1. Therapeutic Targeting of EWS-FLI1: Small Molecule Protein-Protein Interaction Inhibitors Prevent Xenograft Growth Jeffrey Toretsky Department of Oncology and Pediatrics Lombardi Comprehensive Cancer Center Georgetown University Washington, DC

2. E wing’s S arcoma F amily of T umors 1992: Breakpoint of t(11;22) cloned; EWS constitutively expressed –Derived chromosome 22 expressed as EWS-FLI1 11der11 22der22 1983: t(11;22) by standard g-banded karyotype Elimination of EWS-FLI1 from ESFT cells is lethal

3. Transcription Factor Biology Fusion Gene or Message Fusion Protein Cell Membrane Translation Nucleus 22 11 Transformation

4. E9R = PPPLDAVIEA Progression from Oncogene to Protein Partner to Poison EWS-FLI1 RHA Peptide blocks RHA from binding to EWS-FLI EWS-FLI1 RNA Helicase A (RHA) Uren, Biochemistry 2004 Toretsky, Cancer Res 2006

5. 5 Mutant RHA fails to enhance EWS-FLI1 dependent transformation Colony numbers 600 400 200 0 EWS-FLI1 RHA RHA-D827A 150 75 FLAG-RHA EWS-FLI1 Anchorage independent growth

6. RHA peptide inhibits ESFT growth EWS/FLI1 E9R-P EGFP E9R EGFP E9RNES pGE9R pGCE9R

7. P823 P824 P825 L826 D827 A828 V829 I830 E831 A832 Peptide Model: Region of Overlap with Lead Compound GFP-E9R 10 aa peptide NSC635437 in Druggable Space

9. Screening Library for Potential Binding

10. Small molecule synthesis and optimization NCI Compound NSC635437 synthesized at GU Identified Lead with Homology to Peptide E9R Analogs were synthesized YK-4-279 selected based upon binding, EWS- FLI1:RHA disruption, and cytotoxicity profile

11. YK-4-279 blocks RHA binding to EWS-FLI1

12. YK-4-279 displaces E9R from EWS-FLI1

13. YK-4-279 reduces EWS-FLI1 transcription activity

14. YK-4-279 toxicity against EF compared to non-EF cell lines

15. YK-4-279 Inhibits ESFT Xenografts

16. Summary Validated EWS-FLI1:RHA as molecular target SPR screen identified small molecule lead YK-4-279 demonstrates relative specificity with –1 microM IC 50 against ESFT – Xenograft studies Further chemical optimization underway To be followed by ADME/tox Hopeful to advance to clinical trials

17. Collaborators Milton Brown and Yali Kong, GU, LCCC David Loeb, JHH Melinda Merchant, NIH Angela Koehler, Broad Institute Anton Wellstein, GU, LCCC Steve Lessnick and Leah Owen, Utah Olga Tscherkasskaya, GU Jeff Parvin, Harvard Michael Grusby, Harvard Sean Lee, NIH Marc Landanyi, MSKCC Tim Cripe, Cincinnati Children’s

18. Support Children’s Cancer Foundation, Baltimore, MD Dani’s Foundation Go4theGoal Liddy Shriver Sarcoma Initiative Amschwand Sarcoma Foundation CureSearch, NCCF NIH, NCI Burroughs-Wellcome Translational Scientist Award