Abstract 2028: Transcriptional modules involving EWS-FLI1 and SRF in Ewing sarcoma

Abstract

Abstract Ewing Sarcoma (ES) is an aggressive pediatric bone tumor with a high tendency to metastasize. The primary genetic aberration responsible for ES pathogenesis is EWS-FLI1, a chimeric gene, derived from the chromosomal translocation t(11;22)(q24;q12). EWS-FLI1 encodes an aberrant ETS transcription factor. However, due to its lack of enzymatic activity and intrinsically disordered structure it has yet not been successfully targeted. EWS-FLI1 is prone to rapidly engage in protein-protein complexes, enhancing its oncogenic activity. It has been reported to be involved in interactions with regulators of the basal transcriptional machinery, RNA processing and DNA repair. Furthermore, it has been shown that, similar to the ternary complex factors (TCF) SAP1a and ELK1, EWS-FLI1 is able to form a complex with the global transcriptional regulator Serum Response Factor (SRF) on ETS containing promoter elements. SRF binds to CArG motifs in serum response elements (SRE) and requires a transcriptional co-activator, which is commonly provided upstream via the Rho- or the Ras-axis. Upon serum induced Rho activation, polymerization of globular (G-) actin into filamentous (F-) actin takes place, thereby releasing the G-actin bound myocardin-related transcription factors A and B (MRTFA/B) to translocate to the nucleus and concertedly with SRF regulate transcription of target genes. Binding of MRTFs to SRF is mutually exclusive and competed by TCFs, which are activated downstream of Ras, and require presence of an ETS motif adjacent to CArG. In this study we aim to investigate a potential competition of EWS-FLI1 with MRTFs, which might uncouple SRF dependent transcription from upstream G-protein linked regulation and hence interfere with processes such as proliferation, migration and differentiation. To address this question, we performed gene expression profiling in A673 and SKNMC ES cell lines where we combined EWS-FLI1 knockdown with MRTFA/B knockdown under serum starved and serum induced (SI) conditions. We found that transcriptional activity of MRTFA/B is largely repressed by EWS-FLI1 and that upon combined knockdown conditions, EWS-FLI1 transcriptional effects are rescued by MRTFA/B. To further elucidate the underlying mechanisms of EWS-FLI1 and MRTFA/B gene regulation, we performed chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) of MRTFA, MRTFB, SRF and EWS-FLI1 in A673 ES cells. Preliminary results corroborate EWS-FLI1 and MRTF sharing a large number of target genes and indicate that MRTFA binds to DNA motifs, which are associated with activated and repressive EWS-FLI1 signatures. Ongoing experiments aim at a proteomic understanding of the EWS-FLI1 containing SRF complex as a basis for potential therapeutic intervention. This study was supported by the Liddy Shriver Sarcoma Initiative and the 7th framework program of the European Commission (FP7-259348, “ASSET”). Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Dave N t Aryee, Raphaela Schwentner, Elizabeth Lawlor, Heinrich Kovar. Transcriptional modules involving EWS-FLI1 and SRF in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2028.