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. PI3k IFG-1 Growth Factor Receptors Nutrients Hypoxia Stress PTEN IRS Ras bRaf ERK1/2 (p-MAPK) Proliferation Akt p70 s6K Survival and Cell Cycle Progression MEK1/2 TSC 1/2 Rheb AKT 4EBP mTOR TORC1 mTOR TORC2 elF4E uo-126 Rapamycin

5. IC50 Comparing Sensitivity Between Cell Lines

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

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

8. Western blot 4EBP Cont 50 100 200 50 100 200 1hr24 hr p-4EBP 4EBP Cont 50 100 200 50 100 200 1 hr24 hrs p-4EBP1 4EBP1 Cont 50 100 200 50 100 200 1 hr 24 hrs p-4EBP1 4EBP1 COL OS-187 SAOS-2

9. Western blot 70S6k 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. Cnt 1hr 8hr 24hr 1hr 8hr 24hr 1hr 8hr 24hr 50nM100nM200nM p-70 S6k 70 S6k Cont 50 100 200 50 100 200 1 hr 24 hrs p-70 S6k 70 S6k 200 100 50 200 100 50 200 100 50 Cont 24hrs8hrs1hr p-70 S6k 70 S6k COL OS-187 SAOS-2

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

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

21. Thank You