Structural and mechanistic investigation into EWS::FLI1 disruption of EWS function in transcription and DNA repair

PROJECT SUMMARY Ewing sarcoma is a pediatric bone and soft tissue cancer that results from chromosomal translocation, most commonly t(11;22)(q24;q12), which fuses the low-complexity domain (LCD) of the RNA-binding protein EWS (EWS) to the DNA-binding domain (DBD) of Friend leukemia integration 1 transcription factor (FLI1). The resulting oncogenic fusion protein EWS::FLI1 directly interferes with the native functions of EWS, particularly those in transcription and DNA repair, resulting in excessive transcription and sensitivity to DNA damaging agents. EWS::FLI1 prevents BRCA1 re-localization from transcriptional sites to DNA damage, resulting in an apparent BRCA1-deficient phenotype and homologous recombination deficiency. When DNA damage occurs, the EWS RNA-binding domain (EWSRBD) mediates the dissociation of PARP-1 from sites of DNA damage sites, promoting the initiation of DNA repair. EWS::FLI1 severely impairs these interactions, causing PARP-1 buildup on DNA damage sites and PARP-1 inhibitor sensitivity characteristic of Ewing sarcoma. However, the molecular mechanisms underlying EWS::FLI1 interaction with EWS and how those interactions disrupt the native functions of EWS are unknown. Preliminary data has demonstrated how EWSLCD forms self-associative interactions primarily mediated by tyrosines throughout the domain. These EWSLCD tyrosines also play a central role in Ewing sarcoma, mediating self-associative and heterotypic interactions key for neoplastic transformation. EWSLCD also associates with the EWSRBD, driving their co-localization to biomolecular condensates. However, the structural basis of EWSLCD:EWSRBD is unknown and the role of this interaction in the transcriptional and DNA repair functions of EWS remains unknown. The overall goal of this project is to elucidate the structural mechanisms and functional implications of EWS self-association and how they relate to EWS::FLI1-driven oncogenesis. We will test the hypothesis that EWSLCD directly interacts with EWSRBD and enhances the nucleic acid binding propensity of EWS, and that the interplay between EWSLCD and EWSRBD are key to the DNA repair and transcriptional functions of EWS. Enhancing our understanding of the mechanisms underlying EWS::FLI1-driven oncogenesis will enable the development of more targeted therapeutics to Ewing sarcoma and related pediatric malignancies.