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EWS/FLI Regulates NKX2.2 via GLI in a Non-SHH-Dependent Manner

EWS/FLI Regulates NKX2.2 via GLI in a Non-SHH-Dependent Manner. Savita Sankar 1 , Leah A. Owen 1 and Stephen L. Lessnick 1,2

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EWS/FLI Regulates NKX2.2 via GLI in a Non-SHH-Dependent Manner

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  1. EWS/FLI Regulates NKX2.2 via GLI in a Non-SHH-Dependent Manner Savita Sankar1, Leah A. Owen1 and Stephen L. Lessnick1,2 1Department of Oncological Sciences, Center for Children at Huntsman Cancer Institute, and 2Division of Pediatric Hematology/Oncology, University of Utah School of Medicine, Salt Lake City, UT

  2. EWS/FLI is an Aberrant Trancription Factor

  3. EWS/FLI ChIP-chip Formaldehyde crosslinking, DNA shearing Recover DNA, hybridize to Agilent promoter microarray EWS EWS FLI FLI ~900 “direct” EWS/FLI target promoters/genes

  4. EWS/FLI Response Elements high-affinity site E-value = 4.7x10-43 repetitive element E-value = 5x10-175 EWS/FLI response element in NR0B1 promoter

  5. NKX2.2 is Necessary forOncogenic Transformation

  6. Regulation of NKX2.2 • NKX2.2 was not on ChIP-chip list • NKX2.2 promoter does not have obvious EWS/FLI binding site • Implies NKX2.2 is not a direct target of EWS/FLI • Reporter assays also suggest NKX2.2 is not a direct EWS/FLI target • How is NKX2.2 regulated?

  7. NKX2.2 • Involved in CNS development • Involved in pancreatic islet development • Involved in oligodendrocyte development • Regulated by Sonic Hedgehog in CNS • Is NKX2.2 regulated by Sonic Hedgehog in Ewing’s sarcoma?

  8. cyclopamine Sonic Hedgehog Pathway Cyclopamine treatment of Ewing’s sarcoma cell lines did not reduce NKX2.2 expression (with appropriate controls). Other possibilities?

  9. GLI1 GLI1 NKX2.2 Working model GLI1 as a potential NKX2.2 regulator • GLI1 is upregulated by EWS/FLI (on expression microarray dataset) • GLI1 was on ChIP-chip list for EWS/FLI (likely direct target) • GLI consensus binding site in NKX2.2 promoter, conserved across species EWS GLI consensus human: -1966-CCCGGGTCCGACCACCCAGGACGCGCC-1939 mouse: -1976-CCCGGGTCCGACCACCCAGGACGCGCC-1949 zebrafish: -2323-CCCGGGTCCGACCACCCAGGACGCGCC-2296 FLI

  10. GLI1 is Regulated by EWS/FLI

  11. GLI1-Knockdown Reduces NKX2.2 Expression

  12. GLI1 Expression is Required for Oncogenic Transformation

  13. Still to do... • Is NKX2.2 the only critical GLI1-regulated gene, or are others required? • More detailed analysis of NKX2.2 promoter • More detailed analysis of GLI1 promoter

  14. Email: stephen.lessnick@hci.utah.edu Re: International Ewing’s Sarcoma Research Forum Lessnick lab Sam Francis Kunal Gangwal Michelle Kinsey Ashley Kowalewski Wen Luo Savita Sankar Richard Smith Tish Toomey Chi Braunreiter Jeff Hancock Leah Owen Deb Trem Barbara Graves Pete Hollenhorst Cheryl Coffin Lor Randall Ken Boucher Cody Haroldsen Atul Shah Lynn Jorde Scott Watkins Todd Golub Kim Stegmaier Andrew Kung Funding National Cancer Institute American Cancer Society Terri Anna Perine Sarcoma Fund Liddy Shriver Sarcoma Initiative Sunbeam Foundation Department of Pediatrics, U of Utah Huntsman Cancer Foundation Huntsman Cancer Institute CCSG

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