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TARGETING HSP90 IN IM-RESISTANT GIST: KIT DEGRADATION AS A BROADLY RELEVANT SALVAGE STRATEGY

CTOS – Boca Raton, November 21 st , 2005. TARGETING HSP90 IN IM-RESISTANT GIST: KIT DEGRADATION AS A BROADLY RELEVANT SALVAGE STRATEGY. Sebastian Bauer 1,2 , Lynn Yu 1 , George Demetri 3 , Jonathan Fletcher 1 1 Brigham & Women's Hospital, Harvard Medical School, Boston, MA, USA

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TARGETING HSP90 IN IM-RESISTANT GIST: KIT DEGRADATION AS A BROADLY RELEVANT SALVAGE STRATEGY

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  1. CTOS – Boca Raton, November 21st, 2005 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,

  2. Introduction (1) Imatinib in GIST • 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

  3. Introduction (2) KIT signaling in GIST PTEN P SOS P P P SHC FAK p110 PDK1 DOK Adhesion/ Motility PI3K PAK GRB2 p85 Ras AKT Raf ? MTOR JAK MEK p70S6K 4EBP1 STAT MAPK S6 eIF4E Proliferation Survival Transcription NUCLEUS

  4. Introduction (4) HSP90 background , activated! • Chaperone family: • protein folding • translocation • stabilization • prevent aggregation • elimination Workman, TMM 2004

  5. Introduction (5) Rationale for HSP90 inhibition • 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

  6. Introduction (6) Rationale for HSP90 inhibition • Mast cell line harboring IM-resistant D816 KIT mutation sensitive to HSP90 inhibition by 17-AAG HMC-1.1 HMC-1.2 HMC-1.2 HMC-1.2 µM µM 0 .1 1.0 0 1 10 0 .1 .5 1.0 0 2 4 8 24h pKIT pKIT KIT KIT IMATINIB 17-AAG Ma, Blood 2002 Fumo, Blood 2004

  7. Introduction (7) HSP90 inhibition ideal in GIST? • 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

  8. Methods (1) Assays • 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®)

  9. 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

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

  11. Results (2) 17-AAG in IM resistant GIST

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

  13. Results (5) 17-AAG in IM resistant GIST

  14. Results (6) 17-AAG time course in GIST882

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

  16. Results (8a) Effect of 17-AAG on proliferation GIST882 Ex 13 IC50: 320nM

  17. Results (8b) Effect of 17-AAG on proliferation GIST430 Ex 11 Ex 13 IC50: 220nM

  18. Results (8c) Effect of 17-AAG on proliferation GIST48 Ex 11 Ex 17 IC50: 130nM

  19. Results (8d) Effect of 17-AAG on proliferation GIST62 Ex 11 KIT neg IC50: >5000nM

  20. Results (9) Effect of 17-AAG on apoptosis

  21. 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

  22. 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

  23. Acknowledgements • Brigham and Women‘s Hospital • Jonathan Fletcher • Lynn Yu • Meijun Zhu • Wenbin Ou • CJ Chen • Katherine Janeway • Chris Hubert • Leigh Johnson-Abt • Dana-Farber Cancer Institute • George Demetri • Suzanne George • Jeffrey Morgan • Palma Dileo

  24. Results (3) 17-AAG in IM resistant GIST

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