1. High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma Kartik Krishnan, Gaurav Khanna, Stephen Hewitt, Chand Khanna, and Lee J. Helman Pediatric Oncology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland USA

2. Ezrin Expression Correlates with Survival in Osteosarcoma Khanna C, et al., Nature Medicine, 10 (2); 2004

3. Ezrin Expression in Ewings Sarcoma

4. IB: Mouse MAb  Ezrin (Sigma) WV VW LD KAD TC71 RDES 5838 Primary Cultures Cell Lines 202 96 66 45 VW 18 y.o. female presented in 1/01 with widely disseminated disease involving multiple bony sites including femurs, vertebrae, and skull. Diagnosed with Ewing’s sarcoma with Type IV translocation. Progressed on chemo and radiation therapy. Died 8/01. VWCell line Primary xenograft developed tumors rapidly. Stable tissue culture growth established at approximately one year, confirmed presence of type IV EWS-Fli1 translocation. In Mice, orthotopic implantation resulted in tumors with a latency between 21 and 56 days. Experimental metastases developed with intravenous injection between 45 and 80 days.

5. EZRIN  Membrane-cytoskeleton linker protein  Initially identified as a determinant of cell shape  Plays a critical role in cell motility and attachment  Activatation initiated by threonine phosphorylation at position 567  Subsequent tyrosine phosphorylation allows for interaction with signal transduction pathways Band 4.1 homology  -helix N C Y143 T567 Y353 F-actin Binding

6. EZRIN T567A Unselected Batch WB:  -Ezrin WT Ezrin EzrinT567A-GFP Selected Clones VWVW-A VW-GFP WT T567A Dominant Negative Clones CMV GFP

7. Bjornsti and Houghton Nat Rev Cancer Cyclin D1 MYC HIF 1  Ezrin

8. AKT IB:  AKT IB:  P-Ser 473 AKT Serum: - + - + VW-GFP Ezrin T567A

9. 4EBP1 Serum Stimulation (minutes) 0 30 120 VW-GFP Ezrin T567A IB:  -P-Thr 37 -Thr 46 -4EBP1 IB:  -4EBP1

10. p42/44 MAPK Serum: - + - + VW-GFP Ezrin T567A IB:  P-Thr 202 Tyr 204 p42/44 IB:  p42/44

11. Mice 1 X 10 6 cells injected IV Assessed for development of experimental metastases At Day 70: VW-GFP3 / 6 VW-GFP-EzrinT567A0 / 6

12. Conclusions 1.Ezrin is highly and ubiquitously expressed in Ewing’s sarcoma. 2.Expression of a Ezrin containing a non- phosphorylatable mutation at threonine 567 functions as dominant negative. 3.These dominant negative effects are observed on AKT, as well as 4EBP1. 4.Ewing’s sarcoma cells expressing a dominant negative Ezrin form experimental metastases at a lower rate than wild type.

13. Future Directions 1.Expression of Ezrin anti-sense to completely remove Ezrin expression and evaluate growth, survival, and signaling. 2.Analysis of effect of dominant negative and anti-sense Ezrin in other Ewing’s sarcoma cell lines, each with a different base line level of Ezrin expression. 3.Orthotopic implantation of dominant negative expressing cell lines, followed by amputation in mice to evaluate metastatic potential of these cells. 4.Further molecular studies: a.What other signal transduction pathways involve Ezrin? b.What are the Ezrin kinases? Are they suitable targets for small molecule inhibition? c. Proteomics

14. Acknowledgments Lee Helman Choh Yeung Melissa Burgos Shaan Gandhi Pediatric Oncology Branch National Cancer Institute, NIH Tumor and Metastasis Biology Section Pediatric Oncology Branch National Cancer Institute, NIH Chand Khanna Gaurav Khanna Arnulfo Mendoza Tissue Array Research Project National Cancer Institute, NIH Stephen Hewitt

16. Murine Microarray Analysis: Khanna C, et al., Cancer Research, 61 ;2001 Highly Metastatic K7M2 Primary Tumor Less Metastatic K12 Primary Tumor VS EZRIN K7M2 Wild type K12 Wild type

17. Ezrin K7M2 wt K12 wt K7M2/AS Ezrin 13 K7M2/AS Ezrin 1.46 K7M2/AS Ezrin 1.52 K7M2/AS Ezrin 2.12K7M2/AS Ezrin 2.13K7M2/AS Ezrin 2.15 Tubulin Relative Expression 21. 3 1.0 12.4 1.1 12.215.92.8

18. mTOR Signaling Pathway (PIK-related kinase, related to ATM, TRRAP etc.) Bjornsti and Houghton Nat Rev Cancer

19. Osteosarcoma Ezrin

20. P P Threonine 567 Phosphorylation F-Actin CD44, PDGFRA Tyrosine Phosphorylation RhoGDI p85 PI3K AKT Growth, Survival P P