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Disclosure Information CTOS Annual Meeting 2013 Elizabeth R. Lawlor

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.

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Disclosure Information CTOS Annual Meeting 2013 Elizabeth R. Lawlor

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  1. THE EWS-FLI1 ONCOGENE DISRUPTS NORMAL DEVELOPMENTAL REGULATION OF POLYCOMB-MODULATED TRANSCRIPTIONAL PROGRAMSAshley Harris, Natashay Bailey, Laurie SvobodaElizabeth R. LawlorDepartment of PediatricsTranslational Oncology ProgramUniversity of Michigan, Ann Arbor, MI

  2. Disclosure InformationCTOS Annual Meeting 2013Elizabeth 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 H & E BMI-1 Douglas, Cancer Research 2008; Hsu, Oncogene, 2011;von Levetzow, PLoS ONE, 2011 ★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 BMI1 PHC SUZ12 PLC CBX PRC2 PRC1 RING1 A/B EZH2 EED H3K27me3 H2a119Ub H3 H2A Active Chromatin Repressed Chromatin

  5. HOX genes are dynamically expressed during embryonic development • 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

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

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

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

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

  10. Ewing sarcomas have altered HOX profiles A B Abnormal upregulation of posterior HOXD genes PCA Mapping: 37 HOX Genes FDR<0.05 Normal Adult Tissue (N=33) BM-MSC (N=3) NC-MSC (N=3) NCSC (N=3) Ewing Sarcoma (N=32) 24/37 HOX genes are significantly differentially expressed in Ewing sarcoma FDR<0.05 Stem cells Adult tissues ES tumors HOXD9 HOXD10 HOXD11 HOXD13

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

  12. HOXD9 and HOXD13 are down-regulated following EWS-FLI1 knockdown in TC-71 xenografts A. B. 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. Acknowledgements 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 BiopathologyCenter USC/CHLA Tim Triche Rich Sposto UMICH Dafydd Thomas Clinical Faculty & Staff Patients & their Families USC Epigenome Center Peter Laird VasuPunj Dan Weisenberger Martin Brena Vanderbilt Scott Borinstein NIH-R01CA134604 Russell G. AdderleyEndowment UMICH Department of Pediatrics

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