Inhibition of the mTOR and MAPK pathways in the treatment of osteosarcoma

1. Inhibition of the mTOR and MAPK pathways in the treatment of osteosarcoma Kathleen M. Diehl, M.D. FACS Assistant Professor University of Michigan

2. Background • Osteosarcoma cell lines • SAOS-2, COL, OS-187 • Rapamycin • Sirolimus • Natural macrolide antibiotic (anti-fungal) • Binds to FKBP12 inhibiting mTORC1 • Analogues • CCI-779 (Wyeth) • RAD001 (Novartis) • AP23573 (Ariad)

3. Clinical Trials • CCI-779 • I/II lung, breast, neuroendocrine, uterine, cervical, soft tissue sarcomas • III (RCCA) • PR 7-9% • SD 26-36% • RAD001 • I/II, RCCA, solid tumors • PR 5-33% • SD 7.3-23.5% • Very high PR or SD rate soft tissue sarcoma • AP23573 • I/II hematologic, solid tumors, sarcoma • PR 3-11% • SD 25% • 100% of sarcoma patients had PR or SD • 56% clinical improvement

4. Growth Factor Receptors Nutrients Hypoxia Stress IFG-1 Ras Ras IRS PI3k PTEN bRaf Akt Rapamycin MEK1/2 uo-126 TSC 1/2 Rheb ERK1/2 (p-MAPK) mTOR TORC1 mTOR TORC2 4EBP AKT p70s6K Proliferation Survival and Cell Cycle Progression elF4E

5. IC50 Comparing Sensitivity Between Cell Lines

6. Flow Cytometry of Cells Treated for 48 hrs with Rapamycin

7. Control Treated A A C B B C Decrease in cell cycle proteins cyclin D1 and cdk4 in OS-187 cells A = Cyclin D1 B = cdk4 C = Cyclin D3

8. 24 hrs 1 hr COL OS-187 SAOS-2 p-4EBP 4EBP 1 hr 24 hrs Cont 50 100 200 50 100 200 Cont 50 100 200 50 100 200 1hr 24 hr p-4EBP1 p-4EBP1 Cont 50 100 200 50 100 200 4EBP1 4EBP1 Western blot 4EBP

9. COL OS-187 SAOS-2 50nM 100nM 200nM 24hrs 8hrs 1hr Cnt 1hr 8hr 24hr 1hr 8hr 24hr 1hr 8hr 24hr 200 100 50 200 100 50 200 100 50 Cont p-70 S6k p-70 S6k 24hrs 1 hr 70 S6k Cont 50 100 200 50 100 200 Note: lack of activity in COL and OS187 cells confirmed with 2-D gels for T389 and T421-424 at 0-24-48-72 hrs. 70 S6k p-70 S6k 70 S6k Western blot 70S6k

10. Summary Treatment Osteosarcoma Cells with Rapamycin • Concentration dependent decrease in cell growth and proliferation • Associated with G1 arrest but not apoptosis • Cell line dependent decrease in the phosphorylation of proteins of the mTOR pathway • Decrease in cell cycle proteins

11. Proliferation Assays showing effectiveness of uo-126 in decreasing proliferation in these cells COL OS-187 SAOS-2

12. uo-126 • Synthesized, in-vitro use • Inhibits active and inactive MEK1/2 of the Mitogen Activated Protein Kinase Pathway • Cellular proliferation

13. COL

14. OS-187 cells

15. SAOS-2 cells

16. OS-187 Rapa uo-126 OS-187 Rapa OS-187 Control COL Rapa uo-126 COL Rapa COL Control 2-phase Flow Cytometry showing apopotosis with the addition of uo-126 to Rapa in COL and OS-187 cells

17. Summary • The addition of the MAPK pathway inhibitor uo-126 to Rapamycin resulted in • Synergistic decrease in proliferation in COL and OS-187 cells • Additive decrease in proliferation in SAOS-2 cells • Apoptosis

18. Conclusions • The combination of inhibition of the mTOR and MAPK pathways shows promise for the treatment of osteosarcoma

19. Next Steps • Confirmation with in-vivo model • Comparison with inhibitors of other cell survival and proliferation pathways • Comparison with other mTOR inhibitors

20. Laurence Baker Qi Wu Zhiyu Wang Dafydd Thomas Rashmi Chugh Kenine Comstock Carolyn Hoban Scott Schuetze David Lucas Acknowledgements

21. Thank You