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

Savita SankarSavita Sankar11, Leah A. Owen, Leah A. Owen11 and andStephen L. LessnickStephen L. Lessnick1,21,2

11Department of Oncological Sciences, Center for Department of Oncological Sciences, Center for Children at Huntsman Cancer Institute, and Children at Huntsman Cancer Institute, and 22Division of Division of

Pediatric Hematology/Oncology, University of Utah Pediatric Hematology/Oncology, University of Utah School of Medicine, Salt Lake City, UT School of Medicine, Salt Lake City, UT

EWS/FLI is an Aberrant EWS/FLI is an Aberrant Trancription FactorTrancription Factor

EWS/FLI ChIP-chipEWS/FLI ChIP-chip

EWS

FLIFormaldehyde crosslinking, DNA shearing

EWS

FLI

Recover DNA, hybridize to Agilent promoter

microarray

~900 “direct” EWS/FLI target

promoters/genes

high-affinity siteE-value = 4.7x10-43

repetitive elementE-value = 5x10-175

EWS/FLI Response ElementsEWS/FLI Response Elements

EWS/FLI responseelement in NR0B1promoter

NKX2.2 is Necessary forNKX2.2 is Necessary forOncogenic TransformationOncogenic Transformation

Regulation of NKX2.2Regulation of NKX2.2

NKX2.2 was not on ChIP-chip listNKX2.2 was not on ChIP-chip list NKX2.2 promoter does not have obvious NKX2.2 promoter does not have obvious

EWS/FLI binding siteEWS/FLI binding site Implies NKX2.2 is not a direct target of Implies NKX2.2 is not a direct target of

EWS/FLIEWS/FLI Reporter assays also suggest NKX2.2 is Reporter assays also suggest NKX2.2 is

not a direct EWS/FLI targetnot a direct EWS/FLI target How is NKX2.2 regulated?How is NKX2.2 regulated?

NKX2.2NKX2.2

Involved in CNS developmentInvolved in CNS development Involved in pancreatic islet developmentInvolved in pancreatic islet development Involved in oligodendrocyte developmentInvolved in oligodendrocyte development Regulated by Sonic Hedgehog in CNSRegulated by Sonic Hedgehog in CNS Is NKX2.2 regulated by Sonic Hedgehog in Ewing’s Is NKX2.2 regulated by Sonic Hedgehog in Ewing’s

sarcoma?sarcoma?

Sonic Hedgehog PathwaySonic Hedgehog Pathway

cyclopamine

Cyclopamine treatment of Ewing’s sarcoma cell lines did not reduce NKX2.2 expression (with appropriate controls).

Other possibilities?

GLI1 as a potential NKX2.2 GLI1 as a potential NKX2.2 regulatorregulator

GLI consensus human: -1966-CCCGGGTCCGACCACCCAGGACGCGCC-1939mouse: -1976-CCCGGGTCCGACCACCCAGGACGCGCC-1949zebrafish: -2323-CCCGGGTCCGACCACCCAGGACGCGCC-2296

1) GLI1 is upregulated by EWS/FLI (on expression microarray dataset)2) GLI1 was on ChIP-chip list for EWS/FLI (likely direct target)3) GLI consensus binding site in NKX2.2 promoter, conserved across species

EWS

FLIGLI1 NKX2.2

GLI1

Working model

GLI1 is Regulated by EWS/FLIGLI1 is Regulated by EWS/FLI

GLI1-Knockdown Reduces GLI1-Knockdown Reduces NKX2.2 ExpressionNKX2.2 Expression

GLI1 Expression is Required for GLI1 Expression is Required for Oncogenic TransformationOncogenic Transformation

Still to do...Still to do...

Is NKX2.2 the only critical GLI1-regulated Is NKX2.2 the only critical GLI1-regulated gene, or are others required?gene, or are others required?

More detailed analysis of NKX2.2 More detailed analysis of NKX2.2 promoterpromoter

More detailed analysis of GLI1 promoterMore detailed analysis of GLI1 promoter

Lessnick labSam FrancisKunal GangwalMichelle Kinsey Ashley KowalewskiWen LuoSavita SankarRichard SmithTish Toomey

Chi BraunreiterJeff HancockLeah OwenDeb Trem

FundingNational Cancer Institute

American Cancer SocietyTerri Anna Perine Sarcoma Fund

Liddy Shriver Sarcoma InitiativeSunbeam Foundation

Department of Pediatrics, U of UtahHuntsman Cancer Foundation

Huntsman Cancer Institute CCSG

Barbara GravesPete Hollenhorst

Cheryl CoffinLor RandallKen BoucherCody HaroldsenAtul ShahLynn JordeScott Watkins

Todd GolubKim StegmaierAndrew Kung

Email: stephen.lessnick@hci.utah.eduRe: International Ewing’s Sarcoma

Research Forum