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

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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.

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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.

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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.

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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!