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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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3

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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5

0

0.5

1

1.5

2

2.5

RCC4 RCC4/VHL RCC10 RCC10/VHL

% P

rote

in D

egra

datio

n

CtlSTF-62247

** *

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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6

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