1. THE EWS-FLI1 ONCOGENE DISRUPTS NORMAL DEVELOPMENTAL REGULATION OF POLYCOMB- MODULATED TRANSCRIPTIONAL PROGRAMS Ashley Harris, Natashay Bailey, Laurie Svoboda Elizabeth R. Lawlor Department of Pediatrics Translational Oncology Program University of Michigan, Ann Arbor, MI

2. Disclosure Information CTOS Annual Meeting 2013 Elizabeth R. Lawlor I have no financial relationships to disclose. - and - I will not discuss off label use and/or investigational use in my presentation.

3. Polycomb proteins BMI-1 & EZH2 are induced by EWS- FLI1 and function as oncogenes in Ewing sarcoma Douglas, Cancer Research 2008; Hsu, Oncogene, 2011; von Levetzow, PLoS ONE, 2011 H & EBMI-1 ★ EZH2 is an EWS-FLI1 target gene and promotes Ewing sarcoma tumorigenicity Riggi, Cancer Res 2008, Richter, PNAS 2009

4. Polycomb proteins epigenetically regulate gene expression during normal development SUZ12 PLC EED PHC BMI1 CBX RING1 A/B Active ChromatinRepressed Chromatin PRC2 PRC1 EZH2 H3 H2A H3K27me3 H2a119Ub

5. Developmental transcription factors. – Responsible for the anterior-posterior patterning of the central nervous system and proximal-distal axis of the limbs HOX genes are dynamically expressed in embryogenesis in response to polycomb protein regulation – OFF  ON  OFF Deregulation of HOX genes promotes leukemia HOX genes are dynamically expressed during embryonic development

6. Hypothesis Altered expression of polycomb proteins contributes to Ewing sarcoma tumor initiation and maintenance by disrupting expression of developmentally critical transcriptional programs

7. Unique to EWS-FLI1+ Unique to Control 511 231 270 Overlapping Transcripts 219 coordinately regulated B CV EF NCSC 5 days in self- renewal media 6 wks in differentiation media vs. A EWS-FLI1 alters differentiation program of NCSC

8. Unique to EWS-FLI1+ Unique to Control 511 231 270 Overlapping Transcripts 219 coordinately regulated B C EWS-FLI1 shifts transcriptional program to neural from mesenchymal CV EF NCSC 5 days in self- renewal media 6 wks in differentiation media vs. A D

9. EWS-FLI1 alters expression of HOX Genes Fold change in HOX genes after 6 wks in differentiation media HOXA HOXB HOXC HOXD

10. HOXD9 HOXD10HOXD11HOXD13 Ewing sarcomas have altered HOX profiles Normal Adult Tissue (N=33) BM-MSC (N=3) NC-MSC (N=3) NCSC (N=3) Ewing Sarcoma (N=32) PCA Mapping: 37 HOX Genes A Abnormal upregulation of posterior HOXD genes FDR<0.05 Stem cells Adult tissues ES tumors B 24/37 HOX genes are significantly differentially expressed in Ewing sarcoma FDR<0.05

11. Polycomb-mediated H3K27me3 silencing is lost at HOXD13 promoter in Ewing sarcoma cells Ewing sarcoma Fibroblast Neural crest SCMesenchymal SC Fibroblast Neural crest SC Mesenchymal SC Ewing sarcoma H3 H2A H3K27me3 H2a119Ub H3K27me3 ChIP/HOXD13 promoter PCRRNA Polymerase II ChIP/HOXD13 promoter PCR H3 Ewing sarcoma: Active Chromatin state Stem cells & terminally differentiated cells: Repressed Chromatin state H3K4me3

12. B.A. HOXD9 and HOXD13 are down-regulated following EWS-FLI1 knockdown in TC-71 xenografts C.

13. Summary EWS-FLI1 disrupts normal regulation of HOX gene expression during stem cell differentiation Polycomb repressive mark H3K27me3 is absent from the HOXD13 promoter and posterior HOXD genes are abnormally expressed in Ewing sarcoma EWS-FLI1 induces and is necessary to maintain high levels of HOXD9 and HOXD13 These studies implicate altered developmental regulation of HOX genes in ES pathogenesis.

14. USC Epigenome Center Peter Laird Vasu Punj Dan Weisenberger Martin Brena Vanderbilt Scott Borinstein Acknowledgements NIH-R01CA134604 Russell G. Adderley Endowment UMICH Department of Pediatrics Lawlor Lab Merlin Airik Natashay Bailey Ashley Harris Melanie Krook Jack Mosher Beth Pedersen Laurie Svoboda Dortothea Douglas Jessie Hsu Xiaohua (Cynthia) Jiang John van Doorninck Cornelia von Levetzow CHILDREN’S ONCOLOGY GROUP COG Biopathology Center USC/CHLA Tim Triche Rich Sposto UMICH Dafydd Thomas Clinical Faculty & Staff Patients & their Families