STAG2 loss amplifies EWS-FLI1-driven microsatellite enhancer activity promoting Ewing's sarcoma aggressiveness [RNA-Seq]

Ewing sarcomas are driven by chromosomal translocations that fuse a FET RNA‑binding protein to an ETS transcription factor, most commonly generating the EWS-FLI1 fusion oncoprotein. EWS-FLI1 engages GGAA microsatellite repeats to create de novo enhancers and activate oncogenic transcriptional programs—a neomorphic gain-of-function essential for Ewing sarcoma pathogenesis. In addition to the truncal fusion, recurrent loss‑of‑function alterations in the cohesin subunit STAG2 occur in approximately 10–15% of Ewing sarcomas and are associated with adverse clinical outcomes. Yet, how STAG2-cohesin deficiency remodels EWS-FLI1 chromatin occupancy and gene regulatory network remains incompletely understood. Here, using genetic STAG2 loss‑of‑function models combined with functional multi-omic profiling, we show that STAG2-cohesin loss in Ewing sarcoma cells reprograms the EWS-FLI1 cistrome by shifting its binding preference at GGAA microsatellite repeats. Despite increased EWS–FLI1 protein abundance, disruption of STAG2 eliminates more than 40% of EWS-FLI1 binding sites. The lost sites are enriched for elements harboring 1–4 GGAA repeat motifs, with a simultaneous gain in EWS-FLI1 binding at multimeric enhancers containing ≥5 GGAA repeat sequences. Notably, reprogrammed EWS-FLI1 sites show concomitant changes in chromatin accessibility and H3K27ac abundance, which preferentially amplify EWS-FLI1 activity at multimeric enhancers and drive marked up‑regulation of canonical microsatellite‑regulated target genes in STAG2‑null cells. By integrating Hi‑C–derived chromatin interaction maps with altered EWS-FLI1 occupancy, we derive distinct monomeric (1xGGAA) and multimeric (≥10xGGAA) EWS-FLI1 transcriptional signatures and show that STAG2 inactivation selectively augments the multimeric signature while attenuating monomeric activity. We further define a prognostic signature of GGAA-repeat enhancers that is significantly upregulated in patient tumors with aggressive clinical features and deleterious STAG2 alterations. Together, these findings reveal that loss of STAG2–cohesin does not simply attenuate EWS-FLI1 function but reprograms its cistrome toward microsatellite multimeric GGAA neo‑enhancers, thereby amplifying a high‑risk EWS–FLI1 transcriptional state in Ewing sarcoma.