 µM) in duplicate cultures. After four days in culture, cell
growth inhibition was quantified by automated cytometry and percentage growth inhibition was
calculated in comparison to vehicle-only control cells. The IC50 values are from a single
complete study and trends observed are representative of three separate experiments.
115
Figure 4-2. In vitro cytotoxicity of ponatinib against NB cell lines.
Exponentially growing NB cells were incubated with increasing concentrations of ponatinib or
corresponding DMSO controls, in triplicate cultures. After four days, cell viability in each
condition was measured by automated cytometer and percentages of cell survival were calculated
with respect to DMSO controls. Data presented above is representative of three separate
experiments.
116
4.4.4 Target validation of ponatinib in NB cells
Next we aimed to look at the presence of the targets for ponatinib in NB cells that are
sensitive to its effects. The antibody array technique provides an effective tool to screen for
inactivation of receptor tyrosine kinases by targeted therapeutic agents. For screening purposes
one of the cell lines (IMR-5) was selected and treated with ponatinib and evaluated on an RTK
antibody array as described above. The resulting dephosphorylation data of 42 separate receptor
tyrosine kinases were analyzed using ImageJ software. Results presented in Figure 4.3 show the
ponatinib mediated loss of phosphorylation of insulin and IGF-1R family of receptors in these
cells.
4.4.5 Evaluation of ponatinib induced inactivation of IGF-1R and down-stream targets in NB
cells
Antibody array studies have shown that IGF-1R is potentially a target for ponatinib
activity in IMR-5 cells. We then went on to validate the array result by Western blot analysis
against five NB cell lines. Data presented in Figure 4.4A show that ponatinib treatment leads to
IGF-1R dephosphorylation in NB cell lines. We then investigated the downstream consequences
of this effect by looking at the modulation of three downstream components in the signaling
cascades linked to IGF-1R pathway. Data presented in Figure 4.4B show the activity
modulations by decreased phosphorylation of ERK 1/2, AKT and mTOR.
117
Figure 4-3. Identification of potential targets of ponatinib activity on NB cells.
Antibody arrays to a panel of receptor tyrosine kinases were used to screen for
dephosphorylation effects by ponatinib. Proteins were extracted from IMR-5 cells and treated
with 5 µM of ponatinib. Cell lysates were made with lysis buffer containing protease and
phosphatase inhibitors. DMSO treated cells were used as a control. Antibody arrays were
incubated with 200 µg of proteins overnight at 40C with gentle mixing and probed with HRPO
conjugated anti-phosphotyrosine antibodies as per manufacturer’s protocol. A: Antibody array
showing changes in phosphorylation as seen by a decrease or loss of signal. B: A diagrammatic
representation of the array map showing the positions of capture antibodies to different RTKs.
The arrows indicate the position of IGF-1R. Data presented above is representative of two
separate experiments.
118
A.
119
B.
Figure 4-4. Inhibition of IGF-1R and its downstream pathways by ponatinb.
A: Exponentially growing NB cells were treated with 5 µM concentration of ponatinib for 4
hours. Proteins from untreated and ponatinib treated cells were extracted in the presence of
protease and phosphatase inhibitors and the phosphorylation status of IGF-1R in NB cells was
analyzed by western blots. B: A change in activation status of ERK1/2, AKT and mTOR was
measured by western blot analysis using total and phospho-specific antibodies. Data presented
above is representative of three separate experiments.
120
4.4.6 Effect of ponatinib on Src phosphorylation
As Src has been implicated in IGF-1R signaling, we examined the effect of ponatinib on
the activation status of Src on NB cells. IMR-5 cells were treated with increasing concentrations
of ponatinib for four hours and the lysates were probed with phospho-Src specific antibodies
(Figure 4.5A). This effect was consistent in experiments where three other NB cell lines were
treated with ponatinib (5 µM for four hours) (Figure 4.5B). All cell lines showed a reduction in
pSrc, however, the SK-N-AS cells also showed a reduction in total Src level.
4.4.7 Exogenous IGF-1 overcomes the effect of ponatinib on Src dephosphorylation
Next we investigated if Src activation and ponatinib mediated dephosphorylation are
linked to IGF-1R activity. Cells were grown in serum free medium overnight and treated with
rhIGF-1 (100 ng/ ml). Data presented in Figure 4.6 show the expected phosphorylation of Src in
response to its ligand (Lane 7).
However, this activity was decreased by ponatinib in a
concentration dependent manner (Lanes 4, 5 and 6).
121
A.
B.
Figure 4-5. Inhibition of Src phosphorylation by ponatinib in NB cells.
IMR-5 cells were treated with increasing concentrations of ponatinib for four hours and the
phosphorylation status was evaluated by anti-pSrc antibodies. B: Cells from four separate cell
lines were treated with ponatinib (5 µM, 4 hours) and the phosphorylation of Src was identified
as described. Data presented above is representative of three separate experiments.
122
Figure 4-6. Ponatinib blocks the ligand dependent activation of IGF-1R activity.
IMR-5 cells were serum starved and the phosphorylation of IGF-IR was evaluated following the
addition of 100 ng/ml of exogenous IGF-I. Under similar conditions, the phosphorylation of
IGF-IR was also evaluated in the presence of three different concentrations of ponatinib. Loss of
activity of IGF-IR phosphorylation was noted in cells that received 2 µM ponatinib (Lane 6).
Data presented above is representative of two separate experiments.
123
4.4.8 Ponatinib treatment leads to activation of apoptosis in NB cells
Data from cell culture studies indicated cell growth inhibition in NB cells. The following
set of experiments examined the activation of apoptosis by the presence of distinct markers of
apoptosis. IMR-5 cells were treated with two different concentrations of ponatinib (2 µM and 4
µM) for nine hours and probed for the formation of active fragments of PARP and caspase 9
cleavage (Figure 4.7). We have also evaluated the loss of the pro survival protein Mcl-1 under
these conditions (Data not shown). Activation of PARP and caspase 9 appear to be more
prominent in the IMR-5 cell line under these conditions.
4.4.9 Effect of ponatinib on NB cell migration
Previous studies have shown the role of activated Src on tumor cell migration and
metastasis. Although the molecular mechanisms involved in this process are complex and multifactorial, the migration inhibition assay (scratch test) provides a simplified in vitro methodology
to screen for this phenomenon. In addition to the cytotoxic effects observed after four days of
exposure to ponatinib, we evaluated the effects of ponatinib on the migration of SK-N-AS cells
during a shorter time period, conceivably before the onset of cell death. In the scratch assay, the
movement of cells across a scratch line was evaluated as an indication of the capability of an
agent to inhibit cell migration. Photographs presented in Figure 4.8A show a concentrationdependent loss of cell migration over the scratch line (marked with blue lines) when treated with
ponatinib, demonstrating its potential ability to prevent cell migration. To confirm that the
reduction of cells in the scratch zone is not simply because of cell loss due to cell death, the total
number of viable cells in the cell culture dish was measured by Alamar blue assay. A graphical
124
representation of cell viability and corresponding migration into the scratch zone is given in
Figure 4.8B.
125
Figure 4-7. Effect of ponatinib on the markers of apoptosis.
IMR-5 cells were treated with two different concentrations of ponatinib (2 µM and 4 µM) for
nine hours and the changes in caspase 9 and PARP were evaluated by Western blotting using
specific antibodies that recognize native and fragments of these proteins. Data presented above is
representative of three separate experiments.
126
A.
127
B.
Figure 4-8. In vitro cell migration assay.
A: SK-N-AS cells were plated in 12-well plate and a scratch was introduced when cells were
80-90% confluent. Images were acquired at 0h, 8h and 24h following the in vitro scratch assay.
The dotted lines define the areas lacking cells. B: The rate of migration was measured by
quantifying the total distance that cells moved from the edge of the scratch toward the center of
the scratch (marked by imaginary dotted lines). After 24h in culture, cell viability in each
condition was measured by Alamar blue and percentages of cell survival were calculated with
respect to DMSO control wells. Data presented above is representative of three separate
experiments.
128
4.5 Discussion
Although the past two decades have seen a substantial improvement in the overall outcome
in children diagnosed with cancer, those who are diagnosed with high-risk and refractory disease
continue to endure unacceptable rates of morbidity and mortality. Currently, despite the
concentrated efforts of pharmaceutical companies, academic institutions and cooperative groups
to support design and carry out early phase clinical trials, advanced stage childhood cancers
remain mostly incurable. While a number of complex biological, logistical and ethical reasons
may account for the delay in bringing effective new therapeutics for this population, the scarcity
of suitable and available patients for clinical trials remains the most challenging obstacle. This
can lead to early phase clinical trials of drugs with largely unknown toxicity profiles or with
limited supporting data from small adult trials. However, agents that have been developed for
relatively common adult malignancies constitute a group of therapeutics that, with appropriate
biological rationale, can provide a more practical and effective selection for new trial designs in
pediatrics. Our initial in vitro screening of kinase inhibitor library against NB cell lines has
identified candidate protein kinases that interfere with the growth and survival of NB cells (Table
4.1, Figure 4.1). A number of potential kinase targets for NB survival including Aurora kinase,
IGF-1R, MEK1/2 and PLK were identified which have been reported previously and thus
validate the findings of our screening (Grinshtein et al. 2011b). Our study also revealed several
new promising agents, which require further investigation. We next explore the possibility of the
Bcr-Abl targeted agent on NB cell growth and metastasis.
The Bcr-Abl chimeric protein plays a central role in the pathogenesis of Philadelphia (Ph)
chromosome-positive leukemia and, most notably, the more prevalent CML. The malignant
129
transformation and sustained oncogenic activity are defined by the tyrosine kinase activity of the
fusion gene. Mechanistically, blockage of the ATP-binding pocket prevents the phosphorylation
of the fusion protein, ultimately resulting in leukemic cell apoptosis. Recent studies indicate that
many of the Bcr-Abl targeted agents, by virtue of target similarities, also have activity against a
number of other key kinases, and hence carry the potential for significant anti-tumor activities
against many tumor types. For example, dasatinib inhibits other Src family kinases and is
currently being assessed for the treatment of tumors such as melanoma, pancreatic, breast,
ovarian, and head and neck cancers (Montero et al. 2011). A number of preclinical studies have
shown the potent activity of other Bcr-Abl inhibitors such as bosutinib and saracatinib against a
variety of solid tumors (Puls, Eadens and Messersmith 2011).
In this report, we have evaluated a panel of Bcr-Abl targeting agents in a proof-of-concept
study against NB and focused the more detailed evaluation on the drug ponatinib. This agent is
an orally bioavailable Bcr-Abl inhibitor that has shown potent activity against the T315I
gatekeeper mutation and is currently undergoing phase II clinical studies (Cortes et al. 2012).
Furthermore, in preclinical studies, ponatinib has shown activity against a number of potential
targets in solid tumors. For example, DeFalco and colleagues have reported activity of ponatinib
against RET kinase and consequentially the inhibition of RET-driven medullary thyroid
carcinoma cells (De Falco et al. 2013). The combination of ponatinib and ridaforolimus had a
synergistic effect on the in vitro growth of endometrial lines bearing an activating FGFR2
mutation, irrespective of PTEN status (Gozgit et al. 2013). Ponatinib inhibits cell growth in both
established and primary lung cancer cells overexpressing FGFR1 (Ren et al. 2013a).
Neuroblastoma is the most common extracranial solid tumor in children and an advanced
stage disease with metastasis that often presents resistance to conventional chemotherapeutic
130
agents. We looked at the in vitro growth inhibitory effects of a panel of Bcr-Abl targeting
therapeutic agents against NB cell lines. Data presented in Table 4.2 show the wide variability in
the activity of these agents against the different cell lines. Under our particular experimental
conditions, higher concentrations of imatinib and nilotinib were needed to kill any of the NB
cells, whereas the other four agents showed IC50 values mostly in the low micromolar range.
Interestingly, dasatinib was active against three cell lines, but failed to induce cytotoxicity
against SK-N-BE(2) cells. This cell line was derived from an N type, NMYC amplified tumor
with deletion of the short arm of chromosome 1 and alteration in Chromosome 17 (Barnes et al.
1981b). Ponatinib, however, consistently induced cell death in all of the cell lines at about 1.5µM
or lower IC50 values. Our data, as well as the findings from previous reports looking at NB
sensitivity in response to different members of this family of agents, merits analysis in the
context of molecular heterogeneity of NB and the various cell lines used in these studies. In
addition, the potential for off-target effects of the agents themselves should be considered. Vitali
and co-workers evaluated the activity of dasatinib in NB cell lines and found sensitivity in many
of the cell lines at sub micromolar IC50 values (Vitali et al. 2009). In this study, dasatinib
inhibited anchorage independent growth and showed a number of target modulatory activities
including the inhibition of c-Kit and c-Src phosphorylation and down-regulation of ERK1//2 and
AKT activity. Similarly, using different parameters of cell growth inhibition, Timeus and
colleagues have shown the cytostatic and anti cell-migratory effects of dasatinib in NB cell lines
(Timeus et al. 2008). Although early preclinical studies have indicated activity of imatinib
against NB cells, it has failed to show measurable efficacy in clinical studies (Bond et al. 2008).
However, a recent report has indicated the potential for its activity in the subset of subjects with
low bone marrow infiltration as the only site of metastasis (Calafiore et al. 2013). Interestingly, it
131
has been found that prolonged low dose scheduling of imatinib could suppress the growth and
survival of NB cells (Palmberg et al. 2009), indicating the complex relationship between
exposure and cell growth inhibition by TKIs in NB.
The transmembrane receptor tyrosine kinase IGF-1R is normally activated by the ligands
IGF-1 and IGF-2. Its abnormal expression or activity has been implicated in the growth, survival
and metastasis of a number of solid tumors, especially in pediatric embryonal tumors such as
osteosarcoma, rhabdomyosarcoma, Wilms' tumor and NB (El-Badry et al. 1991b; Kim et al.
2009). In one study where IGF-1R expression was evaluated by reverse transcription-PCR
analysis in NB primary tumors, 86% of cases showed positivity (n=43) (Tanno et al. 2006).
Functionally, IGF-1R activation has been shown to induce strong anti-apoptotic signals in NB
cells and has been thought to play a role in the generation of resistance to chemotherapy (van
Golen et al. 2006b; Samani et al. 2007). In preclinical models, antisense IGF-1R has been shown
to inhibit NB tumor growth in vitro and in vivo (Liu et al. 1998). Recently, the humanized
antibody SCH 717454 (Robatumumab, Schering-Plough), which down-regulates the expression
and phosphorylation of IGF-1R, has also been shown to induce growth inhibition of NB,
osteosarcoma, and rhabdomyosarcoma tumor xenografts (Wang et al. 2010). Our screening
studies using the IMR-5 NB cell line showed a decrease in phosphorylated IGF-1R following
treatment with ponatinib (Figure 4.3). Further testing of additional cell lines also showed loss of
phosphorylation in response to ponatinib treatment (Figure 4.4A). Although the expression levels
differed among cell lines, some degree of loss of pIGF-1R was seen in all of the cell lines tested.
This information is consistent with many past reports demonstrating a pivotal role for IGF-1R
mediated pathways in solid tumors. This includes the finding of the pediatric preclinical testing
program (PPTP) in which the anti IGF-1R antibody SCH717454 has shown activity against
132
xenografts of a broad spectrum of solid tumors (Kolb et al. 2011). Similarly, oral administration
of NVP-AEW541, a small molecule inhibitor of IGF-1R activity, suppressed the growth of NB
xenografts in nude mice (Tanno et al. 2006). However, it is interesting to note that ponatinib, in
the initial publication where the potency of ponatinib was assayed in vitro with recombinant
kinase domains and peptide substrates, showed significantly less inhibition (IC50 > 1000-fold
relative to native ABL) (O’Hare et al. 2009). The mechanisms by which ponatinib was still able
to induce measurable dephosphorylation in our experimental model remain to be elucidated.
However, it is possible that the critical dependency of the NB cells on IGF-1R mediated
pathways, in conjunction with yet unclear off-target effects of the agent, may potentially
generate growth inhibition in culture. In the substrate assays, however, ponatinib did show
significant activity against c-Src (IC50, 5.4 nM) (O’Hare et al. 2009).
Signaling through IGF-1R has been shown to involve the activation of phosphatidylinositol
3-kinase (PI3K)/AKT and/or MEK/ERK pathways (Pollak, Schernhammer and Hankinson
2004). We have found that ponatinib treatment leads to significant decreases in phospho- Src,
AKT and mTOR in all of the cell lines (Figure 4.4B). The loss of phospho-AKT is consistent
with the observation by Tanno et al where the treatment of NB cells with the targeted IGF-1R
inhibitor NVP-AEW541 causes a reduction of phosphorylation of AKT (Tanno et al. 2006).
AKT, which is positively regulated by the activation of IGF-1R and Src, is also a key effector of
the PI3K/AKT/mTOR pathway, which is aberrantly activated in the majority of malignancies,
promoting cell survival and proliferation (Ligresti et al. 2009). However, unlike the findings with
NVP-AEW541, ponatinib also led to the loss of phosphorylated ERK1/2 (Figure 4.3B). This
information is suggestive of the capability of ponatinib to block multiple pathways of IGF-1R
signaling that may involve AKT/mTOR, as well as those involving MEK1/2, possibly through
133
the inhibition of the RAS-RAF-MEK pathway. Experiments are currently in progress to further
study the potential for such a mechanism.
In the stem cell leukemia/lymphoma (SCLL) model, previous studies have shown that the
inhibition of FGF-R1 leads to other downstream effectors such as Src (Ren et al. 2013b). Our
investigation of ponatinib treated IMR-5 cells showed concentration dependent loss of
phosphorylated Src (Figure 4.5A). Similarly, Src inactivation was noted in three other cell lines
(Figure 4.5B). The ability of ponatinib to interfere with the activity of the Src family of proteins
has been reported in other experimental systems (Okabe et al. 2013; Ren et al. 2013b). Our next
set of studies also showed that ponatinib induced Src dephosphorylation may be overcome with
exogenous IGF-1 in a competitive manner (Figure 4.6), suggesting a direct link between the two
processes and the ability of ponatinib to block complimentary activities of the two pathways. In
addition to the known Src directed activity of ponatinib (O’Hare et al. 2009), the potential for
dual inhibition of Src and IGF-1R provides a rational therapeutic approach to block both
independent and complementary pathways known to be decisive in tumor growth (Dayyani et al.
