targeting hsp90 in im-resistant gist: kit degradation as a broadly relevant salvage strategy

TARGETING HSP90 IN IM-RESISTANT GIST:

KIT DEGRADATION AS A BROADLY RELEVANT SALVAGE STRATEGY

Sebastian Bauer1,2, Lynn Yu1, George Demetri3, Jonathan Fletcher1

1Brigham & Women's Hospital, Harvard Medical School, Boston, MA, USA 2Westgerman Cancer Center, University of Essen, Germany

3Dana Farber Cancer Institute,

CTOS – Boca Raton, November 21st, 2005

• Biochemical inhibition of oncogenic KIT induces

clinical responses in most GIST patients

• BUT...many patients eventually progress

• Heterogeneous imatinib resistance mechanisms:

secondary KIT/PDGFRA kinase domain mutations genomic amplification activation of alternate oncogenes +/- loss of KIT

• Salvage therapies with alternative KIT/PDGFRA inhibitors = moderate benefit

Introduction (1) Imatinib in GIST

Ex 11

Ex 13

Ex 9

Ex 17

Ex 11

Ex 13

Ex 9

Ex 17

p85

p110

Ex 11

Ex 13

Ex 9

Ex 17

Survival Transcription

RasRaf

MEK

MAPK

GR

B2

Proliferation

AKT

MTOR

p70S6K

S6

4EBP1

eIF4E

PP

P P

PDK1

JAKSTAT

PTEN

?

Ex 11

Ex 13

Ex 9

Ex 17

Ex 11

Ex 13

Ex 9

Ex 17

Adhesion/ Motility

FAK

PAK

NUCLEUS

SOS SHCDOK

Introduction (2) KIT signaling in GIST

PI3K

Workman, TMM 2004

Introduction (4) HSP90 background

Chaperone family:

• protein folding

• translocation

• stabilization

• prevent aggregation

• elimination

, activated!

• 17-AAG inhibition of HSP90 downregulates

crucial tyrosine kinases in various cancers:

• BCR-ABL CML

• ZAP70 CLL

• FLT3 AML

• EGFR lung cancer

• KIT mast cell disease

Introduction (5) Rationale for HSP90 inhibition

• Mast cell line harboring IM-resistant D816 KIT mutation

sensitive to HSP90 inhibition by 17-AAG

Introduction (6) Rationale for HSP90 inhibition

µM

pKIT

KIT

HMC-1.1 HMC-1.2 HMC-1.2 HMC-1.20 .1 .5 1.0 0 2 4 8 24h0 .1 1.0 0 1 10 µM

IMATINIB 17-AAGMa, Blood 2002 Fumo, Blood 2004

pKIT

KIT

• KIT/PDGFRA activation is crucial to transformed

state at PROGRESSION

• Heterogeneous KIT/PDGFRA mutations at

progression

• KIT/PDGFRA small molecule inhibitors

• efficacy depends on mutation type & kinase structure

• HSP90 inhibitors

• efficacy depends on kinase activation

Introduction (7) HSP90 inhibition ideal in GIST?

• Evaluation of 17-AAG mediated HSP90 inhibition in IM-

sensitive and IM-resistant in GIST cell lines

• Biological consequences of HSP90 inhibition were

determined by - immunoblotting

- cell proliferation assays (Cell titer Glo®)

- apoptosis assays (Caspase Glo®)

Methods (1) Assays

Methods (2) Cell line KIT mutations

• GIST882: IM-sensitive / hom K642E

• GIST48: IM-sensitive / hom V560D

IM-resistant / het D820A

• GIST430: IM-sensitive / het JM deletion

IM-resistant / het V654A

• GIST62: IM-resistant / KIT negative

Results (1) Effect of 17-AAG on KIT in GIST

Results (2) 17-AAG in IM resistant GIST

Results (4) Effect of 17-AAG on KIT signaling

Results (5) 17-AAG in IM resistant GIST

Results (6) 17-AAG time course in GIST882

Results (7) Effect of 17-AAG on wt-KIT

Results (8a) Effect of 17-AAG on proliferation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 10 100 1000 10000

17-AAG17-AAG + 100nM IM17-AAG + 1000nM IM

GIST882Ex 13

IC50:320nM

Results (8b) Effect of 17-AAG on proliferation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 10 100 1000 10000

17-AAG17-AAG + 100nM IM17-AAG + 1000nM IM

GIST430Ex 11Ex 13

IC50:220nM

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1 10 100 1000 10000

17-AAG17-AAG + 100nM IM17-AAG + 1000nM IM

Results (8c) Effect of 17-AAG on proliferation

GIST48Ex 11Ex 17

IC50:130nM

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 10 100 1000 10000

17-AAG17-AAG + 100nM IM17-AAG + 1000nM IM

Results (8d) Effect of 17-AAG on proliferation

GIST62Ex 11KIT neg

IC50:>5000nM

Results (9) Effect of 17-AAG on apoptosis

Conclusions (1)

• HSP90-mediated stabilization crucial for KIT

expression and activation in GIST

• 17-AAG has strong antiproliferative and

proapoptotic effects in IM-resistant GISTs at

clinically achievable doses

• HSP90 targeting inhibits KIT oncoproteins with

IM resistance mutations

Conclusions (2)

• Degradation of KIT by 17-AAG depends on KIT

activation irrespective of resistance mutations

• No substantial additive or antagonistic effects

with IM

• Compelling rationale for clinical evaluation of

HSP90 inhibitors in (KIT-positive) GIST

Acknowledgements

Brigham and Women‘s HospitalJonathan FletcherLynn YuMeijun ZhuWenbin OuCJ ChenKatherine JanewayChris HubertLeigh Johnson-Abt

Dana-Farber Cancer InstituteGeorge DemetriSuzanne GeorgeJeffrey MorganPalma Dileo

Results (3) 17-AAG in IM resistant GIST