Ews-Fli1 Represses Rho-Actin Signaling Via Mrtfb/Yap-1/Tead Perturbation In Ewing Sarcoma

Ewing Sarcoma (EwS) is an aggressive pediatric bone tumor driven by the aberrant fusion-oncogene EWS-FLI1, which deregulates hundreds of genes by either activation (EWS-FLI1-correlated genes) or repression (EWS-FLI1-anticorrelated genes). Several EWS-FLI1-anticorrelated genes are involved in cytoskeletal processes and typically regulated by Rho/F-actin signaling. The Rho pathway is a crucial regulator of cell movement and cellular plasticity. Given the fact that EwS cells are highly prone to metastasize we were interested in studying a potential EWS-FLI1-mediated deregulation of Rho signaling. Activation of Rho triggers G- to F-actin polymerization thereby enabling nuclear translocation of the myocardin-related transcription factors MRTFA (MKL-1) and MRTFB (MKL-2). MRTFs typically serve as transcriptional co-activators of the transcription factor SRF regulating several cytoskeletal key players. We used the A673/TR/shEF cell line, harboring a doxycycline inducible sh-EWS-FLI1 plasmid, to interrogate the role of MRTFA/B in the EwS gene regulatory network upon EWS-FLI1-high and -low conditions. Strikingly, MRTFB transcriptional activity was overall repressed by EWS-FLI1. Furthermore, knockdown of MRTFB under EWS-FLI1-low conditions antagonized the re-activation of EWS-FLI1-anticorrelated genes. ChIP-seq revealed a strong overlap of MRTFB and EWS-FLI1 chromatin binding. MRTFB binding was significantly enriched in distal (enhancer) regions of EWS-FLI1-anticorrelated genes, especially upon EWS-FLI1-low conditions. Of note, an overrepresentation of TEAD motifs, but not SRF binding motifs, was observed in these regions suggesting a potential involvement of TEAD transcription factors in the regulation of the MRTFB/EWS-FLI1 reciprocally regulated targets. In line with this finding, target genes of the mechano sensitive YAP/TAZ/TEAD pathway (CTGF, CYR61, SERPINE1) were found among the MRTFB-bound EWS-FLI1-anticorrelated genes. Genome-wide expression profiling upon combined knockdown of EWS-FLI1 and all four TEADs confirmed that TEAD regulates EWS-FLI1 target genes antagonistically. ChIP-qPCR for selected genes validated this finding demonstrating increased TEAD binding to shared MRTFB/EWS-FLI1 target genes upon decreased EWS-FLI1 occupation. Our data suggest that MRTFB/YAP-1/TEAD directly regulate EWS-FLI1-anticorrelated targets. Since, in addition, EWS-FLI1 is known to indirectly perturb F-actin polymerization, we propose a model of dual EWS-FLI1 driven MRTFB/YAP-1/TEAD perturbation by direct and indirect mechanisms downstream of Rho/F-actin signaling. Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Raphaela Schwentner, Eleni M. Tomazou, Markus Linder, Cornelia N. Mutz, Javier Alonso, Dave N. Aryee, Heinrich Kovar. EWS-FLI1 represses Rho-actin signaling via MRTFB/YAP-1/TEAD perturbation in Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3508. doi:10.1158/1538-7445.AM2017-3508