2012). Previous reports have shown that the targeted inhibition of IGF-1R leads to apoptosis as
evidenced by PARP cleavage (Beauchamp et al. 2009). For example, in the lens epithelial
system, PI3K/AKT signaling mediates IGF-1–propagated cell survival, suppression of caspase-3
activation, and prevented PARP degradation in the presence of agents such as staurosporine
(Chandrasekher and Sailaja 2004). PARP cleavage has also been shown to be a marker for
ERK1/2 activation in the protective effect of IGF-1 against drug induced cell death (Hwang,
Kwon and Nam 2007). In NB, an increase in caspase 3 activity and cleavage of PARP have been
known to serve as an early execution phase signal (Bursztajn et al. 2000). Our finding that
ponatinib induces PARP cleavage in IMR-5 cells and activates the pro-apoptotic molecule
134
caspase 9 provides evidence for target modulation and identifies potential cell death pathways
that are activated in response to ponatinib (Figure 4.7).
High risk NB carries the inherent risk of developing distant lesions, in particular, bone
marrow metastasis. Hence, agents that have the capability to interfere with these processes are of
great interest in new drug development for NB. Previous studies have shown IGF-1 to be a
potent stimulator of NB cell motility and invasiveness (Meyer et al. 2005). Furthermore,
molecules such as integrins that are mechanistically involved in this process function through the
recruitment and activation of proteins such as FAK and c-Src (Mitra and Schlaepfer 2006), and
their activation have been shown to stimulate cell migration and invasion (Navarra et al. 2010).
Using an in vitro cell migration inhibition assay, we evaluated the effect of ponatinib on the NB
cells under sub lethal conditions. Data presented in Figure 4.8 show a dose dependent inhibition
of tumor cell migration in the presence of the drug at a time frame when no significant loss of
viability was evident. These data are consistent with the findings previously reported with
dasatinib (Timeus et al. 2008), and provide support for further studies to evaluate the role of such
agents in targeting highly metastatic NB subgroups.
The considerable advances made in the overall outcome of pediatric malignancies in the
past three decades were achieved mostly in low- and standard-risk cancers without any
significant improvement in the survival of those diagnosed with the high-risk and advanced-stage
disease. The agents that have been selected for the treatment of common adult malignancies
often generate usable activity and toxicity data in a relatively short time. With pediatric-tumorfocused preclinical studies, these molecules represent a highly efficient and cost-effective group
for further evaluation. In this report, we have studied the agents that have been used in the
treatment of Ph chromosome positive leukemias. We also described in detail, the in vitro
135
preclinical studies of the multi-target kinase inhibitor ponatinib against NB cell lines. We
provide evidence for its activity and show that this may be through its effect on IGF-1R and Src
functions, leading to the induction of apoptosis in NB cells. The information regarding
cytotoxicity and target modulation analyses provide the essential preliminary data to help
formulate additional clinically relevant studies, including xenograft experiments, with respect to
scheduling and effective drug combinations. The unique target modulatory activities described
herein will help to define effective biological correlative studies as part of future early phase
clinical trials of ponatinib against refractory pediatric solid tumors.
4.6 Acknowledgement
This research was funded in part by the POETIC Foundation, Morgan Adams Foundation,
Alberta Children’s Hospital Foundation and the Kids Cancer Care Foundation of Alberta. AS
received a graduate research fellowship from the Alberta Cancer Foundation.
4.7 References:
References are added to the end in this thesis.
136
Chapter Five: Targeted inhibition of MEK1 by cobimetinib leads to differentiation and
apoptosis in neuroblastoma cells.
In the previous Chapter, we performed an in vitro screen of small molecule kinase
inhibitors (n=151) to identify effective agents for the treatment of refractory NB. During our
study, we observed that MEK inhibitors (n=7) were active against most NB cell lines. Previous
studies have shown that the RAS/MAPK pathway plays a crucial role in NB growth and survival.
Thus, we hypothesized that RAS/MAPK pathway inhibition via a MEK inhibitor would be
effective against NB growth stimulatory mechanisms. In this study, we evaluated the effects of
the targeted MEK inhibitor cobimetinib as a single agent and in combination with cis-RA, on the
growth, survival and differentiation properties in NB cell lines. The following report1 provide
initial proof-of-concept information on the potential utility of cobimetinib as an effective
targeted therapeutic agent against refractory NB.
Author’s Contribution: Anjali Singh performed and analyzed all the experiments with the
support of her supervisor, Dr. Aru Narendran. YR assisted with the original concept
formulation, discussions and manuscript preparation. TT assisted with the reagents and
manuscript preparation. All authors have read and approved the final manuscript. The original
publication has been reproduced as Chapter Five in this thesis. Figures and references have been
137
reformatted according to the guidelines outlined by the University of Calgary Faculty of
Graduate Studies.
1
Anjali Singh, Yibing Ruan, Tanya Tippett and Aru Narendran. Targeted inhibition of MEK1
by cobimetinib leads to differentiation and apoptosis in neuroblastoma cells. Journal of
Experimental and Clinical Cancer Research. 2015; 34(1): 104
5.1 Abstract
Background
Neuroblastoma is one of the most common childhood malignancies. Currently, high risk
NB carries a poor outcome and significant treatment related toxicities and, thus has been a focus
for new therapeutics research in pediatric oncology. In this study, we evaluated the effects of the
MEK inhibitor cobimetinib, as a single agent and in combinations, on the growth, survival and
differentiation properties against a molecularly representative panel of NB cell lines.
Methods
In vitro anti-proliferative activity of cobimetinib alone or in combination was
investigated by cell viability assays and its target modulatory activity was evaluated using
phospho-kinases antibody arrays and western blot analysis. To determine the effect of
combination with cis-RA on differentiation and resulting enhanced cellular cytotoxicity, the
expression of glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2
(MAP2) expression levels were examined by immuno-fluorescence.
Results
138
Our findings show that cobimetinib alone induced a concentration-dependent loss of cell
viability in all NB cell lines. In addition, cobimetinib showed feedback activation of MEK1/2,
and the dephosphorylation of extracellular signal-regulated kinases (ERK1/2) and c-RAF,
providing information on the biological correlates of MEK inhibition in NB. Combined
treatment with cis-RA, led to differentiation and enhanced sensitization of NB cells lines to
cobimetinib.
Conclusion
Collectively, our results provide evidence that cobimetinib, in combination with cis-RA,
represents a feasible option to develop novel treatment strategies for refractory NB.
5.2 Background
Neuroblastoma is a malignancy of the embryonal sympathetic nervous system arising from
neuroblasts and is the most common type of solid tumors in children (Maris et al. 2010).
Although cure rates have improved over the past 20 years, event-free survival is still only about
45 % for patients with high-risk metastatic disease (Huang et al. 2010). Heterogeneity is the
hallmark of NB and its clinical behavior ranges from spontaneous regression to metastatic
disease that is refractory to common therapies. Advanced NB typically metastasizes to regional
lymph nodes, bone marrow, skin and liver (Kamijo and Nakagawara 2012). Distinct prognostic
stages (1, 2A, 2B, 3, 4 and 4S) have been identified for the classification of NB (Cohn et al.
2009). The best-characterized genetic alterations of NB include N-Myc oncogene- amplification
or allelic loss, near triploid karyotype, deletion of short arm of chromosome 1, chromosomal
rearrangements involving chromosome 11q and high expression of tropomyosin receptor kinase
139
A (TrkA) and B (TrkB). To improve the clinical outcome of advanced NB, it is important to
identify the key molecularly defined actionable pathways and targets for novel therapeutics in
these patients.
The Mitogen-activated protein kinases (MAPKs) cascade (RAS/RAF/MEK/ERK) is an
important signal transduction system involved in the control of cell proliferation, survival and
differentiation (Kolch 2005). A wide range of cell-surface molecules activate RAS (KRAS,
NRAS, and HRAS), a family of GTPases, that act as a molecular switch in the activation of
MAPKs cascade (RAF/MEK/ERK) (Yoon and Seger 2006). There are multiple molecular
mechanisms
of
interaction
and
activation
between
the
upstream
nodes
of
the
RAS/RAF/MEK/ERK cascade and other cell signaling pathways, ultimately resulting in ERK
transcription factor activation (Miller, Oliver and Farley 2014). The activation of ERK leads to
cells acquiring many of the hallmarks of cancer such as cell survival, cell migration, and
invasion and inhibitors targeting this pathway have been vigorously developed (Brown et al.
2007; Sette et al. 2013). The MEK inhibitors act on MEK phosphorylation by binding to a
pocket adjacent to the ATP binding site, decreasing both the amount of MEK activity, and the
quantity of activated ERK in the cell. Cobimetinib is a potent and highly selective inhibitor of
MEK1 (Musib et al. 2013).
In this study, we evaluated the expression and activity of MEK1/2 and ERK1/2 in a panel
of NB tumor cell lines and their sensitivity to cobimetinib in vitro. The panel of cell lines used
included the neuroblastic (N), substrate-adherent (S), and intermediate (I) subtypes, classified
based on their morphology, growth patterns, and malignant potential (Ross, Biedler and Spengler
2003a). In addition, in drug combination studies we have also tested the ability of cis-retinoic
acid (cis-RA) that has been known to cause cell growth inhibition and differentiation (Sidell et
140
al. 1983), to enhance the activity of cobimetinib against NB cells. Our data provide initial proofof-concept information on the potential utility of cobimetinib as an effective targeted therapeutic
agent against refractory NB.
5.3 Materials and Methods
5.3.1 Cell lines and cell culture
The following NB cell lines were used: SK-N-AS (ATCC-CRL-2137), SK-N-SH (ATCC
HTB-11), SK-N-BE(2) (ATCC CRL- 2271), IMR-32 (ATCC CCL-127), SHEP, IMR-5. SHEP
and IMR-5 cell lines were a gift from Dr. Herman Yeger (The hospital for Sick Children,
Toronto, ON). These cells were maintained in Opti-MEM media (Gibco, Invitrogen Corporation,
Burlington, ON) supplemented with 5 % fetal bovine serum and 100 units/ml penicillin and 100
units/ml streptomycin (Gibco). Confluent cells were trypsinized with 0.25 % Trypsin-EDTA in
Ca2+ and Mg2+ free balanced salt solution (Gibco) every three to five days. All cell cultures
were maintained in incubators at 37 °C in a humidified atmosphere with 5 % CO2.
The MEK inhibitor cobimetinib (GDC-0973) was kindly provided by Roche (Basel,
Switzerland). Stock solutions of cobimetinib were prepared as 10 mM in DMSO and stored in
aliquots at −20 °C. Cis-RA was obtained from Sigma (Oakville, ON).
5.3.2 Drug cytotoxicity assays
Neuroblastoma cells were trypsinized and placed in 96 well plates (Grenier Bio One,
Monroe, NC) at a concentration of 5 x 103 cells per well. Increasing concentrations of study
agents and a corresponding dilution of DMSO were added to a final volume of 200 µl per well.
After four days in culture, cell survival was quantified by an inverted microscope (Cyntellect Inc,
141
San
Diego,
CA;
http://www.nexcelom.com/Celigo/direct-cell-counting-assays-for-
immunotherapy.php#feature6). The half maximal inhibitory concentration (IC50) values were
calculated for each agent based on individual cytotoxicity plots.
5.3.3 Human phospho-kinase antibody array
Neuroblastoma cells were seeded in six well culture plates (Nunc, Waltham, MA) at 1 x
106 cells/ml and incubated overnight. Fresh culture medium containing cobimetinib or vehicle
control was added and after two hour incubation, cells were washed with ice cold PBS and
treated with lysis buffer (50 mM Tris, 5 mM EDTA, 0.1 % SDS, 1 % Triton X-100, 0.5 %
sodium deoxycholate) containing phosphatase and protease inhibitors (Sigma). Human phopshokinase array (R&D Systems, Inc., Minneapolis, MN) were incubated with cobimetinib-treated
and control cell lysates (150 µg) over-night, washed and probed with horseradish peroxidase
(HRPO) (Sigma) labeled anti-pTyr antibodies according to manufacturer’s protocol. The arrays
were scanned and the spot densities were quantified with ImageJ (National Institutes of Health,
http://rsb.info.nih.gov/ij/ version 1.4.3.67).
5.3.4 Western blot analysis for protein and phosphoprotein detection
Each NB cell line was grown to 70 to 80 % confluence in six well culture plates (Nunc)
and incubated overnight to allow for cell adherence. The cells were then incubated with fresh
culture medium containing cobimetinib or vehicle control as indicated in individual experiments.
After each time period incubation, cells were washed with ice cold PBS and treated with lysis
buffer containing phosphatase and protease inhibitors. Protein concentrations of the lysates were
quantified by BCA Protein Assay (Pierce, Rockford, IL). Proteins were then separated on a 8 %
142
polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC) membranes (BioRad, Mississauga, ON). The membranes were blocked for one hour at room temperature with
5 % skim milk powder in PBS containing 0.1 % Tween-20 (Sigma). The blots were incubated
with primary antibodies (Cell Signaling Technology, Danvers, MA) overnight at 4 °C, washed
and probed with appropriate secondary antibodies conjugated to horseradish peroxidase (HRPO)
(Sigma), followed by a luminal based substrate (Mandel, Guelph, ON) and developed by
exposure to x-ray film (Fisher Scientific, Ottawa, ON).
5.3.5 Annexin V staining for apoptosis
Neuroblastoma cell lines (IMR-32, SHEP and IMR-5) were plated at a concentration of
3  ×  105 cells per well. Following treatment with 1 µM cobimetinib or DMSO (vehicle control)
the cells were incubated for a period of 24 hour prior to FACS analysis. Apoptosis was measured
using the Annexin V-FITC Apoptosis Detection kit (Life Technologies, Carlsbad, CA) according
to the manufacturer’s instructions. In this assay, the apoptotic cells were differentiated from
viable or necrotic cells by the combined application of Annexin V-FITC and propidium iodide
(PI). Briefly, the control and treated cells (1  ×  106) were re-suspended in 500 µl of binding buffer
and incubated with 5 µl of Annexin V-FITC and 1 µl of PI solution for 15 min. The samples
were then analyzed on a BD Facscan Instrument (BD Biosciences, Franklin Lakes, NJ),
measuring the Annexin V-FITC emission at 488 nm and PI emission at 575 nm. Lower right
quadrant (Q4) represent percentage of early apoptotic cells out of total cell population in the
treatment group compared to control.
143
5.3.6 Treatment with cis-RA
Cis-Retinoic acid was added to a final concentration of 10 µM, according to feasible
pharmacological dosages and those used in previous in vitro differentiation studies (Villablanca
et al. 1995; Ratka et al. 1996). To see the combined effect of cis-RA and cobimetinib on cell
growth inhibition, IC25 concentration of cobimetinib (i.e., the amount that induced 25 % cell
death in single drug studies) was added to cultures containing increasing concentrations of cisRA. The number of viable cells present after four days in culture was determined as described.
5.3.7 Immunocytochemical detection of differentiation markers
Neuroblastoma cells were treated with cobimetinib (1 µM) and cis-RA (10 µM) alone or
in combination for 24 hours. Briefly, the cells were fixed with 4 % paraformaldehyde (Sigma)
and permeabilized with 0.05 % Triton X-100 (Sigma). The cells were incubated with antibodies
to Nestin (R&D Systems, 1:1000), GFAP (Sigma, 1:1000) and MAP-2 (Sigma, 1:800) for two
hours at 37 °C. The cells were then washed with PBS and incubated with fluorescence labelled
secondary antibody (Invitrogen, 1:500) at room temperature for one hour. Staining of treated and
untreated cells were then visualized by fluorescence microscopy for detection of differentiation
markers.
5.3.8 Statistical analysis
For two-group comparisons, Student’s t tests using the GraphPad Prism software (version
4.0) were used. The results are considered statistically significant versus the untreated cells, with
a probability level of p  <  0.05 (*), or statistically highly significant with a probability level of
p  <  0.01 (**).
144
5.4 Results
In order to evaluate the cytotoxic effects of cobimetinib against NB, cells from a panel of
cell lines were incubated with increasing concentrations of cobimetinib. After four days in
culture, cell viability was evaluated. Results presented in Fig. 5.1 show that cell line IMR-32 is
highly sensitive to cobimetinib with IC50 of 0.07 µM and IMR-5 was least sensitive for
cobimetinib showing activity at 10 µM. Other NB cell lines fell in between this range of IC50
values showing an intermediate activity. In the next set of experiments, the activation status of
MEK was evaluated by western blot analysis under serum containing and serum free culture
conditions. Findings from these experiments show that cells with increased phosphorylated MEK
were more sensitive to cobimetinib compared to those with less MEK1/2 phosphorylation
(Fig. 5.2). There was no significant difference in MEK1/2 activity between cells grown with
serum and without serum suggesting that the growth factors found in serum do not have
significant influence in the base-line MEK activity found in these cells in culture.
Next, we wanted to evaluate the target modulatory activity of cobimetinib in NB cells.
Antibody arrays to a panel of signaling molecules were used to screen for phosphorylation
effects by cobimetinib. Proteins were extracted from cells treated with 1 µM of cobimetinib and
cell lysates were made with lysis buffer containing protease and phosphatase inhibitors. DMSO
treated cells were used as control. Antibody arrays were incubated with 200 µg of proteins over
night at 4 °C with gentle mixing and probed with HRPO conjugated anti-phospho antibodies.
Figure 5.3A provides a map of the positions of the various kinases on the arrays used.
Figure 5.3B shows changes in phosphorylation as seen by gain or loss of signal on the x-ray film.
A waterfall graph showing the change in optical density of array spots of cobimetinib treated
145
cells compared to control DMSO treated cells is given in Fig. 5.3C. As can be seen in these
figures, there is a gain in MEK phosphorylation whereas a loss in phosphorylation was noticed in
ERK1/2. In addition, c-Jun also showed a gain in phosphorylation while a decrease in
phosphorylation was noted with RSK1/2/3.
Figure 5-1. Cobimetinib mediated cytotoxicity against NB cells.
Triplicate wells of cells from six NB cell lines were treated with increasing concentrations of
cobimetinib or corresponding concentrations of vehicle control (DMSO) and cell viability was
evaluated after four days in culture using automated microscopy. Results presented show cell
146
growth inhibition compared to DMSO control. Data presented above is representative of three
separate experiments.
