TARGETING HSP90 IN IM-RESISTANT GIST: 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,

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 ADD REFERENCE FOR HETEROGENEITY IN CML AND/OR GIST

Introduction (2) KIT signaling in GIST PTEN P P P P SOS SHC FAK p110 PDK1 DOK Adhesion/ Motility PI3K GRB2 PAK p85 Ras AKT Raf PI3K lipid kinase superfamily : 8 isoforms Activated by many (oncogenic) RTKs / cytoplasmatic TKs Effects on: proliferation, survival, protein synthesis, motility Shown to be crucial for oncogenic signal of BCR-ABL PI3K activation by KIT may depend on type of oncogenic mutation and cellular context Binding of p85 to KITactivates PI3K and recruits the complex to the membrane localizing it to its physiological substrate Katalytic (p110) and regulatory subunits (p85) Tissue specific expression of some isoforms ATP competitive Inhibitors of all katalytic isoforms PI3K (α, β, γ, δ): LY294002 and Wortmannin ? MTOR JAK MEK p70S6K 4EBP1 STAT MAPK S6 eIF4E Proliferation Survival Transcription NUCLEUS

Introduction (4) HSP90 background , activated! Chaperone family: protein folding translocation stabilization prevent aggregation elimination What made HSP90 a particularly attractive target in cancer was the observation – that it seems to exist in 2 different states : native – superchaperone complex (diagram of a model for altered states) which have different affinity to the HSP90 inhibitor 17-AAG. Superchaperone complex was mostly found in cells under stress: overexpressed mutated oncoproteins, microenvironmental stress due to hypoxia, Workman, TMM 2004

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

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 0 .1 1.0 0 1 10 µM 0 .1 .5 1.0 0 2 4 8 24h pKIT pKIT KIT KIT IMATINIB 17-AAG Ma, Blood 2002 Fumo, Blood 2004

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

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®)

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 IC50s 100-200nM point at GIST48

Results (4) Effect of 17-AAG on KIT signaling REMOVE TOTAL KIT STAIN Done and Actin added

Results (5) 17-AAG in IM resistant GIST IF POSSIBLE, FORMAT LIKE PREVIOUS SLIDE, WITH LANE HEADINGS OVER LOWER TWO PANELS AS WELL.

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 GIST882 Ex 13 CURVES IN THREE DIFFERENT COLORS??? Difficult, takes longer since curves were modified in Illustrator where you cant change colors but if you would like that for the meeting I could do it tomorrow or on Wednesday… IC50: 320nM

Results (8b) Effect of 17-AAG on proliferation GIST430 Ex 11 Ex 13 CURVES IN THREE DIFFERENT COLORS??? Difficult, takes longer since curves were modified in Illustrator where you cant change colors but if you would like that for the meeting I could do it tomorrow or on Wednesday… IC50: 220nM

Results (8c) Effect of 17-AAG on proliferation GIST48 Ex 11 Ex 17 CURVES IN THREE DIFFERENT COLORS??? Difficult, takes longer since curves were modified in Illustrator where you cant change colors but if you would like that for the meeting I could do it tomorrow or on Wednesday… IC50: 130nM

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

Results (9) Effect of 17-AAG on apoptosis CURVES IN THREE COLORS???

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 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

Results (3) 17-AAG in IM resistant GIST