1. 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,

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
ADD REFERENCE FOR HETEROGENEITY IN CML AND/OR GIST

3. 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): LY and Wortmannin ?
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
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

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 h
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
IC50s nM
point at GIST48

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

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

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
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

17. 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

18. 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

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
CURVES IN THREE COLORS???

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

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