Objective: Ewing's sarcoma is a highly aggressive bone-associated malignancy affecting children and young adults with most cases harboring a somatic translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant transcription factor. Most studies indicate that EWS/FLI functions as a transcriptional activator. However, recent genome-wide transcriptional studies have raised the possibility that EWS/FLI may also repress target genes, although the mechanistic basis for this repression is unknown. We sought to test the hypothesis that EWS/FLI functions as a direct transcriptional repressor at some genetic loci, and that transcriptional repression is required for the oncogenic function of EWS/FLI. Furthermore, we sought to define relevant domains and corepressors that are involved in this activity. Methods: RNAi based microarray combined with genome-wide ChIP-Chip analyses were used to identify direct repressed target genes of EWS/FLI. Soft agar and xenograft tumor formation assays were performed to assess oncogenic transformation. Deletion mutants of EWS/FLI were constructed and used to map the repression domain. Co-immunoprecipitations were performed to validate the interaction of EWS/FLI with transcriptional co-repressors. Luciferase reporter assays and gel shift assays were utilized to characterize the binding site for EWS/FLI at repressed gene promoters. Results: We identified a subset of EWS/FLI-repressed target genes that are directly bound by the protein. Using these target genes, we identified domains in EWS/FLI necessary and sufficient for repression. Interestingly, these repression domains are also necessary for oncogenic transformation. We identified two HDACs that are involved in this repression, and identified a likely candidate transcriptional repression complex that binds EWS/FLI and contributes to its repressive function. Finally, we found that transcriptional repression is absolutely required for the oncogenic function of EWS/FLI, as two downregulated targets function as suppressors of Ewing's sarcoma oncogenesis. Conclusion: Transcriptional deregulation by EWS/FLI is a key oncogenic event in Ewing's sarcoma pathogenesis. Our data demonstrated that, in addition to transcriptional activation, EWS/FLI mediated transcriptional repression is also critical in Ewing's sarcoma oncogenesis. This is the first time that a detailed molecular mechanism of EWS/FLI mediated direct repression is reported, and has important implications for therapeutic targeting of EWS/FLI in Ewing's sarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2219. doi:1538-7445.AM2012-2219