RERE and the Mediator complex cooperate with EWSR1::FLI1 in the reprogramming of Translation and Alternative Splicing, the latter being a therapeutically targetable vulnerability in Ewing sarcoma

Ewing Sarcoma (ES) is an aggressive neoplasm arising in bones and soft tissues driven by the oncogenic fusion EWSR1::FLI1. Through epigenetic deregulation, EWSR1::FLI1 generates de novo super-enhancers that control the expression of key genes for tumor cell maintenance. By an integrative in silico analysis, we identified the subunit of the Mediator complex MED13L and RERE, a member of the atrophin family of arginine-glutamic acid dipeptide repeat-containing proteins, as genes regulated by EWSR1::FLI1-bound super-enhancers. We confirmed that EWSR1::FLI1 regulates MED13L and RERE expression in ES cell lines and showed that these proteins are highly expressed in Ewing primary tumors. Besides the well-established role of the Mediator complex in transcriptional regulation given its association with the RNA polymerase II, in ES cells the DNA binding sites of MED13L overlap with those of RERE and EWSR1::FLI1 in genes that control protein translation and alternative splicing (AS). Accordingly, the expression of various spliceosome components is co-regulated by MED13L, RERE and the oncogene, leading to AS in ES cells. We identified RBM39, a splicing factor downregulated after MED13L and RERE depletion, as a direct transcriptional target of EWSR1::FLI1. Consistently, in vitro viability experiments using indisulam, which induces selective DCAF15-dependent proteosome degradation of RBM39, demonstrate ES cells highly and specifically sensitive to RBM39 inhibition. In vivo experiments with mice xenografted with ES cells show complete tumor regression with indisulam, highlighting the potential of this approach as a novel and promising therapeutic strategy for Ewing sarcoma. STATEMENT OF SIGNIFICANCEEwing sarcoma (ES) is characterized by FET::ETS oncoproteins that act as pioneer transcription factors. Here, we identified two genes controlled by EWSR1::FLI1-bound super-enhancers, MED13L and RERE, and characterized the mechanism by which these proteins cooperate with the oncogene to regulate RNA metabolism and ribosomal processes in ES cells. These findings have led to the identification of the splicing factor RBM39 as a vulnerability in ES, as supported by the extraordinary sensitivity of these tumors to monotherapy with RBM39 degrader indisulam.