147
Figure 5-2. Constitutive phosphorylation status of MEK in NB cells.
NB cells were grown in serum containing and serum free media and collected at their
exponential growth phase (three days after sub-culture). These cells were lysed in buffer
containing protease and phosphatase inhibitors and probed for total and phosphorylated MEK by
western blot analysis. Data presented above is representative of three separate experiments.
148
A.
B.
149
C.
Figure 5-3. Identification of potential targets of cobimetinib activity on NB cells.
Antibody arrays to a panel of signaling molecules were used to screen for dephosphorylation
effects by cobimetinib. Proteins were extracted from IMR-32 cells treated with 1 µM of
cobimetinib and cell lysates were made with lysis buffer containing protease and phosphatase
inhibitors. DMSO treated cells were used as control. Antibody arrays were incubated with
200 µg of proteins over night at 4 °C with gentle mixing and probed with HRPO conjugated antiphospho Tyr/Ser/Thr antibodies as per manufacturer’s protocol. Presented is the map of the
orientation of the antibodies on the array (A), luminographically developed blots of cobimetinib
treated and DMSO treated blots (B) and the waterfall plot showing changes in phosphorylation
status of each signaling molecule on the arrays (C). Data presented above is representative of
two separate experiments.
150
The antibody array findings were then confirmed using western blot analysis (Fig. 5.4).
Three NB cell lines IMR-32, SHEP and IMR-5 representing sensitive, intermediate and resistant
cell toxicity against cobimetinib respectively were used for western blot analysis. The NB cell
lines were incubated with cobimetinib (1 µM) or corresponding DMSO controls for four hours,
harvested and subjected to western blotting. Results showed cobimetinib treatment induced dephosphorylation of c-RAF and ERK and an increase in the phosphorylation of MEK.
The drug sensitivity studies provided evidence for cobimetinib induced reduction in
viable cell numbers after exposure. To further evaluate the potential role of apoptosis in this
process, we examined PARP cleavage induced by cobimetinib (Fig. 5.5). Neuroblastoma cell
lines IMR-32, SHEP and IMR-5 were incubated in the absence or presence of cobimetinib
(1 µM) for 24 hour and cell lysates were analyzed by western blot. IMR-32 and SHEP showed
PARP cleavage as indication of apoptosis induction. To further confirm this finding an Annexin
V/PI binding assay was performed (Fig. 5.6a and b). Our data revealed an increased percentage
of early apoptotic cells (Annexin V+/PI−) after treatment of IMR-32 and IMR-5 cells with
cobimetinib for 24 h. There was no significant difference in the percentage of early apoptotic
cells between the control group and the SHEP cell line.
151
Figure 5-4. Effect of cobimetinib on MAPK cascade pathway.
Changes in activation status of RAF, MEK and ERK were evaluated in three NB cell lines after
treatment with cobimetinib (1 µM) or DMSO for four hours. Cell lysates were made with lysis
buffer containing protease and phosphatase inhibitors and probed for total and phospho specific
antibodies by western blot analysis. Data presented above is representative of three separate
experiments.
152
Figure 5-5. PARP cleavage induced by cobimetinib.
Neuroblastoma cell lines IMR-32, SHEP and IMR-5 were incubated in the absence or presence
of cobimetinib (1 µM) for 24 hour and cell lysates were analyzed by western blotting using
antibodies specific for PARP and its cleaved fragment. Actin was used as loading control. Data
presented above is representative of two separate experiments.
153
Figure 5-6. The flow cytometric analysis of apoptosis in NB cells using FITC-annexin V and
PI double staining.
A. Apoptosis was evaluated after treating NB cells with 1 µM of cobimetinib for 24 hour, and
staining with Annexin-V. Annexin V+/PI− (lower right quadrant) areas represent early apoptotic
cells, and Annexin V+/PI+ (upper right quadrant) areas for late apoptotic or necrotic cells. B.
Quantitative analysis of the percentage of early apoptotic cells in all NB cell lines. Data
presented above is representative of two separate experiments.
154
Next, studies were carried out to examine time dependent changes induced by
cobimetinib with respect to some of the changes in growth regulatory pathways detected in the
western blot analysis (Fig. 5.7a). In these experiments, IMR-32 Cells were harvested after
treatment with or without 1 µM cobimetinib for the indicated periods. The activation of MEK1/2
was noticed as earlier as five minute after treatment. Data from similar analysis on IMR-5 and
SHEP are presented in Fig. 5.7b and c respectively. Both SHEP and IMR-5 cell lines showed
increase of MEK1/2 phosphorylation in the indicated time period.
In the following experiments, we investigated the potential of cis-RA to enhance the
activity of cobimetinib in drug combination. Neuroblastoma cells were incubated with increasing
concentrations of cis-RA plus IC25 concentration of cobimetinib. The IC25 values used were
2 µM, 0.07 µM and 0.04 µM for IMR-5, SHEP and IMR-32 respectively. Data presented in
Fig. 5.8a-c indicate that, under the specific experimental conditions used, cis-RA in combination
with cobimetinib showed a significant decrease in cell growth in all cell lines tested compared to
the drug alone. Next, we explored the induction of differentiation as a potential mechanism in the
enhanced activity seen in this combination. For this purpose, the involvement of cells expressing
the differentiation markers, GFAP and MAP2 were analyzed using immuno-fluorescence. As can
be seen in Fig. 5.9, there was an increase in GFAP (5.9A) and MAP2 (5.9B) expression in cells
treated with cobimetinib and cis-RA alone or in combination, compared to control. These data
suggest that cobimetinib alone was also able to induce differentiation, which was enhanced after
combined treatment with cis-RA. We also tested Nestin to validate differentiation results and
noticed a decrease in Nestin expression after the treatment alone or in combination (Fig. 5.9C).
155
A.
B.
156
C.
Figure 5-7. Time course analysis of cobimetinib on MAPK cascade pathway.
Cells from three NB cell lines (IMR-32 (a), IMR-5 (b) and SHEP (c)) were harvested after
treatment with 1 µM cobimetinib or DMSO control for the indicated periods. Cells were then
lysed in protease and phosphatase containing buffer and probed for total and phosphorylated
MEK by western blot analysis. Data presented above is representative of three separate
experiments.
157
158
Figure 5-8. Drug combination studies of cobimetinib with cis-RA in NB cell lines.
NB cells were incubated with 1 µM of cis-RA acid plus IC25 concentration of cobimetinib for
each cell line as obtained from Fig. 1. The IC25 values used were 2 µM, 0.07 µM and 0.04 µM
for IMR-5, SHEP and IMR-32 respectively. Data shown indicate percent survival with each drug
or in combination compared to corresponding DMSO treatment. Cultures were set up in triplicate
and the data presented are representative of two separate experiments. P values indicate
statistical significance. Figures a, b, and c show findings from the cell lines IMR-32, IMR-5 and
SHEP respectively.
159
A.
160
B.
161
C.
Figure 5-9. Analysis of cellular differentiation induced by cobimetinib, cis-RA or
combination of the two agents.
Exponentially growing NB cells were treated with either agent (1 µM cobimetinib or 10 µM cisRA) or in combination. Cells were then washed and fixed with 4 % paraformaldehyde and
permeabilized with 0.05 % Triton X-100. The cells were then evaluated by conventional
immunohistochemistry using antibodies to Nestin (1:1000), GFAP (1:1000) and MAP2 (1:800)
and fluorescent labelled secondary antibodies. Cells were also counterstained with DAPI and
visualized by fluorescence microscopy and photographed. Presented are randomly picked
microscopic fields for each experimental condition. Staining for GFAP and MAP2 expression
shows an increase and while Nestin expression decreases with differentiation. Changes in
morphology with elongated processes are also visible with increased differentiation of the cells.
Scale bar, 34 µM. Data presented above is representative of three separate experiments.
162
5.5 Discussion
Neuroblastoma is the most common extracranial solid tumor in the pediatric population
and currently, the treatment of high-risk NB with multi-modal therapeutic approaches still results
in less than 50 % 5-year event-free survival (Matthay, George and Yu 2012). Hence, there is a
significant and urgent need to develop mechanism based novel therapeutic approaches and early
phase clinical trials for the treatment of patients with refractory and high-risk disease. Recently,
targeting key receptor tyrosine kinases and their downstream signaling mediators has been
shown to be an effective approach in new therapies development in a number of cancer models.
In NB, abnormal activation of a number of receptor tyrosine kinases (RTKs) has been reported.
These include insulin growth factor 1 (IGF1), c-Kit and the Trk family of receptors. However, as
NB cells appear to be highly heterogeneous in the expression of active RTKs, it remains
unknown if the targeting individual RTKs would be and an efficient approach. For this reason,
the identification of inhibitors for critical downstream signaling nodes that are involved in the
transmission of abnormal survival, proliferation and differentiation signals from affected RTKs
has been suggested as a potentially viable alternative (Boller et al. 2008).
The MAPK signaling pathway has been shown to play a critical role in the transmission
and coordination of diverse extracellular and environmental stimuli to cell growth mechanisms
and MAPK pathway dysregulation has been demonstrated in a variety of human malignancies
(Akinleye et al. 2013b). MEK1 is a dual-specificity tyrosine threonine protein kinase that
occupies a central node in the MAPK signaling pathway (Caunt and Keyse 2013) . MEK1
phosphorylates ERK1 (p44 MAPK) and ERK2 (p42 MAPK ) to activate pathways that regulate
the proliferation and differentiation in cancer cells (Shaul and Seger 2007). A number of
previous studies have shown the important role of MEK1 in NB tumorigenesis. For example, in
163
the NB cell line KP-N-RT, IGF-1 leads to phosphorylation of ERK and that the specific MEK1
inhibitor PD98059 leads to the inhibition of IGF-1 mediated cell cycle progression (Misawa et
al. 2000). In this report we aim to investigate the effects of cobimetinib, a potent, orally
bioavailable, small-molecule inhibitor of MEK1, against a panel of NB cell lines in order to
investigate its feasibility in future therapeutic interventions. Cytotoxicity data presented in
Fig. 5.1 shows effective cell killing, as determined by IC50 values, of SK-N-BE(2), SHEP and
IMR-32 cells and intermediate activity against SK-N-AS and SK-N-SH cells. The IMR-5 cells
appear to be resistant under our experimental conditions. Previously, Eppstein and colleagues
have investigated the effects of the MEK inhibitor U0126 against three NB cell lines
representing the three types of cells; SK-N-AS (S-type), SH-SY5Y (N-type) and BE(2)-C (Itype) (Eppstein et al. 2006). Their findings showed that although all three cell lines exhibit
decrease in pERK with MEK inhibition only the I type cell BE(2)–C had decreased proliferation
showing heterogeneity in MEK mediated growth inhibition. Next, we examined the levels of
constitutive pMEK in each cell line with the aim of analyzing a potential correlation to their
response to cobimetinib (Fig. 5.2). Interestingly, the IMR-5 cells that appear to have the lowest
amounts of pMEK also appear to be resistant to growth inhibition by cobimetinib. This indicates
the potential of using constitutive activation status of MEK1 as a biomarker for efficacy in
treatment protocols using cobimetinib. However, due to the limited sample size of the cell lines
used, studies using a larger cell line panel and/or primary samples are needed to confirm this
utility.
To further expand the target modulatory effects of cobimetinib in NB cells, we used
antibody arrays to identify changes in various intracellular signaling molecules (Fig. 5.3). As
expected, there is an increase in MEK and decrease in ERK activity was noted. However, we
164
also detected an increase in c-Jun, which is normally a target of ERK activity, and a decrease in
the activity of RSK 1/2/3. The RSK family of proteins function downstream of ERK and are
considered to be an important conveyer of ERK signaling (Čáslavský, Klímová and Vomastek
2013). In addition we also examined changes in c-RAF, MEK and ERK status in relation to their
sensitivity to cobimetinib by evaluating the levels of activation in IMR-32 (sensitive), SHEP
(intermediate) and IMR-5 (resistant) cells (Fig. 5.4). From these data we observed that the
dephosphorylation pattern of c-RAF corresponds well with cell growth inhibition as it is most
prominent in IMR-32 and next SHEP cells with IMR-5 showing no change. A similar pattern is
also seen with ERK activity. However, conversely, IMR-32 cells showed the most increase in
MEK activation indicating a consistent pattern of intracellular target modulation and inhibition
of proliferation by cobimetinib in distinct NB cell lines. Previously, using the proteomic
profiling approach, the potential biological pathways that may confer sensitivity to MEK
inhibition in NB subtypes have been investigated by Sandoval and colleagues (Sandoval et al.
2006). Their data suggested that the inhibition of MEK leads to differential intracellular stress
response in different NB subtypes and the most resistant cell lines generated unique patterns in
protein profiling indicating the utility of these proteins as biomarkers for therapeutic response. In
our studies PARP cleavage data provides a biomarker of apoptosis that corresponds to the
activity of cobimetinib against the three NB subtypes (Fig. 5.5). In FACS analysis, cobimetinib
treatment has also shown an increased in the number of early apoptotic cells in the IMR-32 and
IMR-5 cell line after 24 hour treatment (Fig. 5.6). We also found that although all three cell lines
generated pMEK in response to cobimetinib treatment, the resistant cell line IMR-5 also took the
longest time to show this response (30 min to 2 hours), compared to the sensitive cell lines
165
(5 min) (Fig. 5.7). These data provide dosing and scheduling guidelines for future studies to
further investigate the activity of cobimetinib in vivo.
Our initial cytotoxicity and target modulation studies described above suggested potential
antitumor activity of cobimetinib against the NB cells although there is a range in the sensitivity
exhibited by different cell lines. In order to optimize the utility of cobimetinib across all NB
subtypes and to enhance the overall effectiveness in future therapeutic regimens, we then
explored possible drug combinations that can further enhance the overall effectiveness of
cobimetinib against NB. Previous studies have shown that treatment of NB cells with retinoic
acid leads to differentiation, cell growth arrest and the decrease in MYCN expression (di Masi et
al. 2014). Cis-RA has also been found to provide survival advantage in combination with
immunotherapy with anti-GD2 antibody, combined with GM-CSF or IL-2 (Yu et al. 2010). It
has been suggested that RA combination with other drugs may lead to synergistic effects
allowing the potential to deliver RA at low concentrations to minimize non-specific side effects
(di Masi et al. 2014). Based on this premise we explored the ability of cis-RA to enhance the
activity of cobimetinib against the NB cell lines. Interestingly, this combination resulted in
increased cell killing in all three cell lines (Fig. 5.8). Furthermore, morphological and
immunohistochemical analysis of the three cell lines treated with this combination showed
evidence for morphological differentiation and the expression of markers involved in this
process. Unexpectedly we also found that cobimetinib itself is capable of inducing differentiation
in NB cells. At present, minimal residual disease is prevented by repeated courses of the RA
treatment; however this treatment only improves survival by 35 % in children with metastatic
neuroblastoma (Matthay et al. 1999). In NB cell differentiation, cobimetinib alone showed less
potency; however, cobimetinib was found to enhance the differentiation induced by RA,
166
suggesting that these compounds could interact cooperatively to improve the NB differentiation
and cell killing (Fig. 5.9). Studies are currently in progress to identify the mechanism of this
effect, particularly to understand the influence MEK targeting agents in NB differentiation.
5.6 Conclusion
Taken together, our in vitro findings from a panel of NB cell lines suggest that the targeted
inhibition of MEK1 by cobimetinib holds the potential to induce potent antitumor activity
although there are subsets of cells that may be affected by this treatment. We have also provided
key biological markers for this activity that can be used to identify the patient population that
may benefit the most by this treatment, furthermore we also provide evidence for an effective
combination of cobimetinib with cis-RA would to enhance antitumor activity in all NB cells.
Additional in vivo studies in NB xenograft are needed to confirm and further develop these
finding for the formulation of effective early phase clinical trials for the treatment of refractory
NB patients in the future.
5.7 Acknowledgements
We acknowledge Drs. Raphaël F. Rousseau, Hubert Caron, Stephen Simko and Romina Genhart
(Genentech-Roche) for their insight and helpful discussions with respect to this study. This
research was funded in part by the POETIC Foundation, Morgan Adams Foundation, Alberta
Children’s Hospital Foundation and the Kids Cancer Care Foundation of Alberta. AS received a
graduate research fellowship from the Alberta Cancer Foundation.
167
5.8 References
References are added to the end in this thesis.
168
Chapter Six: General Discussion and Insights
6.1 Summary
The search for novel cancer chemotherapeutics focuses on screening and identifying
compounds that can target ‘cancer-specific’ biological processes while causing minimal toxicity
to normal cells. Recent insights into the molecular basis of cancer have led to a transition from
cytotoxic chemotherapy to molecularly-targeted cancer drug discovery. Thus far, however, the
identification of effective targeted therapeutic agents for pediatric cancers has been more
difficult compared to the progress made in common adult cancers because of the paucity of
information on specific driver alterations in childhood malignancies. The lack of candidate
molecular drug targets in pediatric cancers has triggered a number of research groups to adopt an
alternative strategy when developing new treatments for childhood cancer. Such an alternative
approach that is currently being explored is the potential repurposing of established anti-cancer
agents, which have been approved for clinical use in the past. In short, this approach investigates
whether validated molecular targets of adult cancers might also be used in the management of
pediatric cancers. Initial proof-of-concept screening using cell lines has been effective in many
cases and has led to the identification a number of agents for further studies. In addition,
rationally designed small molecule inhibitors against some of the fundamental biological
processes have also found utility in new therapies discovery (Hoelder, Clarke and Workman
2012).
Based on this premise, we have explored the use of diverse panels of cell lines against
169
libraries of small molecule inhibitors, including those that were considered to be specific for
other tumor types, for new drug discovery against AT/RT and NB.
