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Systematic interactome mapping of acute lymphoblastic leukemia
cancer gene products reveals EXT-1 tumor suppressor as a Notch1
and FBW7 common interactor
Despoina Kerselidou1, Sarah Daakour1 ,Leon Juvenal Hajingabo2, Aurelie Devresse1, Nicolas Simonis2, Franck Dequiedt1 and Jean-Claude Twizere1
1 Laboratory of Protein Signaling and Interactions, GIGA-Research, University of Liège, B-4000 Liège, Belgium
2 Laboratoire de Bioinformatique des Génomes et des Réseaux (BiGRe), Université Libre de Bruxelles (ULB), B-1050 Bruxelles, Belgium
I. Introduction
Perturbed genotypes in cancer can now be identified by whole genome sequencing of large number of diverse tumor samples, and observed gene
mutations can be used for prognosis and classification of cancer subtypes. Although mutations in a few causative genes are directly linked to key
signaling pathways perturbation, a global understanding of how known cancer genes drive oncogenesis in human is difficult to assess. Here we have
mapped inter-connections between 116 cancer census gene products associated with Acute Lymphoblastic Leukemia.
II. Materials
Combining protein-protein interactions data and specific gene mutations information, we highlighted exclusive and co-existested genetic alterations in
key signaling pathways including the Notch, AKT and JAK pathways. We observed that 54% of ALL census genes products are inter-connected and
identified 189 (162 LCI, 27 Y2H) human proteins interacting with at least 2 ALL census genes products, which were not previously linked to ALL via
mutations. Finally, we showed that EXT1, a tumor suppressor implicated in chondrosarcomagenesis, is a common interactor of Notch1 and FBW7. We
speculate that EXT1 is a novel ALL cancer gene controlling Notch1 activity in an FBW7-dependend manner.
IV. Results
1.Interactome map of proteins involved in T-ALL
III. Objectives
3.EXT1 regulates NOTCH1 degradation through
2.EXT1
depletion
promotes
NOTC1 transcriptional activity
FBWX7
Figure1. Cancer census gene encoded proteins are
represented in grey and their partners in green.
4. Transcriptome
comparison of cells depleted
for EXT1 and FBWX7
A
B
Figure 2. (a) Luciferase reporter assay using TP1-luciferase
A
construct in HeLaNotch∆E-eGFP cell line transfected with
EXT1 siRNA or control siRNA. The relative luciferase values
are normalized using a Renilla luciferase construct. (b)
mRNA expression level
C of cMYC and EXT1 were analysed by
qPCR after EXT1 knock down in HeLaNotch∆E-eGFP cell
line. (c) A zebrafish transgenic line Tg(Tp1bglob:eGFP)um13,
reporter for Notch1 transcriptional activity was treated with
control or EXT1 a and b ortholog-targeted morpholinos. Left
panel represents TP1 bglob:hngb1-eGFP construct.D The
graph represents the percentage eGFP cells sorted by FACS.
Data represent the means ±SEs of three independent
experiments.
(a)
(b)
(c)
(d)
Figure 3. (a) EXT1 over-expression decreases the amount of
NICD FLAG tagged protein. (b) The degradation of NICD
following EXT1 over-expression is proteasomal-dependent.
(c) The degradation of NICD following EXT1 overexpression is FBXW7-dependent. (d) Relative mRNA
expression levels of FBXW7 analyzed by qPCR. Data
represent the means ±SEs of three independent experiments,
each performed in triplicate.
5. EXT1 depletion sensitizes TC
lympocytes to DAPT treatment
E
F
Figure 4. (a) Circles represent genes that are differentially
expressed in both siEXT1 and siFBXW7 conditions compared
to control siRNA condition in HeLaNotch∆E-eGFP cells
subjected to RNAseq analyses. (b) Relative mRNA expression
levels of EXT1 and FBXW7 analyzed by qPCR. (c) Molecular
functions associated with genes co-regulated by EXT1 and
FBXW7.
A
IV. Discussion
VI. Conclusion
The Notch Meeting IX, 4-8 October 2015, Athens-Greece
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