THE EWS-FLI1 ONCOGENE DISRUPTS NORMAL DEVELOPMENTAL REGULATION OF POLYCOMB-MODULATED TRANSCRIPTIONAL PROGRAMS

 Ashley Harris, Natashay Bailey, Laurie Svoboda

Elizabeth R. LawlorDepartment of Pediatrics

Translational Oncology ProgramUniversity of Michigan, Ann Arbor, MI

Disclosure InformationCTOS 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.

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 & E BMI-1

★EZH2 is an EWS-FLI1 target gene and promotes Ewing sarcoma tumorigenicity

Riggi, Cancer Res 2008, Richter, PNAS 2009

Polycomb proteins epigenetically regulate gene expression during normal development

SUZ12PLCEED

PHC BMI1

CBXRING1 A/B

Active Chromatin Repressed Chromatin

PRC2PRC1

EZH2

H3 H2A

H3K27me3 H2a119Ub

• 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

Hypothesis

Altered expression of polycomb proteins contributes to Ewing sarcoma tumor initiation and maintenance by 

disrupting expression of developmentally critical transcriptional programs

Unique to EWS-FLI1+

Unique to Control

511231

270

Overlapping Transcripts 219 coordinately regulated

BCV CV

EF EF

NCSC

5 days in self-renewal media

6 wks in differentiation

media

vs.

A

EWS-FLI1 alters differentiation program of NCSC

Unique to EWS-FLI1+

Unique to Control

511231

270

Overlapping Transcripts 219 coordinately regulated

Upregulated in EWS-FLI1+ cellsGO:0007155~cell adhesionGO:0022610~biological adhesionGO:0030182~neuron differentiationGO:0048666~neuron developmentGO:0031175~neuron projection developmentGO:0007409~axonogenesisGO:0048667~cell morphogenesis involved in neuron differentiationGO:0048812~neuron projection morphogenesisGO:0042127~regulation of cell proliferationGO:0000904~cell morphogenesis involved in differentiation

Down-regulated in EWS-FLI1+ cellsGO:0048706~embryonic skeletal system developmentGO:0001501~skeletal system developmentGO:0009952~anterior/posterior pattern formationGO:0007155~cell adhesionGO:0022610~biological adhesionGO:0003002~regionalizationGO:0048562~embryonic organ morphogenesisGO:0007389~pattern specification processGO:0048568~embryonic organ developmentGO:0048705~skeletal system morphogenesis

B

C

EWS-FLI1 shifts transcriptional program to neural from mesenchymal

CV CV

EF EF

NCSC

5 days in self-renewal media

6 wks in differentiation

media

vs.

A

D

EWS-FLI1 alters expression of HOX Genes

Fold change in HOX

genes after 6 wks in

differentiation media

HOXA HOXB HOXC HOXD

HOXD

9

HOXD

10

HOXD

11

HOXD

13

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 cellsAdult tissuesES tumors

B

24/37 HOX genes are significantly differentially expressed in Ewing sarcomaFDR<0.05

Polycomb-mediated H3K27me3 silencing is lost at HOXD13 promoter in Ewing sarcoma cells

Ewing sarcomaFibroblast

Neural crest SC

Mesenchymal SC

Fibroblast

Neural crest SC

Mesenchymal SCEwing sarcoma

H3 H2A

H3K27me3 H2a119Ub

H3K27me3 ChIP/HOXD13 promoter PCR RNA Polymerase II ChIP/HOXD13 promoter PCR

H3

Ewing sarcoma: Active Chromatin stateStem cells & terminally differentiated cells: Repressed Chromatin state

H3K4me3

B.A.

HOXD9 and HOXD13 are down-regulated following EWS-FLI1 knockdown in TC-71 xenografts

C.

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. 

USC Epigenome CenterPeter LairdVasu PunjDan WeisenbergerMartin Brena

VanderbiltScott Borinstein

Acknowledgements

NIH-R01CA134604Russell G. Adderley EndowmentUMICH Department of Pediatrics

Lawlor LabMerlin AirikNatashay BaileyAshley HarrisMelanie KrookJack MosherBeth PedersenLaurie Svoboda

Dortothea DouglasJessie Hsu Xiaohua (Cynthia) JiangJohn van DoorninckCornelia von Levetzow

CHILDREN’S ONCOLOGY GROUPCOG 

Biopathology Center

USC/CHLATim Triche Rich Sposto

UMICHDafydd Thomas

Clinical Faculty & StaffPatients & their Families