6.2 Drug screening to identify potential targets for therapeutics for AT/RT
`AT/RT is a rare tumor with limited therapeutic options. Given the rarity and the sparsity
of the information available about the biological aspects of this tumor, finding new therapeutic
agents has been challenging. The first aim of this graduate thesis was to identify the potential
therapeutic targets for the treatment of AT/RT and we used a systematic research approach to
achieve this objective (Figure 6.1). In this study, we performed an in vitro drug screen to
examine the effect of 129 small molecule kinase inhibitors on AT/RT cell lines (Chapter 3). To
our knowledge, this is the first study to use a large library of clinically feasible drugs to test for
anti-proliferative effect in AT/RT cell lines. In our initial approach, we screened agents at
multiple concentrations ranging from 10-10-2 µM. By employing this approach, we found many
agents across different therapeutic categories and modes of action that had an anti-cancer effect
(Table 3.1). Many of the agents used in this study are standard chemotherapeutic drugs used in
clinics today (for example, doxorubicin and bortezomib) (Hande 1998; Field-Smith, Morgan and
Davies 2006). This screening also identified many new actionable targets for AT/RT such as
mTOR, Src and PLK kinases, which can be explored further in the future studies. Inhibitors with
the IC50 value of the arbitrary value of less than 1µM were considered a primary hit in first
screening. The compounds found to have potent activity in our screen represent possible
opportunities to repurpose these drugs for the treatment of patients with aggressive recurrent or
metastatic AT/RT cancer.
Among the potential targets identified in this study, we selected EGFR/HER-2 for further
170
evaluation for a number of reasons. We noticed that 80% of the EGFR/HER-2 inhibitors (six out
of 8 inhibitors) were effective against the majority of AT/RT cell lines tested in these
experiments. Amplification or over-expression of EGFR/HER-2 genes occur in many cancers
and are associated with poor prognosis (Fukushige et al. 1986; Field-Smith, Morgan and Davies
2006; Reichelt et al. 2007; Iqbal and Iqbal 2014). In a previous study, the over-expression of
EGFR was shown to correlate with poor outcomes in pediatric CNS tumors (Liu et al. 2014).
Similarly, in a recent report, HER-2 was found to be over-expressed in a considerable number of
AT/RT specimens (6/7), which suggests that HER-2 may represent a potential target for novel
therapies (Patereli et al. 2010). To validate EGFR/HER-2 as a potential therapeutic target in
AT/RT, we further explored the role of lapatinib as an EGFR/HER-2 inhibitor in this project.
Lapatinib, which is currently in clinical use for HER-2 positive breast cancers, has shown
clinical activity in patients with CNS brain metastases (Metro et al. 2011). The ability of
lapatinib to cross the BBB makes it an ideal candidate for the treatment of brain tumors (Gril et
al. 2008b). We also demonstrated that EGFR/HER-2 molecules were the primary target of
lapatinib and the activity was inhibited in AT/RT cells following treatment. In addition, lapatinib
was also able to inhibit both phosphorylation and activation of the downstream RAS/MAPKs
and PI3K-Akt signaling and this confirms the target modulation effects of lapatinib on AT/RT
cells. These results are consistent with the findings in the breast cancer model where lapatinib
has been shown to decrease HER-2 and EGFR phosphorylation leading to inhibition of
downstream growth regulators (Rusnak et al. 2001b).
In this study, we also examined the effects of lapatinib on the migration of AT/RT cells
in vitro and found that it interferes with the cellular migration mechanism. These data also
171
validate the efficacy of lapatinib for the inhibition of metastatic colonization by cancer cells (Gril
et al. 2008b). Because the in vitro studies were carried out for a short-term period (4 days), we
further evaluated the long-term effect of lapatinib in vivo. Subsequently, we demonstrated that
administration of lapatinib as a single agent substantially suppressed tumor growth in vivo.
Together, our data suggest that; i) in vitro drug screen can be used to identify new therapeutic
targets; ii) lapatinib has antitumor effects in vitro and in vivo; and iii) lapatinib may provide
useful clinical benefits to AT/RT patients.
172
Figure 6-1. A detailed design of the experimental procedure used in this research.
6.3 Drug screening to identify potential targets for therapeutics for NB
Neuroblastoma is the most common extracranial solid tumor in children and patients with
the high-risk disease have less than 50% chance of survival (Matthay et al. 1999). In the recent
years, research has increased our understanding of NB biology and helped us discover many key
pathways for the development of NB therapeutics. However, children with high-risk NB still
173
have poor outcomes (Weinstein 2003). Thus, there is an urgent need to develop novel therapies
that are more effective and tolerable. Therefore, we have conducted an in vitro drug screen on six
different NB cell lines to identify new therapeutic agents against NB (Chapter 4). In our second
aim, we applied the same approach as used in the earlier AT/RT model (Figure 6.1) and we
performed a drug screen using small molecule kinase inhibitor library. The compounds included
in our drug library are known to have diverse mechanisms of action and are known to be active
against at least 65 individual kinases. Through this screen, we also validated the agents that are
currently in use for NB treatment such as doxorubicin, topotecan and etoposide (Hadjidaniel and
Reynolds 2010). The aforementioned agents were active against all six NB cell lines and thus
validate our findings. In addition to identifying agents that have been previously utilized or are
currently going under clinical investigations, we also identified a number of agents that have not
been investigated in NB including ponatinib, cobimetinib and many others (Chapter 4; table 4.1).
Human cancers frequently display tumor heterogeneity and this can have an important
role in drug sensitivity (Marusyk and Polyak 2010). Neuroblastoma is a heterogeneous tumor
and demonstrates clinical and biological heterogeneity (Brodeur 1995). The heterogeneity also
plays a role in the prediction of a response to treatment, as well as the outcome of the patient
(Brodeur & Nakagawara 1992). Neuroblastoma also displays cellular heterogeneity as well and it
can be identified whether the cells originate from neuronal, melanocytic, or glial/schwannian
lineage (Ross, Biedler and Spengler 2003b). This cellular heterogeneity is present in vitro as
well, where neuroblastic (N), flat or substrate adherent (S) and intermediate (I) cell types can be
identified (Ciccarone et al. 1989; Ross, Biedler and Spengler 2003b). It has been hypothesized
that the sensitivity to drugs is at least partially, dependent on the different cell phenotypes (Ross,
174
Biedler and Spengler 2003b). A better understanding of the molecular and cellular heterogeneity
of human NB tumors is an important prerequisite when it comes to developing more effective
therapies for this potentially fatal disease. Therefore, in this study we examined the responses of
three different types of cell lines (SK-N-AS (S-type) and IMR-5 (N-type) and IMR-32 (I type)).
These cell lines also reflect the molecular heterogeneity found in NB patient specimens such as
MycN amplification, 1p allelic loss and gain of chromosome arm 17q (Abel et al. 1999). In this
study, heterogeneity of drug sensitivities was also noted amongst these six NB cell lines. Thus,
this study has revealed that cell heterogeneity can potentially affect the response to treatment
with anticancer agents and subsequently contribute to the nature of clinical treatment responses
in patients.
We have also reported the evaluation of a panel of Bcr-Abl targeting agents for antitumor activities. The Bcr-Abl targeting agents have shown a broad range of inhibitory activity
against a number of other tyrosine kinases including PDGFR, c-KIT, Src and Src-family kinases
(Giles, O’Dwyer and Swords 2009). Previous studies have also shown that Bcr-Abl targeted
agents also exert antiproliferative effects on NB cell lines (Lupino et al. 2014). In this study, we
explored the role of Bcr-Abl targeting agents as multi-target kinase inhibitors in NB and focused
on the drug ponatinib. Since there was no known target of ponatinib in NB, we used the antibody
array approach to identify the potential targets of this drug. We observed that IGF-1R
phosphorylation was significantly reduced in the IMR-5 cell line after the treatment, which was
also further validated by western blotting. Previous studies have shown that IGF-IR expression
and activation regulate NB cell proliferation, motility, invasion, and survival (Coulter et al.;
Kim, van Golen and Feldman 2004b; van Golen et al. 2006a). Thus, these studies demonstrate
175
the potential benefits of targeting IGF-1R in NB to develop novel therapies to treat this tumor.
The effects of ponatinib on the downstream signaling of IGF-1R, such as ERK1/2 and
AKT/mTOR pathways were also investigated in this study.
Previous reports have suggested that Src may be involved either directly or indirectly in
signal transduction through the Bcr-Abl signaling pathway (Warmuth et al. 1997; Plattner et al.
1999). Since ponatinib was originally developed as a Bcr-Abl targeted agent, we set out to
determine whether ponatinib affects the activity of Src in NB cell lines. We observed that
ponatinib indeed inhibited phosphorylation of Src in all NB cell lines. It has been known that
Src, a non-receptor protein tyrosine kinase, can directly phosphorylate IGF-1R (Peterson et al.
1996; Yeatman 2004). It has also been shown that Src can also be activated by other membrane
receptors including IGF-1R (Zhang and Yu 2012). Hence, it is likely that Src has a dual function
in this scenario, as a downstream node that links signalings among several collateral membrane
associated receptors or as an upstream node to activate the receptors. Further, to understand the
possible relationship between Src and IGF-1R, we demonstrated that exogenous IGF-1 is able to
activate Src in serum starved NB cells and subsequently, ponatinib was able to inhibit this
activation in a dose-dependent manner. To further validate these findings, siRNA-mediated IGF1R inhibition can be explored in the future.
Previous studies have shown that the cell migration can be induced by IGF-1 and/or
IGF-2 by activating IGF-1R signaling (Shigematsu et al. 1999; Mancini et al. 2014). It is known
that high risk NB carries the risk of metastasis (Maris 2010). It will be interesting to observe if
targeted inhibition of IGF-1R has the potential to inhibit NB cell migration. In this study, we
used an in vitro cell migration inhibition assay to evaluate the effect of ponatinib on NB
176
migration. Consequently, we showed that ponatinib was able to inhibit NB cell migration in a
dose dependent manner.
6.4 Inhibiting MAP kinase signaling in NB
Receptor tyrosine kinases such as EGFR, IGF-1R, VEGFR, PDGFR, and MET all activate
the MAPK pathway. This pathway is a key integration point along the signal transduction
cascade that links diverse extracellular stimuli to intracellular cascades, thus, affecting the
survival, proliferation and differentiation of cells. In the MAPK pathway, MEK1/2 is a key
kinase that forwards upstream signals from RAS and RAF via activation of ERK. Activated ERK
phosphorylates a number of substrates, affecting key cellular functions, which could potentially
lead to the progression of cancer if ERK is abnormally activated (Samatar and Poulikakos 2014).
Recent whole-genome sequencing data from the relapse samples of NB patients identified a
strong enrichment for mutations in genes associated with MAPK signaling (Eleveld et al. 2015).
In this study, 78% of samples from relapsed NB patients contained somatic mutations the MAPK
pathway, and all alterations were consistent with pathway activation. This has led to the
hypothesis that mutations activating RAS-MAPK signaling exhibit relapse-specific enrichment
due to treatment. Thus, these findings suggest that constitutive activation of MAPK signaling is
an independent predictor of poor response and MEK1/2 inhibition might be of clinical
significance when used in the treatment for relapsed NB. Our data have shown that the disruption
of the MEK/MAPK module has functional consequences in NB cell lines, thus providing the preclinical rationale for the development of MAPK-targeted therapeutic strategies in NB (Chapter
5). In our initial drug screen, we observed that all seven MEK inhibitors had an effect in NB cell
lines tested, except IMR-5. As discussed earlier, possibly due to tumor heterogeneity, we also
177
observed differences in sensitivity to the MEK inhibitors in the various cell lines tested. For
further studies, we investigated the effects of cobimetinib on a panel of NB cell lines along with
a control cell line (NRAS positive RD (rhabdomyosarcoma) cell line) and observed a variability
in response (ranging from most sensitive to least sensitive) (Appendix 1). Based on the
cytotoxicity data and activation status of MEK1/2, we were able to provide evidence that
cobimetinib affects the growth of NB cell lines with constitutive MAPK activation, but had little
to no effect on cells with low MAPK activity. A previous study has also observed a significant
correlation between constitutive MAPK phosphorylation and sensitivity to MEK1/2 blockadeinduced growth inhibition in both AML cell lines and primary AML samples (Milella et al.
2001). Along these lines, the lack of constitutive MAPK activation in IMR-5 may explain why it
is the least sensitive to MEK inhibition, as observed here and in previously published studies
(Sebolt-Leopold et al. 1999; Milella et al. 2001).
We also demonstrated that cobimetinib has a potent inhibitory effect on ERK
phosphorylation but also induced MEK1/2 phosphorylation. We further confirmed this
phenomenon in a time-dependent inactivation analysis where we studied the effect of
cobimetinib on the induction of MEK1/2 phosphorylation. This observation is consistent with the
previous reports that cobimetinib and other allosteric MEK1/2 inhibitors, including PD0325901
and selumetinib have such activities (Hatzivassiliou et al. 2013b; Ishii et al. 2013; Lito et al.
2014a). One explanation for this phenomenon is that inhibition of ERK1/2 disables several
negative-feedback loops to the upstream pathway components such as RAS, RAF and SOS,
thereby permitting stronger signaling to MEK1/2 (Sale and Cook 2014). A recent study has
suggested that interaction with Ser212 is critical for the binding of MEK1/2 inhibitors to
178
MEK1/2. This interaction is likely to be the modulator that results in the attenuation of MEK1/2
phosphorylation by some drugs, but not others (Lito et al. 2014b). It has been shown that
MEK1/2 inhibitors have potential to either promote or disrupt the interaction between RAF and
MEK1/2 and this might influence the MEK1/2 phosphorylation event by RAF although no
simple correlation is apparent (Hatzivassiliou et al. 2013a). Different MEK1/2 inhibitors may,
however, disrupt MEK1/2 phosphorylation through distinct interactions with MEK1/2 but the
structural basis for this phenomenon is currently unclear.
In addition to published data, we also explored the efficacy of cobimetinib as an inhibitor
of cell migration inhibitor in NB cell lines. We also validated our findings of in the RD cell line
(Appendix 1). Our results suggest that RD cell line has constitutive activation of ERK1/2 due to
a RAS mutation and this activity of ERK1/2 is inhibited upon treatment of cobimetinib.
Cobimetinib also inhibited cell migration as a single agent as well as in combination with cis-RA
in both NB and RD cell lines.
Results of this study provide a rationale for targeting MEK1 kinase in NB to maximize the
therapeutic benefit for patients. In summary, advantages of targeted therapeutics, such as MEK
inhibition, will ultimately depend upon the ability to prospectively select those patients who are
most likely to benefit from a specific therapeutic intervention because of a molecular aberration
present in their tumors.
6.5 Drug combination studies
Cancer is an extraordinarily complex disease with numerous genetic alterations and a
considerable amount of genetic heterogeneity, not only between different tumors but also within
179
an individual tumor (McDermott, Downing and Stratton 2011). Due to heterogeneous
populations of the cells, cancers are more likely to become resistant to treatment over time and
often require drug combinations to overcome this problem (Hoelder, Clarke and Workman
2012). Recent innovations have enabled us to use cell line-based screening platforms to explore
drug combinations in a high-throughput fashion. Before selecting a drug to be used in
combination, we need to address a few questions; 1) What would be the rationale for selecting a
target? 2) Should we target vertical (inhibiting two targets in the same pathway) or parallel
pathways? 3) Should combinations be made with conventional chemotherapeutic agents or with
new agents? and 4) Is there preclinical evidence of synergy between the compounds?
It has been reported that constantly dividing cancer cells often switch cellular pathways
and tend to rely on redundant pathways to support survival once one signaling pathway is
abolished by a targeted therapeutic agent (Hanahan and Weinberg 2000). Therefore, inhibition of
parallel pathways should be an effective strategy in combination therapy. In our first aim, we
explored the effect of inhibiting two parallel pathways (HER-2 and IGF-1R) in AT/RT cell lines
(Chapter 3). The rationale for combination regimens was also based on following observations.
First, the results of our antibody array experiments showed the increased expression of IGF-1R
in AT/RT cells, which has been reported in previous studies as well (Ogino, Cohen and AbdulKarim 1999; Shim et al. 2013). The HER-2 inhibitors combined with IGF-1R inhibitors have
been shown to be synergistic in in vitro and the combination effects were replicated in vivo
(Chakraborty, Zerillo and DiGiovanna 2015; Urtasun et al. 2015). Lapatinib in combination with
other chemotherapeutic agents, such as trastuzumab and capecitabine has shown a synergistic
and antiproliferative effect in many cancers (Blackwell et al. 2012; Hecht et al. 2015). We have
180
also reported that lapatinib synergizes with IGF-1R inhibitor in the AT/RT cell lines tested.
Interestingly, we also found that this combination is equally effective in the AT/RT cell line
(BT12), which was less sensitive to lapatinib alone, possibly due to its lower Erb expression.
Based on these results, we rationalized that co-inhibiting HER-2 along with IGF-1R may
represent a novel therapeutic strategy for AT/RT malignancies.
The question remains whether MAPK pathway inhibitors are effective single-agent
cancer therapeutics. It is unclear whether down-regulation of MEK activity alone will have an
anti-tumorigenic effect in NB patients. One potential reason for the limited efficacy of singleagent MAPK pathway inhibitors is the presence of signaling feedback loops in cells. Inhibition
of MEK might alleviate the repression of other pro-survival pathways. For example, MEK
inhibition leads to activation of MAPK signaling through a feedback loop and this has been
proposed as a possible reason for the limited efficacy of MEK inhibitors in RAS mutated cancer
cells (Migliardi et al. 2012; Lamba et al. 2014). These studies suggest that combining MAPK
pathway inhibitors with other therapies might improve their efficacy. The most “classic” drug
combination therapy explored to date in the clinical settings is based on therapeutic synergism
between established chemotherapeutic agents and agents representing other classes (Emil Frei
and Eder 2003). The current treatment protocol for NB includes the differentiating agent cis-RA,
which has been effective at increasing event-free survival in children with high-risk NB
(Matthay et al. 2009). Thus, we hypothesized that cobimetinib combined with cis-RA would be
effective against the NB tumor models. In our study, the addition of cobimetinib with cis-RA
was more effective in reducing tumor growth than either treatment alone (Chapter 5). We also
observed that cobimetinib was able to induce differentiation by itself and was able to enhance the
181
differentiation induced by cis-RA in NB cells. Moreover, cobimetinib in along with cis-RA had
an inhibitory effect on cell migration (Appendix 1).
6.6 Future directions
When compared to the adult disease, less is known about the molecular alterations that
cause childhood cancers. However, with the ongoing research in cancer biology, new targetbased therapies are making their way to the clinic for the treatment of pediatric malignancies.
