The Ews-Fli1 Oncogene Disrupts Normal Developmental Regulation Of Polycomb-Modulated Transcriptional Programs

Disordered control of developmentally regulated polycomb target genes leads to severe abnormalities in embryonic development. Pediatric cancers often arise as a consequence of disordered embryonic development and thus, we hypothesize that disruption of polycomb-regulated transcriptional programs may be a key driver of pediatric tumor initiation. Ewing sarcoma (ES) is a bone and soft tissue tumor that predominantly affects children and young adults. Significantly, established ES constitutively over-express EZH2 and BMI-1 and both of these polycomb proteins have been implicated in ES pathogenesis. ES is initiated by EWS-ETS fusion oncogenes that result from chromosomal translocation events. In the current study we used in vitro models of human neural crest-derived stem cells, ES cell lines and xenografts and established primary ES tumors to define the pattern of polycomb target gene expression in ES and determine whether EWS-FLI1 disrupts normal developmental regulation of polycomb target gene expression during stem cell differentiation. Neuromesenchymal stem cells (NMSC), candidate cells of ES origin, were generated from human embryonic stem cells and then transduced with an EWS-FLI1-EGFP (EF) or EGFP (control) lentivirus. Western blot analysis of transduced cells after six weeks in differentiation media demonstrated persistently high expression of BMI-1 and EZH2 in the EF transduced cells. Comparison of whole genome expression profiles of the cells before and after exposure to differentiation media for six to thirteen weeks revealed that while control cells activated a transcriptional program consistent with mesenchymal differentiation, the mesenchymal program was inhibited in EF cells and a neuronal transcriptional program was instead dominant. Significantly, although expression of polycomb target genes was altered in both conditions following exposure to differentiation media, the pattern of expression was notably divergent in EF cells. In particular, HOX gene expression was found to be reproducibly and globally altered in EF cells. Evaluation of primary ES tumors by gene expression microarrays confirmed global dysregulation of polycomb target gene expression in tumor cells and again revealed a marked variance in HOX gene profiles compared to both normal stem cells as well as normal terminally differentiated adult tissues. Although the HOX gene expression pattern in ES was found to be globally abnormal, HOXD13 was reproducibly highly elevated in all EWS-FLI1+ samples and this was confirmed by RT-PCR of ES cell lines. To determine if abnormal polycomb target gene expression is dependent on EWS-FLI1 in established ES we knocked down EWS-FLI1 in ES cell line xenografts using a doxycline-inducible shRNA. Knockdown of EWS-FLI1 led to marked inhibition of tumor growth and this was associated with downregulation of HOXD13 . Studies to further delineate the mechanistic contribution of HOX gene dysregulation to ES pathogenesis are now underway. Together these studies demonstrate that disordered regulation of polycomb target genes is ubiquitous in ES and results from EWS-FLI1-mediated disruption of polycomb-modulated transcriptional programs during stem cell differentiation. In addition, these studies for the first time implicate altered expression of HOX genes in ES tumor initiation and progression. Citation Format: Ashley Harris, Natashay Bailey, Elizabeth R. Lawlor. The EWS-FLI1 oncogene disrupts normal developmental regulation of polycomb-modulated transcriptional programs. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A26.