Madrid-S Turcotte-F - VHL Alliance · 2016. 4. 26. · Turcotte and Giaccia, Am Soc Clin Oncol Ed...
Transcript of Madrid-S Turcotte-F - VHL Alliance · 2016. 4. 26. · Turcotte and Giaccia, Am Soc Clin Oncol Ed...
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Exploiting synthetic lethalityin kidney cancer to target
the loss of VHL for targeted therapy
Sandra Turcotte, PhD
Department of chemistry and biochemisty, Université de Moncton,Atlantic Cancer Research Institute
11th International VHL Symposium24th October 2014
# of people diagnosed
# of people dead
Worldwide 200,000 100,000Canada 6,100 1,800USA 63,920 13,860
Kidney Cancer
Linehard et al., 2003JCO, 2009
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Genetic interaction between two genes.
When either gene ismutated, the cell remainsalive.
However, when bothgenes are mutated the cell will die.
Chan and Giaccia, 2011
Targeting VHL-deficient cells through synthetic lethality
To develop a targeted therapy that kills cancer cellsand spare normal tissu
Turcotte and Giaccia, Am Soc Clin Oncol Ed Book 2008
Incubate withcompound library
Loss of VHL
Wild-type VHL
Seed cells onto384 well plate
Screening
Label cellsMeasure
fluorescence
Validation
Loss of VHL
Wild-type VHL
Clonogenic AssayXTT
Loss of VHL
Wild-typeVHL
Cel
lSur
viva
l(c
olon
yas
say)
0.001
0.01
0.1
1
0 0.3125 uM 0.625 uM 1.25 uM 2.5 uM 5uM
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02468
101214161820
0 3 6 9 12 15
VehicleSTF-62247(8 mg/kg)
02468
101214
1 3 5 7 9
VehicleSTF-62247 (2.7 mg/kg)STF-62247 (8 mg/kg)
00.5
11.5
22.5
33.5
1 3 5 7 9 11 13 15
VehicleSTF-62247 (2.7 mg/kg)STF-62247 (8 mg/kg)
STF-Vehicle 62247
SN12C+shVHL
SN12C
Tum
or V
olum
e (fo
ld)
Days
786-0
DaysTurcotte et al.
Cancer Cell 14:90-102, 2008
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Hay, Turcotte et al.,J. Med. Chem. 53, 787-97, 2010
HIF-independent (shHIF-1a, shHIF-2a)
Modulates autophagy and increases autophagosomeformation
Autophagy is important for selective cytotoxicity
Autophagic flux is not block
Target lysosome???
Auto «self» phagy «eat»
Lysosomal degradation process to remove misfolded proteins, aggregate or organelle
Important for balancing sources of energy
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Autophagosome
Fusion
Autolysosome
LC3-lipidationLC3I LC3II
ClassIII PI3KBeclin-1(Atg6)VPS34
3-MALY294002
Lysosome
Atg9Atg12-Atg5 complexMAP-LC3
Atg12 (ub-like)Atg7 (E1-like)Atg10 (E2-like)Atg5
Atg12-Atg5
LC3 (Atg8) (ub-like)Atg7 (E1-like)Atg3 (E2-like)Atg4
Reyjal, Cormier and TurcotteAdv. Exp. Med Biol 772:167-88 2014
Bafilomycin A,ChloroquineE64d
Autolysosome(Degradation)
Maturation
LysosomeLamp1 Lamp2
Autophagosome
VHL
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0
0.5
1
1.5
2
2.5
RCC4 RCC4/VHL RCC10 RCC10/VHL
% P
rote
in D
egra
datio
n
CtlSTF-62247
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0102030405060708090
100
0 2 4 6
Lyso
som
al m
embr
ane
perm
eabi
lity
(% L
MP)
Days
RCC4 1.25uM STF-62247RCC4 2.5 uM STF-62247VHL 1.25uM STF-62247
VHL 2.5uM STF-62247
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02468
101214161820
Ctl
STF-
6224
7
Suni
tinib
Cip
roflo
xaci
n
Cip
ro+S
uniti
nib
Baf
A1 CQ
Rap
amyc
ine
EBSS
% L
MP
RCC4RCC4/VHL
0
20
40
60
80
100
120
0 0,1 0,3 0,5 1 2 4 8 16
Concanamycine A (nM)
Surv
ie (%
)
RCC4
RCC4-VHL
To study the transcriptomic of RCC in response to STF-62247 To understand the STF-62247 signalling To identify targets of the small molecule To evaluate the contribution of glutamate/glutamine in RCC
Genomic Transcriptomic Proteomic Metabolomic
VHL mutationNext-generation
sequencing
microRNANext-generation
SequencingmRNA microarray
AutophagyTarget of STF-62247
Fluorescence MicroscopyMass spectrometry
Glutamate/glutamineNMR
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SILAC
N=3
N=2N=194%
81% 91%78%
2819 3271
2862
INV=3
INV=2INV=192%
86% 88%79%
3052 3031
3199
INVERT
We demonstrated the possibility of targeting VHL-deficient cellsspecifically using small molecule
Mechanistically, STF-62247 modulates autophagy
Lysosome behavior is an important step in the decision between cell death or survival in response to STF-62247
This work identified a function for VHL at a late stage of autophagy
The use of synthetic lethality in cancer therapeutics is very promising. This research offers an attractive opportunity insofar tas our fundamental
research lead to the development of VHL targeted treatment for RCC
Kevin Cormier, MScJulie Reyjal, MScAlexandre CoholanNadia BouhamdaniSonia DastousSarah RobichaudMaxime CahuzacJessica SchraderJérémie BannisterArij Zhani
Stanford UniversityDr Amato J GiacciaDr Denise Chan
University of AucklandDr Michael HayDr William Denny
Financial Support
Institut du Cancer de MontrealDr Richard BertrandDr Anne-Marie Mes-Masson