These agents differ greatly from the majority of the conventional chemotherapeutic drugs
because they are rationally designed to mechanistically inhibit a specific protein target that is
hypothesized to be critical for tumor growth and progression. Although these novel agents offer
great promise for significant improvements in cancer chemotherapy, they have also brought new
challenges for cancer treatment. The major obstacle is the identification of useful and predictive
biomarkers in different cancers. The most direct and relevant measure of target inhibition is to
use pre- and post-treatment tumor samples to validate the biomarker of interest. The obvious
limitation of this approach is that tumor tissue is not readily accessible in most childhood
cancers, especially in the rare tumors like AT/RT. The second obstacle is the identification of
specific alterations that define the patient population that will respond to a given target based
therapy. Targeted therapies such as small-molecule MEK inhibitors are likely to benefit only a
subset of patients.
In vitro drug screens using monolayer or suspension cultures still reflect a highly
artificial cellular environment and may have limited predictive value for the clinical efficacy of
anti-cancer agents in patients. Hence, efforts to establish and optimize new tools for advanced
cell-based in vitro screening are necessary. Tumors are not isolated masses of malignant cells but
182
are also composed of non-malignant cells that create the tumor microenvironment. These include
transformed vasculature, immune cells and stromal elements, which contribute to the
development, growth and spread of cancers (Hanahan and Weinberg 2000). Over the past few
years, the importance of the tumor microenvironment has prompted efforts to model these
features of tumor cell growth in vitro more precisely. Tumor cells growing in 3D cultures are
generally believed to more closely mimic in vivo behavior of most cell types and are believed to
uncover aspects of tumor biology and tumor metastases (for example, invasive potential, changes
in polarity, matrix-independent survival, and sensitivity to drugs and radiation). Such an
experimental model will be helpful in optimizing the pre-clinical selection of the most effective
molecules from the large and growing pool of drug candidates. The most common 3-D cell
culture systems employed in basic and applied tumor biology are multilayer cell systems, matrixembedded cultures, hollow-fiber bioreactor, ex vivo tumor cultures and multicellular tumor
spheroids (Kunz-Schughart et al. 2004).
To understand the migration of cancer cell, many in vitro assays (2D assays) have been
developed to study the underlying molecular mechanism and to screen agents that would inhibit
the cancer migration process. Compared with 2D cellular migration, 3D assays could provide a
more realistic microenvironment and better information on the in vivo cell migration behavior
and will be effective in studying the effect of agents that inhibit cancer cell migration. Thus, it
will be interesting to further study and validate the efficacy of lapatinib, ponatinib and
cobimetinib as inhibitors of cell migration. In addition, these 3D systems will also be valuable in
studying combinations before testing them in xenograft models.
Progress in anti-cancer drug development has been obstructed by a lack of pre-clinical
183
models that can reliably predict the clinical activity of novel agents in cancer patients. In an
effort to address these shortcomings, we have used the patient-derived tumor xenografts
(subcutaneous tumors) engrafted into immunodeficient CD-1 nude mice for preclinical studies.
However, whereas subcutaneous growth is an in vivo condition, it may not simulate an orthotopic
environment relevant for tumor-host interactions and disease-relevant metastases. The orthotopic
xenograft is an alternative model where cancer cells are implanted into the organs from which
they are originated. Orthotopic brain tumor xenograft establishment provides an appropriate
microenvironment for modeling CNS cancer to be tested for the therapeutic response (Ozawa
and James 2010). It is more physiologically relevant and is suitable for studying metastasis
because subcutaneous tumors rarely metastasize. A recent study has compared both
subcutaneous xenograft and orthotopic models of glioma and found that orthotopic growth
conditions induce a different set of modifications compared to subcutaneous xenografts
(Camphausen et al. 2005). Thus, the results presented in this study provide the basis for
investigations aimed at defining the specific aspects of the CNS microenvironment, which would
provide a more appropriate model for investigating therapeutic agents against such tumors.
Orthotopic models, however, often require complex surgical procedures and are costly and timeconsuming. However, they are useful for confirming the activity of drugs or for studying antimetastatic therapy. Similarly, we need to validate findings of NB study in an in vivo model.
Additional studies using relevant xenograft models are needed to evaluate the clinical suitability
of ponatinib and cobimetinib in the treatment of NB. Future studies will also need to focus on
evaluating the effect of the drug combination in such model systems.
184
6.7 Conclusion
As we discover the underlying mechanisms responsible for the initiation and progression
of human cancers, we transition the focus from classic cytotoxic agents to the rationally designed
of small molecule targeted anticancer therapeutics. The work presented in this thesis establishes
the preclinical data describing the potential effects of targeting selective receptor tyrosine kinases
in NB and AT/RT. The advantage of rationally targeted therapeutics, particularly those directed
against oncogenic kinases, is their specificity and consequently high tolerability in patients. At
the level of basic biological mechanisms, the effects of small molecule targeted inhibitors can
help us to understand the processes and players involved in cancer growth and metastasis.
185
Figure 6-2. Targeting selective receptor tyrosine kinases in NB and AT/RT
186
APPENDIX A: EXAMINATION OF THE EFFECTS OF COBIMETINIB ON RAS
MUTATED CELL LINE
RD is a human rhabdomyosarcoma cell line and has embryonal histology (Hinson et al.
2013). RD cell line has been shown to have amplification of the MYCN oncogene and mutation
of NRAS gene (Missiaglia et al. 2009). Initially, MEK inhibitors were developed for cancers that
have dependency on MAPK signaling for growth and survival. These cancers have constitutive
activation of MAPK signaling due to mutation in either RAS or RAF proteins and showed high
sensitivity towards MEK inhibitors such as malignant melanoma (Tran et al. 2016).
Amplification of the MYCN oncogene is found in approximately 25% of cases of NB and
correlates with high-risk disease and poor prognosis (Huang and Weiss 2013). Thus, these
features make RD cell line an ideal control to study MEK inhibition and NB malignancies.
The cytotoxic effect of cobimetinib was examined on NB and RD cell lines. In this
study, NB and the control cell line RD were treated with increasing concentrations of
cobimetinib and cell viability was evaluated after four days in culture. Results presented in
Figure A1 show that cell line RD is sensitive to cobimetinib with an IC50 of 0.7 µM. Next, we
wanted to evaluate the target modulatory activity of cobimetinib in RD cells. RD cells were
incubated with cobimetinib (1 µM) or corresponding DMSO control for four hours, harvested
and subjected to western blotting. Results in Figure A2 show cobimetinib treatment induced
dephosphorylation of c-RAF and ERK1/2 and induced phosphorylation of MEK as observed in
NB cell lines (Chapter 5).
187
Appendix A-1 In vitro cytotoxicity of cobimetinib against NB and RD cell lines.
Exponentially growing NB and control RD cells were incubated with increasing concentrations
of cobimetinib or corresponding DMSO control in triplicate. After four days in culture, cell
viability in each condition was measured by automated cytometer and percentages of cell
survival were calculated with respect to DMSO control wells. Data presented above is
representative of three separate experiments.
188
Appendix A-2. Effect of cobimetinib on MAPK cascade pathway in RD cell line.
Changes in activation status of RAF, MEK1/2 and ERK1/2 were evaluated in RD cell line after
treatment with cobimetinib (1µM) or DMSO for four hours. Cell lysates were made with lysis
buffer containing protease and phosphatase inhibitors and probed with total and phosphor
specific antibodies by western blot analysis. Data presented above is representative of three
separate experiments.
189
In addition to the cytotoxicity effects observed after cobimetinib treatment, we also
evaluated the effects of cobimetinib on cell migration either alone or in combination with cis-RA
in both NB and RD cells. In the scratch assay, the movement of cells across a scratch line is
evaluated as an indication of the capability of an agent to inhibit cell migration. Results
presented in Figure A3 show a concentration-dependent loss of cell migration of SHEP cells over
the scratch line when treated with cobimetinib alone or in combination. These data suggest that
cobimetinib alone is also able to prevent cell migration, which is enhanced after combined
treatment with cis-RA. The similar trend was observed in RD cell line (Figure A4). Cell
migration into the detection zone was quantified by counting cell number using ImageJ software.
190
A.
24h
0h
Cobimetinib
Cis-RA
Cis-RA
+
Cobimetinib
Control
191
B.
SHEP
Cell number (Average)
300
200
0h
24h
100
0
Control
cis-RA
Cobimetinib
cis-RA +
Cobimetinib
Appendix A-3. In vitro cell migration assay in SHEP cell line.
A: SHEP cells were plated in 6-well plates and a scratch was introduced when cells were 70-80%
confluent. Cells were treated with 10 µM cis-RA and 1 µM cobimetinib alone or in combination
compared to corresponding DMSO treatment. Images were acquired at 0 h, and 24 h following
the in vitro scratch assay. The black lines define the areas lacking cells. B: Cell migration was
measured by quantifying the total number of cells that moved from the edge of the scratch
toward the center of the scratch. Data presented above is representative of three separate
experiments.
192
A.
24h
0h
RD
Cobimetinib
Cis-RA
Cis-RA
+
Cobimetinib
Control
193
B.
RD
Cell Number (Average)
300
200
0h
24h
100
0
Control
cis-RA
Cobimetinib
cis-RA + Cobimetinib
Appendix A-4. In vitro cell migration assay in RAS mutated RD cell line.
A: RD cells were plated in 6-well plate and a scratch was introduced when cells were 70-80%
confluent. Cells were treated with 10 µM cis-RA and 1 µM cobimetinib alone or in combination
compared to corresponding DMSO treatment. Images were acquired at 0 h, and 24 h following
the in vitro scratch assay. The black lines define the areas lacking cells. B: Cell migration was
measured by quantifying the total number of cells that moved from the edge of the scratch
toward the center of the scratch. Data presented above is representative of three separate
experiments.
194
195
References
Abel F, Ejeskär K, Kogner P et al. Gain of chromosome arm 17q is associated with
unfavourable prognosis in neuroblastoma, but does not involve mutations in the somatostatin
receptor 2(SSTR2) gene at 17q24. Br J Cancer 1999;81:1402–9.
Abraham J, Prajapati SI, Nishijo K et al. Evasion mechanisms to Igf1r inhibition in
rhabdomyosarcoma. Mol Cancer Ther 2011;10:697–707.
Akinleye A, Furqan M, Mukhi N et al. MEK and the inhibitors: from bench to bedside. J
Hematol Oncol 2013b;6:27.
Alessi DR, Saito Y, Campbell DG et al. Identification of the sites in MAP kinase kinase-1
phosphorylated by p74raf-1. EMBO J 1994;13:1610–9.
Andreoli F, Barbosa AJM, Parenti MD et al. Modulation of epigenetic targets for anticancer
therapy: clinicopathological relevance, structural data and drug discovery perspectives. Curr
Pharm Des 2013;19:578–613.
Arcaro A, Doepfner KT, Boller D et al. Novel role for insulin as an autocrine growth factor
for malignant brain tumour cells. Biochem J 2007;406:57–66.
Bacac M, Stamenkovic I. Metastatic cancer cell. Annu Rev Pathol 2008;3:221–47.
Bai X, Yan Y, Coleman M et al. Tracking long-term survival of intramyocardially delivered
human adipose tissue-derived stem cells using bioluminescence imaging. Mol Imaging Biol
2011;13:633–45.
Barnes EN, Biedler JL, Spengler BA et al. The fine structure of continuous human
neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC. In Vitro 1981a;17:619–31.
Baselga J. Targeting tyrosine kinases in cancer: the second wave. Science 2006;312:1175–8.
Baserga R. The IGF-I receptor in cancer research. Exp Cell Res 1999a;253:1–6.
Batzer AG, Rotin D, Ureña JM et al. Hierarchy of binding sites for Grb2 and Shc on the
epidermal growth factor receptor. Mol Cell Biol 1994;14:5192–201.
Beauchamp M-C, Knafo A, Yasmeen A et al. BMS-536924 sensitizes human epithelial
ovarian cancer cells to the PARP inhibitor, 3-aminobenzamide. Gynecol Oncol
2009;115:193–8.
Beckwith JB, Palmer NF. Histopathology and prognosis of Wilms tumors: results from the
196
First National Wilms’ Tumor Study. Cancer 1978;41:1937–48.
Beech DJ, Parekh N, Pang Y. Insulin-like growth factor-I receptor antagonism results in
increased cytotoxicity of breast cancer cells to doxorubicin and taxol. Oncol Rep 8:325–9.
Beppu K, Jaboine J, Merchant MS et al. Effect of imatinib mesylate on neuroblastoma
tumorigenesis and vascular endothelial growth factor expression. J Natl Cancer Inst
2004;96:46–55.
Beschorner R, Mittelbronn M, Koerbel A et al. Atypical teratoid-rhabdoid tumor spreading
along the trigeminal nerve. Pediatr Neurosurg 2006;42:258–63.
Biegel JA, Fogelgren B, Zhou JY et al. Mutations of the INI1 rhabdoid tumor suppressor
gene in medulloblastomas and primitive neuroectodermal tumors of the central nervous
system. Clin Cancer Res 2000;6:2759–63.
Biegel JA. Molecular genetics of atypical teratoid/rhabdoid tumor. Neurosurg Focus
2006;20:E11.
Bikowska B, Grajkowska W, Jóźwiak J. Atypical teratoid/rhabdoid tumor: short clinical
description and insight into possible mechanism of the disease. Eur J Neurol 2011;18:813–8.
Bjørge T, Engeland A, Tretli S et al. Birth and parental characteristics and risk of
neuroblastoma in a population-based Norwegian cohort study. Br J Cancer 2008;99:1165–9.
Blackwell KL, Burstein HJ, Storniolo AM et al. Overall survival benefit with lapatinib in
combination with trastuzumab for patients with human epidermal growth factor receptor 2positive metastatic breast cancer: final results from the EGF104900 Study. J Clin Oncol
2012;30:2585–92.
Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411:355–65.
Bodey B, Kaiser HE, Siegel SE. Epidermal growth factor receptor (EGFR) expression in
childhood brain tumors. In Vivo 19:931–41.
Boller D, Schramm A, Doepfner KT et al. Targeting the phosphoinositide 3-kinase isoform
p110delta impairs growth and survival in neuroblastoma cells. Clin Cancer Res
2008;14:1172–81.
Bond M, Bernstein ML, Pappo A et al. A phase II study of imatinib mesylate in children with
refractory or relapsed solid tumors: a Children’s Oncology Group study. Pediatr Blood
Cancer 2008;50:254–8.
Bozzuto G, Ruggieri P, Molinari A. Molecular aspects of tumor cell migration and invasion.
Ann dell’Istituto Super di sanità 2010;46:66–80.
197
Brignole C, Pagnan G, Marimpietri D et al. Targeted delivery system for antisense
oligonucleotides: a novel experimental strategy for neuroblastoma treatment. Cancer Lett
2003;197:231–5.
Brodeur GM, Iyer R, Croucher JL et al. Therapeutic targets for neuroblastomas. Expert Opin
Ther Targets 2014.
Brodeur GM, Minturn JE, Ho R et al. Trk receptor expression and inhibition in
neuroblastomas. Clin Cancer Res 2009;15:3244–50.
Brodeur GM, Nakagawara A. Molecular basis of clinical heterogeneity in neuroblastoma. Am
J Pediatr Hematol Oncol 1992;14:111–6.
Brodeur GM, Pritchard J, Berthold F et al. Revisions of the international criteria for
neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993;11:1466–77.
Brodeur GM. Molecular basis for heterogeneity in human neuroblastomas. Eur J Cancer
1995;31A:505–10.
Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer
2003;3:203–16.
Brown AP, Carlson TCG, Loi C-M et al. Pharmacodynamic and toxicokinetic evaluation of
the novel MEK inhibitor, PD0325901, in the rat following oral and intravenous
administration. Cancer Chemother Pharmacol 2007;59:671–9.
Brunton VG, Frame MC. Src and focal adhesion kinase as therapeutic targets in cancer. Curr
Opin Pharmacol 2008;8:427–32.
Burger PC, Yu IT, Tihan T et al. Atypical teratoid/rhabdoid tumor of the central nervous
system: a highly malignant tumor of infancy and childhood frequently mistaken for
medulloblastoma: a Pediatric Oncology Group study. Am J Surg Pathol 1998;22:1083–92.
Burness ML, Grushko TA, Olopade OI. Epidermal growth factor receptor in triple-negative
and basal-like breast cancer: promising clinical target or only a marker? Cancer J 16:23–32.
Bursztajn S, Feng JJ, Berman SA et al. Poly (ADP-ribose) polymerase induction is an early
signal of apoptosis in human neuroblastoma. Brain Res Mol Brain Res 2000;76:363–76.
Calafiore L, Amoroso L, Della Casa Alberighi O et al. Two-stage phase II study of imatinib
mesylate in subjects with refractory or relapsing neuroblastoma. Ann Oncol Off J Eur Soc
Med Oncol / ESMO 2013;24:1406–13.
Camirand A, Pollak M. Co-targeting IGF-1R and c-kit: synergistic inhibition of proliferation
198
and induction of apoptosis in H 209 small cell lung cancer cells. Br J Cancer 2004;90:1825–
9.
Camphausen K, Purow B, Sproull M et al. Influence of in vivo growth on human glioma cell
line gene expression: convergent profiles under orthotopic conditions. Proc Natl Acad Sci U
S A 2005;102:8287–92.
Čáslavský J, Klímová Z, Vomastek T. ERK and RSK regulate distinct steps of a cellular
program that induces transition from multicellular epithelium to single cell phenotype. Cell
Signal 2013;25:2743–51.
Caunt CJ, Keyse SM. Dual-specificity MAP kinase phosphatases (MKPs): shaping the
outcome of MAP kinase signalling. FEBS J 2013;280:489–504.
Caunt CJ, Sale MJ, Smith PD et al. MEK1 and MEK2 inhibitors and cancer therapy: the long
and winding road. Nat Rev Cancer 2015;15:577–92.
Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011;331:1559–
64.
Chakraborty AK, Zerillo C, DiGiovanna MP. In vitro and in vivo studies of the combination
of IGF1R inhibitor figitumumab (CP-751,871) with HER2 inhibitors trastuzumab and
neratinib. Breast Cancer Res Treat 2015;152:533–44.
Chandrasekher G, Sailaja D. Phosphatidylinositol 3-kinase (PI-3K)/Akt but not PI-3K/p70 S6
kinase signaling mediates IGF-1-promoted lens epithelial cell survival. Invest Ophthalmol
Vis Sci 2004;45:3577–88.
