1. EWS:ERG PREDICTS RESPONSE AND PTEN LOSS PREDICTS RESISTANCE TO SELECTIVE IGF1R INHIBITION IN EWING’S SARCOMA William D. Tap, Judy Dering, Charles Ginther, Daniel Wai, Pedro Beltran, Sarah Dry, Nicholas Bernthal, Kareem Clarke, Christine Tung, Amrita Desai, Jonathan Nakashima, Hong Wu, Frank Calzone, Timothy Triche, Dennis Slamon, Fritz C. Eilber. UCLA, Dept of Medicine, Pathology, Surgery CHLA, Dept Pathology Amgen Pharmaceuticals

2. INSRIGFR HR INS IGF2 IGF1 IGF2 INS IGF1 IGF2 INS Adapted: Frasca et al. Archiv Physiol Biochem, 2008; 114(1) INSRIGFR HR IGF1 IGF2 Adapted: Beltran et al. Mol Cancer Ther, 2009; 8(5) Insulin Family of Receptors and AMG479

3. Cell Line Characterization 21% EWS:ERG 14% PTEN mutated, inactive ERG – PTEN mutually exclusive

4. AMG479 against Ewing’s sarcoma family of tumor panel (N=28) % inhibition as compared to the control Sensitive AMG479 - 100µg/ml IGF1 – 2nm Resistant

5. TC174 SKNMC RDES S34 TTC487 CHLA-32 Cntrl IGF1 AMG Combo pAKT-ser473 Cntrl IGF1 AMG Combo Total AKT Increase pAKT after IGF stimulation No change pAKT resistant cell lines

6. No Change in pERK in Non-responders A673 6647 SKES SKNEP TC253 TC32 SKNMC CHP100S CHLA10 TTC475 TTC466 Cntrl IGF1 AMG Combo pERK-Thr202/Tyr200 Cntrl IGF1 AMG Combo Total ERK

7. Meso Scale Discovery System SECTOR ® Imager 6000 High throughput immunoassay that uses electrochemiluminescence to detect phosphoproteins SensitiveResistant

8. P-AKT SensitiveResistant Sensitive Cell Lines: Increased p-AKT after exposure to IGF P-AKT/IGF SensitiveResistant

9. p-GSKbp-AKTp-IRS1p-IGFR p-GSKbp-AKTp-IRS1p-IGFR

10. p-GSKbp-AKT p-IRS1 p-IGFR Phosphoprofile PTEN Null ES Cell Lines

11. AMG479/IGF1RAD001

12. mTOR Signaling Pathway Combination AMG/RAD

13. Combination RAD001/AMG479

14. Cell lines resistant to AMG479/RAD001 Capture PTEN Null

15. Conclusions: Ewing’s Sarcoma is a diverse malignancy. 15/28 ES Cell Lines (54%) do not respond to IGF stimulation and are resistant to IGF1R blockade. IGF/PI3K/mTOR pathway active majority ES cell lines with different levels input and control. The majority of ES cell lines 25/28 (90%) respond to inhibiting IGF/PI3K/mTOR signaling pathway some form (IGF1R, mTOR, PI3K inhibition – single agent and combination).

16. Conclusions: Screening for PTEN should be performed in ES and possibly direct patients to PI3K/mTOR inhibition. Continued molecular work on ES samples (cell lines, murine models, patient samples) should be performed help stratify patients prior enrollment onto clinical trials with agents that disrupt the IGF/PI3K/mTOR signaling pathway.

17. Thank You

18. PTEN Characterization IHC – sample 1 pS6 - negative pERK - negative 20x pPTEN – pos?

19. PTEN Characterization IHC – sample 2 20x pPTEN – negative? pERK - positive pS6 - positive

20. CGH – PTEN Null Patient (Sample 2) EWS:FLI1 Y- PTEN

21. Insulin Growth Factor Pathway – AMG479

22. 88 Human Sarcoma Cell Lines 34 Soft tissue sarcoma 53 Bone sarcoma 14 Osteosarcoma 34 Ewing’s 20 Rhabdomyosarcoma Timothy Triche, MD PhD 68 pediatric sarcomas