A critical dependency in Ewing sarcoma: PAX7 attenuates EWS::FLI1 activity at de novo microsatellite enhancers to maintain oncogenesis

The pediatric cancer Ewing sarcoma is driven by the fusion oncoprotein EWS::FLI1, which retains the DNA-binding domain of FLI1. Rather than canonical ETS sites, EWS::FLI1 is retargeted to GGAA-microsatellite loci, where it remodels chromatin to establish de novo enhancer elements that drive oncogenic transcription. Because EWS::FLI1 lacks intrinsic chromatin-remodeling activity, it depends on recruited proteins to regulate chromatin. Furthermore, precise regulation of EWS::FLI1 activity is critical: both over and under expression of EWS::FLI1 result in cell cycle arrest. However, mechanisms of EWS::FLI1 regulation at non-canonical GGAA-mSats remain unknown. We used a proximity-labelling proteomic approach to identify EWS::FLI1-associated proteins in a native chromatin context. We discovered that the myogenic transcription factor PAX7 selectively interacts with EWS::FLI1 but not wild-type FLI1. PAX7 silencing impaired cell proliferation, indicating an essential role. We found that PAX7 is itself a direct target gene of EWS::FLI1, associated with high, ubiquitous PAX7 expression in EwS tumors. Mechanistically, EWS::FLI1 drives the relocalization of PAX7 to GGAA-mSats and ETS-sites, where PAX7 modulates chromatin accessibility and attenuates EWS::FLI1-driven transcription. At GGAA-mSats, PAX7 depletion results in chromatin opening, whereas at ETS sites, chromatin accessibility is deregulated. PAX7 depletion results in and an exaggerated EWS::FLI1 transcriptional response. Mutational analysis of PAX7 revealed that its Paired DNA binding domain and OAR-domain are required for interaction with EWS::FLI1 and that interaction between the two transcription factors is required for cell proliferation. These findings define PAX7 as a context-specific regulator of EWS::FLI1 activity at non-canonical GGAA-microsatellite enhancers and identify a previously unrecognized vulnerability in Ewing sarcoma that could be exploited for future therapeutic intervention.