Chattopadhyay A, Vecchi M, Ji Q s et al. The role of individual SH2 domains in mediating
association of phospholipase C-gamma1 with the activated EGF receptor. J Biol Chem
1999;274:26091–7.
Chen HX, Sharon E. IGF-1R as an anti-cancer target--trials and tribulations. Chin J Cancer
2013;32:242–52.
Chi SN, Zimmerman MA, Yao X et al. Intensive multimodality treatment for children with
newly diagnosed CNS atypical teratoid rhabdoid tumor. J Clin Oncol 2009;27:385–9.
Childhood cancer incidence and mortality in Canada. 2015.
Chou T-C. Drug combination studies and their synergy quantification using the Chou-Talalay
method. Cancer Res 2010;70:440–6.
Ciccarone V, Spengler BA, Meyers MB et al. Phenotypic diversification in human
neuroblastoma cells: expression of distinct neural crest lineages. Cancer Res 1989;49:219–
199
25.
Cohen P. The role of protein phosphorylation in human health and disease. The Sir Hans
Krebs Medal Lecture. Eur J Biochem 2001;268:5001–10.
Cohn SL, Pearson ADJ, London WB et al. The International Neuroblastoma Risk Group
(INRG) classification system: an INRG Task Force report. J Clin Oncol 2009;27:289–97.
Cole KA, Maris JM. New strategies in refractory and recurrent neuroblastoma: translational
opportunities to impact patient outcome. Clin Cancer Res 2012;18:2423–8.
Cooper GM. The Development and Causes of Cancer. 2000.
Cortes JE, Kantarjian H, Shah NP et al. Ponatinib in refractory Philadelphia chromosomepositive leukemias. N Engl J Med 2012;367:2075–88.
Coulter DW, Blatt J, D’Ercole AJ et al. IGF-I receptor inhibition combined with rapamycin
or temsirolimus inhibits neuroblastoma cell growth. Anticancer Res 28:1509–16.
Cruickshanks N, Hamed HA, Bareford MD et al. Lapatinib and obatoclax kill tumor cells
through blockade of ERBB1/3/4 and through inhibition of BCL-XL and MCL-1. Mol
Pharmacol 2012;81:748–58.
D’cunja J, Shalaby T, Rivera P et al. Antisense treatment of IGF-IR induces apoptosis and
enhances chemosensitivity in central nervous system atypical teratoid/rhabdoid tumours
cells. Eur J Cancer 2007;43:1581–9.
Daud A, Bastian BC. Beyond BRAF in melanoma. Curr Top Microbiol Immunol
2012;355:99–117.
Davidoff AM. Neuroblastoma. Semin Pediatr Surg 2012;21:2–14.
Davies H, Bignell GR, Cox C et al. Mutations of the BRAF gene in human cancer. Nature
2002;417:949–54.
Dayyani F, Parikh NU, Varkaris AS et al. Combined Inhibition of IGF-1R/IR and Src family
kinases enhances antitumor effects in prostate cancer by decreasing activated survival
pathways. PLoS One 2012;7:e51189.
Denduluri SK, Idowu O, Wang Z et al. Insulin-like growth factor (IGF) signaling in
tumorigenesis and the development of cancer drug resistance. Genes Dis 2015;2:13–25.
Dhillon AS, Hagan S, Rath O et al. MAP kinase signalling pathways in cancer. Oncogene
2007;26:3279–90.
Dimitropoulos K, Giannopoulou E, Argyriou AA et al. The effects of anti-VEGFR and anti200
EGFR agents on glioma cell migration through implication of growth factors with integrins.
Anticancer Res 2010;30:4987–92.
Dolloff NG, Mayes PA, Hart LS et al. Off-target lapatinib activity sensitizes colon cancer
cells through TRAIL death receptor up-regulation. Sci Transl Med 2011;3:86ra50.
Douziech N, Calvo E, Lainé J et al. Activation of MAP kinases in growth responsive
pancreatic cancer cells. Cell Signal 1999;11:591–602.
Downward J, Yarden Y, Mayes E et al. Close similarity of epidermal growth factor receptor
and v-erb-B oncogene protein sequences. Nature 307:521–7.
Dunn SE, Hardman RA, Kari FW et al. Insulin-like growth factor 1 (IGF-1) alters drug
sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by
diverse anticancer drugs. Cancer Res 1997;57:2687–93.
Eccles SA. The epidermal growth factor receptor/Erb-B/HER family in normal and
malignant breast biology. Int J Dev Biol 2011;55:685–96.
El-Badry OM, Helman LJ, Chatten J et al. Insulin-like growth factor II-mediated
proliferation of human neuroblastoma. J Clin Invest 1991a;87:648–57.
El-Badry OM, Romanus JA, Helman LJ et al. Autonomous growth of a human
neuroblastoma cell line is mediated by insulin-like growth factor II. J Clin Invest
1989;84:829–39.
Eleveld TF, Oldridge DA, Bernard V et al. Relapsed neuroblastomas show frequent RASMAPK pathway mutations. Nat Genet 2015;47:864–71.
Emil Frei I, Eder JP. Combination Chemotherapy. 2003.
Eppstein AC, Sandoval JA, Klein PJ et al. Differential sensitivity of chemoresistant
neuroblastoma subtypes to MAPK-targeted treatment correlates with ERK, p53 expression,
and signaling response to U0126. J Pediatr Surg 2006;41:252–9.
Erickson SL, O’Shea KS, Ghaboosi N et al. ErbB3 is required for normal cerebellar and
cardiac development: a comparison with ErbB2-and heregulin-deficient mice. Development
1997;124:4999–5011.
De Falco V, Buonocore P, Muthu M et al. Ponatinib (AP24534) is a novel potent inhibitor of
oncogenic RET mutants associated with thyroid cancer. J Clin Endocrinol Metab
2013;98:E811–9.
Farabaugh SM, Boone DN, Lee A V. Role of IGF1R in Breast Cancer Subtypes, Stemness,
and Lineage Differentiation. Front Endocrinol (Lausanne) 2015;6:59.
201
Feduska JM, Garcia PL, Brennan SB et al. N-glycosylation of ICAM-2 is required for
ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma
cells. BMC Cancer 2013;13:261.
Fernández-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer
2011;2:344–58.
Ferrís i Tortajada J, Ortega García JA, García i Castell J et al. [Risk factors for
neuroblastoma]. An Pediatr (Barcelona, Spain 2003) 2005;63:50–60.
Fidler IJ. The pathogenesis of cancer metastasis: the “seed and soil” hypothesis revisited. Nat
Rev Cancer 2003;3:453–8.
Field-Smith A, Morgan GJ, Davies FE. Bortezomib (Velcadetrade mark) in the Treatment of
Multiple Myeloma. Ther Clin Risk Manag 2006;2:271–9.
Foster K, Wang Y, Zhou D et al. Dependence on PI3K/Akt signaling for malignant rhabdoid
tumor cell survival. Cancer Chemother Pharmacol 2009;63:783–91.
Foster KSJ, McCrary WJ, Ross JS et al. Members of the hSWI/SNF chromatin remodeling
complex associate with and are phosphorylated by protein kinase B/Akt. Oncogene
2006;25:4605–12.
Fouladi M, Stewart CF, Blaney SM et al. Phase I trial of lapatinib in children with refractory
CNS malignancies: a Pediatric Brain Tumor Consortium study. J Clin Oncol 2010;28:4221–
7.
Freudenberg JA, Wang Q, Katsumata M et al. The role of HER2 in early breast cancer
metastasis and the origins of resistance to HER2-targeted therapies. Exp Mol Pathol
2009;87:1–11.
Friedl P, Wolf K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol
2010;188:11–9.
Frühwald MC, Biegel JA, Bourdeaut F et al. Atypical teratoid/rhabdoid tumors-current
concepts, advances in biology, and potential future therapies. Neuro Oncol 2016:nov264 – .
Fujisawa H, Misaki K, Takabatake Y et al. Cyclin D1 is overexpressed in atypical
teratoid/rhabdoid tumor with hSNF5/INI1 gene inactivation. J Neurooncol 2005;73:117–24.
Fukushige S, Matsubara K, Yoshida M et al. Localization of a novel v-erbB-related gene, cerbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line. Mol Cell
Biol 1986;6:955–8.
202
Gains J, Mandeville H, Cork N et al. Ten challenges in the management of neuroblastoma.
Future Oncol 2012;8:839–58.
Gassmann M, Casagranda F, Orioli D et al. Aberrant neural and cardiac development in mice
lacking the ErbB4 neuregulin receptor. Nature 1995;378:390–4.
Gatta G, Ferrari A, Stiller CA et al. Embryonal cancers in Europe. Eur J Cancer
2012;48:1425–33.
Gheeya JS, Chen Q-R, Benjamin CD et al. Screening a panel of drugs with diverse
mechanisms of action yields potential therapeutic agents against neuroblastoma. Cancer Biol
Ther 2009;8:2386–95.
Ghigo A, Li M. Phosphoinositide 3-kinase: friend and foe in cardiovascular disease. Front
Pharmacol 2015;6:169.
Gilbertson RJ, Perry RH, Kelly PJ et al. Prognostic significance of HER2 and HER4
coexpression in childhood medulloblastoma. Cancer Res 1997;57:3272–80.
Gilbertson RJ. ERBB2 in pediatric cancer: innocent until proven guilty. Oncologist
2005;10:508–17.
Giles FJ, O’Dwyer M, Swords R. Class effects of tyrosine kinase inhibitors in the treatment
of chronic myeloid leukemia. Leukemia 2009;23:1698–707.
Ginn KF, Gajjar A. Atypical teratoid rhabdoid tumor: current therapy and future directions.
Front Oncol 2012;2:114.
Gisselsson D, Lundberg G, Ora I et al. Distinct evolutionary mechanisms for genomic
imbalances in high-risk and low-risk neuroblastomas. J Carcinog 2007;6:15.
van Golen CM, Schwab TS, Kim B et al. Insulin-like growth factor-I receptor expression
regulates neuroblastoma metastasis to bone. Cancer Res 2006a;66:6570–8.
Gooch JL, Van Den Berg CL, Yee D. Insulin-­‐like growth factor (IGF)-­‐I rescues breast cancer
cells from chemotherapy-­‐induced cell death – proliferative and anti-­‐apoptotic effects. Breast
Cancer Res Treat 1999;56:1–10.
Gorlick R, Kolb EA, Houghton PJ et al. Initial testing (stage 1) of lapatinib by the pediatric
preclinical testing program. Pediatr Blood Cancer 2009;53:594–8.
Gozgit JM, Squillace RM, Wongchenko MJ et al. Combined targeting of FGFR2 and mTOR
by ponatinib and ridaforolimus results in synergistic antitumor activity in FGFR2 mutant
endometrial cancer models. Cancer Chemother Pharmacol 2013;71:1315–23.
203
Gozgit JM, Wong MJ, Moran L et al. Ponatinib (AP24534), a multitargeted pan-FGFR
inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol Cancer
Ther 2012;11:690–9.
Gozgit JM, Wong MJ, Wardwell S et al. Potent activity of ponatinib (AP24534) in models of
FLT3-driven acute myeloid leukemia and other hematologic malignancies. Mol Cancer Ther
2011;10:1028–35.
Gril B, Palmieri D, Bronder JL et al. Effect of lapatinib on the outgrowth of metastatic breast
cancer cells to the brain. J Natl Cancer Inst 2008a;100:1092–103.
Grinshtein N, Datti A, Fujitani M et al. Small molecule kinase inhibitor screen identifies
polo-like kinase 1 as a target for neuroblastoma tumor-initiating cells. Cancer Res
2011a;71:1385–95.
Guidi CJ, Sands AT, Zambrowicz BP et al. Disruption of Ini1 leads to peri-implantation
lethality and tumorigenesis in mice. Mol Cell Biol 2001;21:3598–603.
Guy CT, Cardiff RD, Muller WJ. Activated neu induces rapid tumor progression. J Biol
Chem 1996;271:7673–8.
Hadjidaniel MD, Reynolds CP. Antagonism of cytotoxic chemotherapy in neuroblastoma cell
lines by 13-cis-retinoic acid is mediated by the antiapoptotic Bcl-2 family proteins. Mol
Cancer Ther 2010;9:3164–74.
Hallett A, Traunecker H. A review and update on neuroblastoma. Paediatr Child Health
(Oxford) 2012;22:103–7.
Hanahan D, Weinberg RA. The Hallmarks of Cancer. Cell 2000;100:57–70.
Hande KR. Clinical applications of anticancer drugs targeted to topoisomerase II. Biochim
Biophys Acta - Gene Struct Expr 1998;1400:173–84.
Hasselblatt M, Isken S, Linge A et al. High-resolution genomic analysis suggests the absence
of recurrent genomic alterations other than SMARCB1 aberrations in atypical
teratoid/rhabdoid tumors. Genes Chromosomes Cancer 2013;52:185–90.
Hatzivassiliou G, Haling JR, Chen H et al. Mechanism of MEK inhibition determines
efficacy in mutant KRAS- versus BRAF-driven cancers. Nature 2013a;501:232–6.
Hecht JR, Bang Y-J, Qin SK et al. Lapatinib in Combination With Capecitabine Plus
Oxaliplatin in Human Epidermal Growth Factor Receptor 2-Positive Advanced or Metastatic
Gastric, Esophageal, or Gastroesophageal Adenocarcinoma: TRIO-013/LOGiC- A
Randomized Phase III Trial. J Clin Oncol 2015:JCO.2015.62.6598 – .
204
Heck JE, Ritz B, Hung RJ et al. The epidemiology of neuroblastoma: a review. Paediatr
Perinat Epidemiol 2009;23:125–43.
Hegde PS, Rusnak D, Bertiaux M et al. Delineation of molecular mechanisms of sensitivity
to lapatinib in breast cancer cell lines using global gene expression profiles. Mol Cancer Ther
2007;6:1629–40.
Heidegger I, Pircher A, Klocker H et al. Targeting the insulin-like growth factor network in
cancer therapy. Cancer Biol Ther 2011;11:701–7.
Hemmings BA, Restuccia DF. PI3K-PKB/Akt pathway. Cold Spring Harb Perspect Biol
2012;4:a011189.
Hinson ARP, Jones R, Crose LES et al. Human rhabdomyosarcoma cell lines for
rhabdomyosarcoma research: utility and pitfalls. Front Oncol 2013;3:183.
Hirsch FR, Varella-Garcia M, Bunn PA et al. Epidermal growth factor receptor in non-smallcell lung carcinomas: correlation between gene copy number and protein expression and
impact on prognosis. J Clin Oncol 2003;21:3798–807.
Ho DM, Hsu CY, Wong TT et al. Atypical teratoid/rhabdoid tumor of the central nervous
system: a comparative study with primitive neuroectodermal tumor/medulloblastoma. Acta
Neuropathol 2000;99:482–8.
Hoelder S, Clarke PA, Workman P. Discovery of small molecule cancer drugs: successes,
challenges and opportunities. Mol Oncol 2012;6:155–76.
von Hoff K, Hinkes B, Dannenmann-Stern E et al. Frequency, risk-factors and survival of
children with atypical teratoid rhabdoid tumors (AT/RT) of the CNS diagnosed between
1988 and 2004, and registered to the German HIT database. Pediatr Blood Cancer
2011;57:978–85.
Hogarty MD, Norris MD, Davis K et al. ODC1 is a critical determinant of MYCN
oncogenesis and a therapeutic target in neuroblastoma. Cancer Res 2008;68:9735–45.
HOLT R. Fetal programming of the growth hormone–insulin-like growth factor axis. Trends
Endocrinol Metab 2002;13:392–7.
Huang M, Weiss WA. Neuroblastoma and MYCN. Cold Spring Harb Perspect Med
2013;3:a014415.
Huang W-S, Metcalf CA, Sundaramoorthi R et al. Discovery of 3-[2-(imidazo[1,2b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of
breakpoint cluster region-abelson (BCR-ABL) kinase including th. J Med Chem
205
2010;53:4701–19.
Hwang H-J, Kwon M-J, Nam T-J. Chemoprotective effect of insulin-like growth factor I
against acetaminophen-induced cell death in Chang liver cells via ERK1/2 activation.
Toxicology 2007;230:76–82.
Iqbal N, Iqbal N. Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers:
Overexpression and Therapeutic Implications. Mol Biol Int 2014;2014:852748.
Ishii N, Harada N, Joseph EW et al. Enhanced inhibition of ERK signaling by a novel
allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.
Cancer Res 2013;73:4050–60.
Jabbour E, Kantarjian H. Chronic myeloid leukemia: 2016 update on diagnosis, therapy, and
monitoring. Am J Hematol 2016;91:252–65.
Jayanthan A, Bernoux D, Bose P et al. Multi-tyrosine kinase inhibitors in preclinical studies
for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition. Cancer
Cell Int 2011;11:44.
Jin Q, Esteva FJ. Cross-talk between the ErbB/HER family and the type I insulin-like growth
factor receptor signaling pathway in breast cancer. J Mammary Gland Biol Neoplasia
2008;13:485–98.
Kaleko M, Rutter WJ, Miller AD. Overexpression of the human insulinlike growth factor I
receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol 1990;10:464–
73.
Kamijo T, Nakagawara A. Molecular and genetic bases of neuroblastoma. Int J Clin Oncol
2012;17:190–5.
Kembhavi SA, Shah S, Rangarajan V et al. Imaging in neuroblastoma: An update. Indian J
Radiol Imaging 2015;25:129–36.
Keshelava N, Seeger RC, Reynolds CP. Drug resistance in human neuroblastoma cell lines
correlates with clinical therapy. Eur J Cancer 1997;33:2002–6.
Khanna C, Jaboin JJ, Drakos E et al. Biologically relevant orthotopic neuroblastoma
xenograft models: primary adrenal tumor growth and spontaneous distant metastasis. In Vivo
2002;16:77–85.
Kieran MW, Roberts CWM, Chi SN et al. Absence of oncogenic canonical pathway
mutations in aggressive pediatric rhabdoid tumors. Pediatr Blood Cancer 2012;59:1155–7.
Kim B, van Golen CM, Feldman EL. Insulin-like growth factor-I signaling in human
neuroblastoma cells. Oncogene 2004a;23:130–41.
