The hegemonic EWSR1::ETS oncoprotein overrules core regulatory circuitry principles in Ewing sarcoma.

Ewing sarcoma is an aggressive bone tumor of adolescence characterized by a hallmark EWSR1::ETS fusion oncogene. The resulting chimeric oncoprotein drives tumorigenesis by reshaping transcriptional and epigenetic landscapes. However, how it is transcriptionally regulated and whether additional master transcription factors (MTFs) form a core regulatory circuit (CRC) in Ewing sarcoma remain unclear. Using an extensive panel of Ewing sarcoma cell lines and primary tumors, we mapped super-enhancers and identified enrichment of GGAA microsatellites, confirming their specificity to Ewing sarcoma as compared to other pediatric cancers and normal tissues. Integrating transcriptomic, epigenetic, 3D chromatin conformation, and dependency data, we predicted a set of MTFs potentially forming a CRC. However, functional validation demonstrated that these MTFs neither establish auto-regulatory loops nor confer robust proliferative dependencies typical of CRCs in other pediatric tumors. Instead, EWSR1::FLI1 emerged as an "hegemonic" oncoprotein, regulating expression of these MTFs without reciprocal regulation. Knockdown of EWSR1::FLI1 strongly shifted H3K27ac profiles toward mesenchymal states, whereas silencing individual or combined MTFs did not alter cell growth or EWSR1::FLI1 expression. These findings highlight the absence of a classical CRC in Ewing sarcoma and emphasize EWSR1::FLI1 as the dominant oncoprotein and a major vulnerability in this disease.