206
Kim SY, Toretsky JA, Scher D et al. The role of IGF-1R in pediatric malignancies.
Oncologist 2009;14:83–91.
Kim SY, Wan X, Helman LJ. Targeting IGF-1R in the treatment of sarcomas: past, present
and future. Bull Cancer 96:E52–60.
Kiyonari S, Kadomatsu K. Neuroblastoma models for insights into tumorigenesis and new
therapies. Expert Opin Drug Discov 2014.
Klapper LN, Glathe S, Vaisman N et al. The ErbB-2/HER2 oncoprotein of human
carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth
factors. Proc Natl Acad Sci U S A 1999;96:4995–5000.
Knipstein JA, Birks DK, Donson AM et al. Histone deacetylase inhibition decreases
proliferation and potentiates the effect of ionizing radiation in atypical teratoid/rhabdoid
tumor cells. Neuro Oncol 2012;14:175–83.
Kohn EC, Francis EA, Liotta LA et al. Heterogeneity of the motility responses in malignant
tumor cells: a biological basis for the diversity and homing of metastatic cells. Int J Cancer
1990;46:287–92.
Kolb EA, Gorlick R, Lock R et al. Initial testing (stage 1) of the IGF-1 receptor inhibitor
BMS-754807 by the pediatric preclinical testing program. Pediatr Blood Cancer
2011;56:595–603.
Kolch W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev
Mol Cell Biol 2005;6:827–37.
Konecny GE, Pegram MD, Venkatesan N et al. Activity of the dual kinase inhibitor lapatinib
(GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells.
Cancer Res 2006;66:1630–9.
Kuan CT, Wikstrand CJ, Bigner DD. EGFRvIII as a promising target for antibody-based
brain tumor therapy. Brain Tumor Pathol 2000;17:71–8.
Kunz-Schughart LA, Freyer JP, Hofstaedter F et al. The use of 3-D cultures for highthroughput screening: the multicellular spheroid model. J Biomol Screen 2004;9:273–85.
Kuwahara Y, Hosoi H, Osone S et al. Antitumor activity of gefitinib in malignant rhabdoid
tumor cells in vitro and in vivo. Clin Cancer Res 2004;10:5940–8.
LaBonte MJ, Wilson PM, Fazzone W et al. The dual EGFR/HER2 inhibitor lapatinib
synergistically enhances the antitumor activity of the histone deacetylase inhibitor
panobinostat in colorectal cancer models. Cancer Res 2011;71:3635–48.
207
Lamba S, Russo M, Sun C et al. RAF suppression synergizes with MEK inhibition in KRAS
mutant cancer cells. Cell Rep 2014;8:1475–83.
Larsson O, Girnita A, Girnita L. Role of insulin-like growth factor 1 receptor signalling in
cancer. Br J Cancer 2005a;92:2097–101.
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell
2010;141:1117–34.
Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature
1998;396:643–9.
Liang C-C, Park AY, Guan J-L. In vitro scratch assay: a convenient and inexpensive method
for analysis of cell migration in vitro. Nat Protoc 2007a;2:329–33.
Light JE, Koyama H, Minturn JE et al. Clinical significance of NTRK family gene
expression in neuroblastomas. Pediatr Blood Cancer 2012;59:226–32.
Ligresti G, Militello L, Steelman LS et al. PIK3CA mutations in human solid tumors: role in
sensitivity to various therapeutic approaches. Cell Cycle 2009;8:1352–8.
Lin NU, Amiri-Kordestani L, Palmieri D et al. CNS metastases in breast cancer: old
challenge, new frontiers. Clin Cancer Res 2013;19:6404–18.
Liotta LA, Kohn EC. Tumor Cell Migration. 2003.
Lito P, Saborowski A, Yue J et al. Disruption of CRAF-mediated MEK activation is required
for effective MEK inhibition in KRAS mutant tumors. Cancer Cell 2014a;25:697–710.
Liu JP, Baker J, Perkins AS et al. Mice carrying null mutations of the genes encoding
insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59–72.
Liu W, Zhang S, Zhang L et al. A prognostic analysis of pediatrics central nervous system
small cell tumors: evaluation of EGFR family gene amplification and overexpression. Diagn
Pathol 2014;9:132.
Liu X, Turbyville T, Fritz A et al. Inhibition of insulin-like growth factor I receptor
expression in neuroblastoma cells induces the regression of established tumors in mice.
Cancer Res 1998;58:5432–8.
Lo H-W, Xia W, Wei Y et al. Novel prognostic value of nuclear epidermal growth factor
receptor in breast cancer. Cancer Res 2005;65:338–48.
London WB, Castel V, Monclair T et al. Clinical and biologic features predictive of survival
208
after relapse of neuroblastoma: a report from the International Neuroblastoma Risk Group
project. J Clin Oncol 2011;29:3286–92.
Lonergan GJ, Schwab CM, Suarez ES et al. Neuroblastoma, ganglioneuroblastoma, and
ganglioneuroma: radiologic-pathologic correlation. Radiographics 2002;22:911–34.
Lopez T, Hanahan D. Elevated levels of IGF-1 receptor convey invasive and metastatic
capability in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 2002;1:339–53.
Louis DN, Ohgaki H, Wiestler OD et al. The 2007 WHO classification of tumours of the
central nervous system. Acta Neuropathol 2007;114:97–109.
Lun X, Alain T, Zemp FJ et al. Myxoma virus virotherapy for glioma in immunocompetent
animal models: optimizing administration routes and synergy with rapamycin. Cancer Res
2010;70:598–608.
Lupino E, Ramondetti C, Buccinnà B et al. Exposure of neuroblastoma cell lines to imatinib
results in the upregulation of the CDK inhibitor p27(KIP1) as a consequence of c-Abl
inhibition. Biochem Pharmacol 2014;92:235–50.
MacDonald T. Pediatric cancer: A comprehensive review. Part I: Biology, epidemiology,
common tumours, principles of treatment and late effects. Can Pharm J 2010;143:176–83.
Maloney EK, McLaughlin JL, Dagdigian NE et al. An anti-insulin-like growth factor I
receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res
2003;63:5073–83.
Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3:459–
65.
Mancini M, Gariboldi MB, Taiana E et al. Co-targeting the IGF system and HIF-1 inhibits
migration and invasion by (triple-negative) breast cancer cells. Br J Cancer 2014;110:2865–
73.
Maris JM, Morton CL, Gorlick R et al. Initial testing of the aurora kinase A inhibitor
MLN8237 by the Pediatric Preclinical Testing Program (PPTP). Pediatr Blood Cancer
2010;55:26–34.
Maris JM. The biologic basis for neuroblastoma heterogeneity and risk stratification. Curr
Opin Pediatr 2005;17:7–13.
Maris JM. Recent advances in neuroblastoma. N Engl J Med 2010;362:2202–11.
Martin TA, Ye L, Sanders AJ et al. Cancer Invasion and Metastasis: Molecular and Cellular
Perspective. 2000.
209
Martini M, Santis MC De, Braccini L et al. PI3K/AKT signaling pathway and cancer: an
updated review. Ann Med 2014.
Marusyk A, Polyak K. Tumor heterogeneity: causes and consequences. Biochim Biophys
Acta 2010;1805:105–17.
di Masi A, Leboffe L, De Marinis E et al. Retinoic acid receptors and cancer: From
molecular mechanisms to therapy. Mol Aspects Med 2014;41:1–115.
Matthay KK, George RE, Yu AL. Promising therapeutic targets in neuroblastoma. Clin
Cancer Res 2012;18:2740–53.
Matthay KK, Reynolds CP, Seeger RC et al. Long-term results for children with high-risk
neuroblastoma treated on a randomized trial of myeloablative therapy followed by 13-cisretinoic acid: a children’s oncology group study. J Clin Oncol 2009;27:1007–13.
Matthay KK, Villablanca JG, Seeger RC et al. Treatment of high-risk neuroblastoma with
intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cisretinoic acid. Children’s Cancer Group. N Engl J Med 1999;341:1165–73.
McCubrey JA, Steelman LS, Chappell WH et al. Roles of the Raf/MEK/ERK pathway in cell
growth, malignant transformation and drug resistance. Biochim Biophys Acta
2007;1773:1263–84.
McDermott U, Downing JR, Stratton MR. Genomics and the continuum of cancer care. N
Engl J Med 2011;364:340–50.
Meco D, Riccardi A, Servidei T et al. Antitumor activity of imatinib mesylate in
neuroblastoma xenografts. Cancer Lett 2005;228:211–9.
Metro G, Foglietta J, Russillo M et al. Clinical outcome of patients with brain metastases
from HER2-positive breast cancer treated with lapatinib and capecitabine. Ann Oncol
2011;22:625–30.
Meyer G, Kim B, van Golen C et al. Cofilin activity during insulin-like growth factor Istimulated neuroblastoma cell motility. Cell Mol life Sci C 2005;62:461–70.
Middleton J, Arnott N, Walsh S et al. Osteoblasts and osteoclasts in adult human osteophyte
tissue express the mRNAs for insulin-like growth factors I and II and the type 1 IGF
receptor. Bone 1995;16:287–93.
Miettinen PJ, Berger JE, Meneses J et al. Epithelial immaturity and multiorgan failure in
mice lacking epidermal growth factor receptor. Nature 1995;376:337–41.
210
Migliardi G, Sassi F, Torti D et al. Inhibition of MEK and PI3K/mTOR suppresses tumor
growth but does not cause tumor regression in patient-derived xenografts of RAS-mutant
colorectal carcinomas. Clin Cancer Res 2012;18:2515–25.
Milella M, Kornblau SM, Estrov Z et al. Therapeutic targeting of the MEK/MAPK signal
transduction module in acute myeloid leukemia. J Clin Invest 2001;108:851–9.
Miller CR, Oliver KE, Farley JH. MEK1/2 inhibitors in the treatment of gynecologic
malignancies. Gynecol Oncol 2014;133:128–37.
Miller EE, Evans AE, Cohn M. Inhibition of rate of tumor growth by creatine and
cyclocreatine. Proc Natl Acad Sci U S A 1993;90:3304–8.
Min Y, Adachi Y, Yamamoto H et al. Insulin-like growth factor I receptor blockade
enhances chemotherapy and radiation responses and inhibits tumour growth in human gastric
cancer xenografts. Gut 2005;54:591–600.
Misawa A, Hosoi H, Arimoto A et al. N-Myc induction stimulated by insulin-like growth
factor I through mitogen-activated protein kinase signaling pathway in human neuroblastoma
cells. Cancer Res 2000;60:64–9.
Missiaglia E, Selfe J, Hamdi M et al. Genomic imbalances in rhabdomyosarcoma cell lines
affect expression of genes frequently altered in primary tumors: an approach to identify
candidate genes involved in tumor development. Genes Chromosomes Cancer 2009;48:455–
67.
Mitra SK, Schlaepfer DD. Integrin-regulated FAK-Src signaling in normal and cancer cells.
Curr Opin Cell Biol 2006;18:516–23.
Monclair T, Brodeur GM, Ambros PF et al. The International Neuroblastoma Risk Group
(INRG) staging system: an INRG Task Force report. J Clin Oncol 2009;27:298–303.
Montero JC, Seoane S, Ocaña A et al. Inhibition of SRC family kinases and receptor tyrosine
kinases by dasatinib: possible combinations in solid tumors. Clin cancer Res an Off J Am
Assoc Cancer Res 2011;17:5546–52.
Mosesson Y, Shtiegman K, Katz M et al. Endocytosis of receptor tyrosine kinases is driven
by monoubiquitylation, not polyubiquitylation. J Biol Chem 2003;278:21323–6.
Mughal A, Aslam HM, Khan AMH et al. Bcr-Abl tyrosine kinase inhibitors- current status.
Infect Agent Cancer 2013;8:23.
Mukohara T. Role of HER2-Targeted Agents in Adjuvant Treatment for Breast Cancer.
Chemother Res Pract 2011;2011:730360.
211
Musib L, Choo E, Deng Y et al. Absolute bioavailability and effect of formulation change,
food, or elevated pH with rabeprazole on cobimetinib absorption in healthy subjects. Mol
Pharm 2013;10:4046–54.
Musumeci F, Schenone S, Brullo C et al. An update on dual Src/Abl inhibitors. Future Med
Chem 2012;4:799–822.
Nabzdyk CS, Chun M, Pradhan L et al. High throughput RNAi assay optimization using
adherent cell cytometry. J Transl Med 2011;9:48.
Nahta R, Yuan LXH, Du Y et al. Lapatinib induces apoptosis in trastuzumab-resistant breast
cancer cells: effects on insulin-like growth factor I signaling. Mol Cancer Ther 2007;6:667–
74.
Narendran A, Coppes L, Jayanthan A et al. Establishment of atypical-teratoid/rhabdoid
tumor (AT/RT) cell cultures from disseminated CSF cells: a model to elucidate biology and
potential targeted therapeutics. J Neurooncol 2008;90:171–80.
Navarra M, Celano M, Maiuolo J et al. Antiproliferative and pro-apoptotic effects afforded
by novel Src-kinase inhibitors in human neuroblastoma cells. BMC Cancer 2010;10:602.
Nicholas MK, Lukas R V, Jafri NF et al. Epidermal growth factor receptor - mediated signal
transduction in the development and therapy of gliomas. Clin Cancer Res 2006;12:7261–70.
Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human
malignancy. Cell Signal 2002;14:381–95.
Nicholson R., Gee JM., Harper M. EGFR and cancer prognosis. Eur J Cancer 2001;37:9–15.
Normanno N, Bianco C, De Luca A et al. Target-based agents against ErbB receptors and
their ligands: a novel approach to cancer treatment. Endocr Relat Cancer 2003;10:1–21.
Normanno N, De Luca A, Bianco C et al. Epidermal growth factor receptor (EGFR)
signaling in cancer. Gene 2006;366:2–16.
O’Donovan N, Byrne AT, O’Connor AE et al. Synergistic interaction between trastuzumab
and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells. Invest
New Drugs 2011;29:752–9.
O’Hare T, Shakespeare WC, Zhu X et al. AP24534, a pan-BCR-ABL inhibitor for chronic
myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based
resistance. Cancer Cell 2009;16:401–12.
Oakman C, Pestrin M, Zafarana E et al. Role of lapatinib in the first-line treatment of
patients with metastatic breast cancer. Cancer Manag Res 2010;2:13–25.
212
Ogino S, Cohen ML, Abdul-Karim FW. Atypical teratoid/rhabdoid tumor of the CNS:
cytopathology and immunohistochemistry of insulin-like growth factor-II, insulin-like
growth factor receptor type 1, cathepsin D, and Ki-67. Mod Pathol 1999;12:379–85.
Ogino S, Kubo S, Abdul-Karim FW et al. Comparative immunohistochemical study of
insulin-like growth factor II and insulin-like growth factor receptor type 1 in pediatric brain
tumors. Pediatr Dev Pathol 4:23–31.
Ohren JF, Chen H, Pavlovsky A et al. Structures of human MAP kinase kinase 1 (MEK1)
and MEK2 describe novel noncompetitive kinase inhibition. Nat Struct Mol Biol
2004;11:1192–7.
Okabe S, Tauchi T, Tanaka Y et al. Efficacy of ponatinib against ABL tyrosine kinase
inhibitor-resistant leukemia cells. Biochem Biophys Res Commun 2013;435:506–11.
Ozawa T, James CD. Establishing intracranial brain tumor xenografts with subsequent
analysis of tumor growth and response to therapy using bioluminescence imaging. J Vis Exp
2010.
Palazzo A, Iacovelli R, Cortesi E. Past, present and future of targeted therapy in solid tumors.
Curr Cancer Drug Targets 2010;10:433–61.
Palmberg E, Johnsen JI, Paulsson J et al. Metronomic scheduling of imatinib abrogates
clonogenicity of neuroblastoma cells and enhances their susceptibility to selected
chemotherapeutic drugs in vitro and in vivo. Int J Cancer 2009;124:1227–34.
Pandya K, Meeke K, Clementz AG et al. Targeting both Notch and ErbB-2 signalling
pathways is required for prevention of ErbB-2-positive breast tumour recurrence. Br J
Cancer 2011;105:796–806.
Papaioannou G, McHugh K. Neuroblastoma in childhood: review and radiological findings.
Cancer Imaging 2005;5:116–27.
Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and treatment. Hematol
Oncol Clin North Am 2010;24:65–86.
Patereli A, Alexiou GA, Stefanaki K et al. Expression of epidermal growth factor receptor
and HER-2 in pediatric embryonal brain tumors. Pediatr Neurosurg 2010;46:188–92.
Pattengale PK, Stewart TA, Leder A et al. Animal models of human disease. Pathology and
molecular biology of spontaneous neoplasms occurring in transgenic mice carrying and
expressing activated cellular oncogenes. Am J Pathol 1989;135:39–61.
Paul MK, Mukhopadhyay AK. Tyrosine kinase - Role and significance in Cancer. Int J Med
213
Sci 2004;1:101–15.
Pestalozzi BC, Brignoli S. Trastuzumab in CSF. J Clin Oncol 2000;18:2349 – b – 2351.
Peterson JE, Kulik G, Jelinek T et al. Src Phosphorylates the Insulin-like Growth Factor
Type I Receptor on the Autophosphorylation Sites: REQUIREMENT FOR
TRANSFORMATION BY src. J Biol Chem 1996;271:31562–71.
Peyssonnaux C, Provot S, Felder-Schmittbuhl MP et al. Induction of postmitotic neuroretina
cell proliferation by distinct Ras downstream signaling pathways. Mol Cell Biol
2000;20:7068–79.
Philip T. Overview of current treatment of neuroblastoma. Am J Pediatr Hematol Oncol
1992;14:97–102.
Plattner R, Kadlec L, DeMali KA et al. c-Abl is activated by growth factors and Src family
kinases and has a role in the cellular response to PDGF. Genes Dev 1999;13:2400–11.
Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer
2008;8:915–28.
Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Nat
Rev Cancer 2004;4:505–18.
Pui C-H, Gajjar AJ, Kane JR et al. Challenging issues in pediatric oncology. Nat Rev Clin
Oncol 2011;8:540–9.
Puls LN, Eadens M, Messersmith W. Current status of SRC inhibitors in solid tumor
malignancies. Oncologist 2011;16:566–78.
Rabinovich BA, Ye Y, Etto T et al. Visualizing fewer than 10 mouse T cells with an
enhanced firefly luciferase in immunocompetent mouse models of cancer. Proc Natl Acad
Sci U S A 2008;105:14342–6.
Radi M, Brullo C, Crespan E et al. Identification of potent c-Src inhibitors strongly affecting
the proliferation of human neuroblastoma cells. Bioorg Med Chem Lett 2011;21:5928–33.
Radpour R, Barekati Z, Kohler C et al. Integrated epigenetics of human breast cancer:
synoptic investigation of targeted genes, microRNAs and proteins upon demethylation
treatment. PLoS One 2011;6:e27355.
Raggi C, Mousa HS, Correnti M et al. Cancer stem cells and tumor-associated macrophages:
a roadmap for multitargeting strategies. Oncogene 2015, DOI: 10.1038/onc.2015.132.
Rajwanshi A, Srinivas R, Upasana G. Malignant small round cell tumors. J Cytol 2009;26:1–
10.
214
Ratka A, Flores BM, Mambourg SE et al. Luteinizing hormone-releasing hormone in
undifferentiated and differentiated SK-N-SH human neuroblastoma cells. Neuropeptides
1996;30:87–94.
Reddy AT. Atypical teratoid/rhabdoid tumors of the central nervous system. J Neurooncol
2005;75:309–13.
Reddy UR, Venkatakrishnan G, Roy AK et al. Characterization of Two Neuroblastoma Cell
Lines Expressing Recombinant Nerve Growth Factor Receptors. J Neurochem 1991;56:67–
74.
Regad T. Targeting RTK Signaling Pathways in Cancer. Cancers (Basel) 2015;7:1758–84.
Reichelt U, Duesedau P, Tsourlakis MC et al. Frequent homogeneous HER-2 amplification
in primary and metastatic adenocarcinoma of the esophagus. Mod Pathol 2007;20:120–9.
Ren M, Hong M, Liu G et al. Novel FGFR inhibitor ponatinib suppresses the growth of nonsmall cell lung cancer cells overexpressing FGFR1. Oncol Rep 2013a;29:2181–90.
Ren M, Qin H, Ren R et al. Ponatinib suppresses the development of myeloid and lymphoid
malignancies associated with FGFR1 abnormalities. Leukemia 2013b;27:32–40.
Resnicoff M, Sell C, Rubini M et al. Rat glioblastoma cells expressing an antisense RNA to
the insulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce regression
of wild-type tumors. Cancer Res 1994;54:2218–22.
Reynolds CP, Kang MH, Carol H et al. Initial testing (stage 1) of the phosphatidylinositol 3’
kinase inhibitor, SAR245408 (XL147) by the pediatric preclinical testing program. Pediatr
Blood Cancer 2013;60:791–8.
Reynolds CP, Matthay KK, Villablanca JG et al. Retinoid therapy of high-risk
neuroblastoma. Cancer Lett 2003;197:185–92.
Richman J, Martin-Liberal J, Diem S et al. BRAF and MEK inhibition for the treatment of
advanced BRAF mutant melanoma. Expert Opin Pharmacother 2015.
Riedemann J, Macaulay VM. IGF1R signalling and its inhibition. Endocr Relat Cancer
2006;13 Suppl 1:S33–43.
Roberts CW, Galusha SA, McMenamin ME et al. Haploinsufficiency of Snf5 (integrase
interactor 1) predisposes to malignant rhabdoid tumors in mice. Proc Natl Acad Sci U S A
2000;97:13796–800.
Roberts CWM, Orkin SH. The SWI/SNF complex — chromatin and cancer. Nat Rev Cancer
215
2004;4:133–42.
Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade
for the treatment of cancer. Oncogene 2007;26:3291–310.
Robson DK. Pathology & Genetics. Tumours of the Nervous System. World Health
Organisation Classification of Tumours. P. Kleihues and k. Cavenee (eds). IARC Press,
Lyon, 2000. No. of pages: 314. ISBN: 92 832 2409 4. J Pathol 2001;193:276–276.
Rorke LB, Packer R, Biegel J. Central nervous system atypical teratoid/rhabdoid tumors of
infancy and childhood. J Neurooncol 1995;24:21–8.
Rorke LB, Packer RJ, Biegel JA. Central nervous system atypical teratoid/rhabdoid tumors
of infancy and childhood: definition of an entity. J Neurosurg 1996;85:56–65.
Roskoski R. The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys
Res Commun 2004;319:1–11.
Roskoski R. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res
2014;79:34–74.
Ross RA, Biedler JL, Spengler BA. A role for distinct cell types in determining malignancy
in human neuroblastoma cell lines and tumors. Cancer Lett 2003a;197:35–9.
Rusnak DW, Lackey K, Affleck K et al. The effects of the novel, reversible epidermal
growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human
normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 2001a;1:85–94.
Sale MJ, Cook SJ. Intrinsic and acquired resistance to MEK1/2 inhibitors in cancer. Biochem
Soc Trans 2014;42:776–83.
Saletta F, Wadham C, Ziegler DS et al. Molecular profiling of childhood cancer: Biomarkers
and novel therapies. BBA Clin 2014;1:59–77.
Samani AA, Yakar S, LeRoith D et al. The role of the IGF system in cancer growth and
metastasis: overview and recent insights. Endocr Rev 2007;28:20–47.
Samatar AA, Poulikakos PI. Targeting RAS–ERK signalling in cancer: promises and
challenges. Nat Rev Drug Discov 2014;13:928–42.
Sánchez-Martín M, Pandiella A. Differential action of small molecule HER kinase inhibitors
on receptor heterodimerization: therapeutic implications. Int J Cancer 2012;131:244–52.
Sandoval JA, Eppstein AC, Hoelz DJ et al. Proteomic analysis of neuroblastoma subtypes in
response to mitogen-activated protein kinase inhibition: profiling multiple targets of cancer
216
kinase signaling. J Surg Res 2006;134:61–7.
Schlenska-Lange A, Knüpfer H, Lange TJ et al. Cell proliferation and migration in
glioblastoma multiforme cell lines are influenced by insulin-like growth factor I in vitro.
Anticancer Res 28:1055–60.
Scotlandi K, Manara MC, Nicoletti G et al. Antitumor activity of the insulin-like growth
factor-I receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors. Cancer Res
2005;65:3868–76.
Sebolt-Leopold JS, Dudley DT, Herrera R et al. Blockade of the MAP kinase pathway
suppresses growth of colon tumors in vivo. Nat Med 1999;5:810–6.
Ségaliny AI, Tellez-Gabriel M, Heymann M-F et al. Receptor tyrosine kinases:
Characterisation, mechanism of action and therapeutic interests for bone cancers. J bone
Oncol 2015;4:1–12.
Seidel J, Kunc K, Possinger K et al. Effect of the tyrosine kinase inhibitor lapatinib on CUBdomain containing protein (CDCP1)-mediated breast cancer cell survival and migration.
Biochem Biophys Res Commun 2011;414:226–32.
Seitz C, Hugle M, Cristofanon S et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 and
chloroquine synergize to trigger apoptosis via mitochondrial-lysosomal cross-talk. Int J
Cancer 2013;132:2682–93.
Sell C, Dumenil G, Deveaud C et al. Effect of a null mutation of the insulin-like growth
factor I receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell
Biol 1994;14:3604–12.
Sette G, Fecchi K, Salvati V et al. Mek inhibition results in marked antitumor activity against
metastatic melanoma patient-derived melanospheres and in melanosphere-generated
xenografts. J Exp Clin Cancer Res 2013;32:91.
Shaul YD, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions.
Biochim Biophys Acta 2007;1773:1213–26.
Shigematsu S, Yamauchi K, Nakajima K et al. IGF-1 regulates migration and angiogenesis
of human endothelial cells. Endocr J 1999;46 Suppl:S59–62.
Shim K-W, Xi G, Farnell B-M et al. Epigenetic modification after inhibition of IGF-1R
signaling in human central nervous system atypical teratoid rhabdoid tumor (AT/RT). Childs
Nerv Syst 2013;29:1245–51.
Sidell N, Altman A, Haussler MR et al. Effects of retinoic acid (RA) on the growth and
phenotypic expression of several human neuroblastoma cell lines. Exp Cell Res
217
1983;148:21–30.
Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 62:10–29.
Sikkema AH, Diks SH, den Dunnen WFA et al. Kinome profiling in pediatric brain tumors
as a new approach for target discovery. Cancer Res 2009;69:5987–95.
Singleton JR, Randolph AE, Feldman EL. Insulin-like growth factor I receptor prevents
apoptosis and enhances neuroblastoma tumorigenesis. Cancer Res 1996;56:4522–9.
Slamon DJ, Clark GM, Wong SG et al. Human breast cancer: correlation of relapse and
survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–82.
Smith ME, Das BC, Kalpana G V. In vitro activities of novel 4-HPR derivatives on a panel
of rhabdoid and other tumor cell lines. Cancer Cell Int 2011;11:34.
Sohara Y, Shimada H, DeClerck YA. Mechanisms of bone invasion and metastasis in human
neuroblastoma. Cancer Lett 2005;228:203–9.
Souki C, Abdel-Rhaman M, Qasem A et al. Atypical teratoid rhabdoid tumor in adulthood.
Clin Neuropathol 33:245–50.
de Souza JA, Davis DW, Zhang Y et al. A phase II study of lapatinib in recurrent/metastatic
squamous cell carcinoma of the head and neck. Clin Cancer Res 2012;18:2336–43.
Stamos J, Sliwkowski MX, Eigenbrot C. Structure of the epidermal growth factor receptor
kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J Biol Chem
2002;277:46265–72.
Stiller CA. Epidemiology and genetics of childhood cancer. Oncogene 2004;23:6429–44.
Strother D. Atypical teratoid rhabdoid tumors of childhood: diagnosis, treatment and
challenges. Expert Rev Anticancer Ther 2005;5:907–15.
Studebaker AW, Kreofsky CR, Pierson CR et al. Treatment of medulloblastoma with a
modified measles virus. Neuro Oncol 2010;12:1034–42.
Tagliabue E, Agresti R, Carcangiu ML et al. Role of HER2 in wound-induced breast
carcinoma proliferation. Lancet (London, England) 2003;362:527–33.
Tang J, Li J, Zeng G et al. Antisense oligonucleotide suppression of human IGF-1R inhibits
the growth and survival of in vitro cultured epithelial ovarian cancer cells. J Ovarian Res
2013;6:71.
Tanno B, Mancini C, Vitali R et al. Down-regulation of insulin-like growth factor I receptor
218
activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in
vivo. Clin cancer Res an Off J Am Assoc Cancer Res 2006;12:6772–80.
Targeted immunotherapy for high-risk neuroblastoma--the role of monoclonal antibodies.
Ann Pharmacother 2013;47:210–8.
Taskar KS, Rudraraju V, Mittapalli RK et al. Lapatinib distribution in HER2 overexpressing
experimental brain metastases of breast cancer. Pharm Res 2012;29:770–81.
Tekautz TM, Fuller CE, Blaney S et al. Atypical teratoid/rhabdoid tumors (ATRT):
improved survival in children 3 years of age and older with radiation therapy and high-dose
alkylator-based chemotherapy. J Clin Oncol 2005;23:1491–9.
Timeus F, Crescenzio N, Fandi A et al. In vitro antiproliferative and antimigratory activity of
dasatinib in neuroblastoma and Ewing sarcoma cell lines. Oncol Rep 2008;19:353–9.
Tolcher AW, Sarantopoulos J, Patnaik A et al. Phase I, pharmacokinetic, and
pharmacodynamic study of AMG 479, a fully human monoclonal antibody to insulin-like
growth factor receptor 1. J Clin Oncol 2009;27:5800–7.
Torre LA, Bray F, Siegel RL et al. Global cancer statistics, 2012. CA Cancer J Clin
2015;65:87–108.
Tran KA, Cheng MY, Mitra A et al. MEK inhibitors and their potential in the treatment of
advanced melanoma: the advantages of combination therapy. Drug Des Devel Ther
2016;10:43–52.
Tsatsanis C, Spandidos DA. Oncogenic kinase signaling in human neoplasms. Ann N Y Acad
Sci 2004;1028:168–75.
Tsikitis M, Zhang Z, Edelman W et al. Genetic ablation of Cyclin D1 abrogates genesis of
rhabdoid tumors resulting from Ini1 loss. Proc Natl Acad Sci U S A 2005;102:12129–34.
Tulla M, Berthold F, Graf N et al. Incidence, Trends, and Survival of Children With
Embryonal Tumors. Pediatrics 2015;136:e623–32.
Urtasun N, Vidal-Pla A, Pérez-Torras S et al. Human pancreatic cancer stem cells are
sensitive to dual inhibition of IGF-IR and ErbB receptors. BMC Cancer 2015;15:223.
Venneti S, Le P, Martinez D et al. p16INK4A and p14ARF tumor suppressor pathways are
deregulated in malignant rhabdoid tumors. J Neuropathol Exp Neurol 2011;70:596–609.
Villablanca JG, Khan AA, Avramis VI et al. Phase I trial of 13-cis-retinoic acid in children
with neuroblastoma following bone marrow transplantation. J Clin Oncol 1995;13:894–901.
219
Vitali R, Cesi V, Nicotra MR et al. c-Kit is preferentially expressed in MYCN-amplified
neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571. Int J
Cancer 2003;106:147–52.
Vitali R, Mancini C, Cesi V et al. Activity of tyrosine kinase inhibitor Dasatinib in
neuroblastoma cells in vitro and in orthotopic mouse model. Int J Cancer 2009;125:2547–55.
Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer.
Nat Rev Cancer 2002;2:489–501.
Wan PTC, Garnett MJ, Roe SM et al. Mechanism of activation of the RAF-ERK signaling
pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–67.
Wang D, Boerner SA, Winkler JD et al. Clinical experience of MEK inhibitors in cancer
therapy. Biochim Biophys Acta 2007;1773:1248–55.
Wang Y, Lipari P, Wang X et al. A fully human insulin-like growth factor-I receptor
antibody SCH 717454 (Robatumumab) has antitumor activity as a single agent and in
combination with cytotoxics in pediatric tumor xenografts. Mol Cancer Ther 2010;9:410–8.
Wang Y, Yuan JL, Zhang YT et al. Inhibition of both EGFR and IGF1R sensitized prostate
cancer cells to radiation by synergistic suppression of DNA homologous recombination
repair. PLoS One 2013;8:e68784.
Ward E, DeSantis C, Robbins A et al. Childhood and adolescent cancer statistics, 2014. CA
Cancer J Clin 64:83–103.
Warmuth M, Bergmann M, Priess A et al. The Src family kinase Hck interacts with Bcr-Abl
by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr. J Biol
Chem 1997;272:33260–70.
Warshamana-Greene GS, Litz J, Buchdunger E et al. The insulin-like growth factor-I
receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the
effects of chemotherapy. Clin Cancer Res 2005;11:1563–71.
Weinstein JL. Advances in the Diagnosis and Treatment of Neuroblastoma. Oncologist
2003;8:278–92.
Wieduwilt MJ, Moasser MM. The epidermal growth factor receptor family: biology driving
targeted therapeutics. Cell Mol Life Sci 2008;65:1566–84.
Wood ER, Truesdale AT, McDonald OB et al. A unique structure for epidermal growth
factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation,
inhibitor off-rate, and receptor activity in tumor cells. Cancer Res 2004;64:6652–9.
220
Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. Curr Opin Cell Biol
2005;17:559–64.
Yan S, Li Z, Thiele CJ. Inhibition of STAT3 with orally active JAK inhibitor, AZD1480,
decreases tumor growth in Neuroblastoma and Pediatric Sarcomas In vitro and In vivo.
Oncotarget 2013;4:433–45.
Yang M, Chen X, Wang N et al. Primary atypical teratoid/rhabdoid tumor of central nervous
system in children: a clinicopathological analysis and review of literature in China. Int J Clin
Exp Pathol 2014;7:2411–20.
Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and
therapeutic opportunities. Eur J Cancer 2001;37 Suppl 4:S3–8.
Yeatman TJ. A renaissance for SRC. Nat Rev Cancer 2004;4:470–80.
Yoon KJ, Phelps DA, Bush RA et al. ICAM-2 expression mediates a membrane-actin link,
confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in
neuroblastoma. PLoS One 2008;3:e3629.
Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate
diverse cellular functions. Growth Factors 2006;24:21–44.
Yu AL, Gilman AL, Ozkaynak MF et al. Anti-GD2 antibody with GM-CSF, interleukin-2,
and isotretinoin for neuroblastoma. N Engl J Med 2010;363:1324–34.
Zage PE, Louis CU, Cohn SL. New aspects of neuroblastoma treatment: ASPHO 2011
symposium review. Pediatr Blood Cancer 2012;58:1099–105.
Zhang D, Pal A, Bornmann WG et al. Activity of lapatinib is independent of EGFR
expression level in HER2-overexpressing breast cancer cells. Mol Cancer Ther 2008;7:1846–
50.
Zhang S, Yu D. Targeting Src family kinases in anti-cancer therapies: turning promise into
triumph. Trends Pharmacol Sci 2012;33:122–8.
Zhang Z-K, Davies KP, Allen J et al. Cell cycle arrest and repression of cyclin D1
transcription by INI1/hSNF5. Mol Cell Biol 2002;22:5975–88.
Zhao L, Wientjes MG, Au JL-S. Evaluation of combination chemotherapy: integration of
nonlinear regression, curve shift, isobologram, and combination index analyses. Clin Cancer
Res 2004;10:7994–8004.
Zhou T, Commodore L, Huang W-S et al. Structural mechanism of the Pan-BCR-ABL
inhibitor ponatinib (AP24534): lessons for overcoming kinase inhibitor resistance. Chem Biol
221
Drug Des 2011;77:1–11.
Zhu H, Acquaviva J, Ramachandran P et al. Oncogenic EGFR signaling cooperates with loss
of tumor suppressor gene functions in gliomagenesis. Proc Natl Acad Sci U S A
2009;106:2712–6.
Ziegler AN, Levison SW, Wood TL. Insulin and IGF receptor signalling in neural-stem-cell
homeostasis. Nat Rev Endocrinol 2015;11:161–70.
222