KDM6 Demethylases Contribute to EWSR1::FLI1-Driven Oncogenic Reprogramming in Ewing Sarcoma

Ewing sarcoma is a highly aggressive tumor arising in bones and soft tissues, driven by the fusion oncoprotein EWSR1::FLI1. This aberrant transcription factor binds to GGAA microsatellites, causing epigenetic reprogramming through the formation of active neoenhancers in a permissive cellular context. Inhibition of the oncogene remains challenging, and current efforts seek to exploit emergent epigenetic treatments targeting EWSR1::FLI1 cofactors. In Ewing sarcoma, neoenhancers are characterized by strong enrichment of the active H3K27ac mark and a concomitant lack of the repressive H3K27me3 mark. These regions are typically decorated with high levels of H3K27me3 prior to EWSR1::FLI1 activation. In this study, upon expression of EWSR1::FLI1 in human pediatric mesenchymal stem cells, considered the putative cell of origin of Ewing sarcoma, we unraveled the genome-wide redistribution of H3K27me3. Stemming from these results, we elucidated the contribution of the H3K27me3 demethylases KDM6A/UTX and KDM6B/JMJD3 in the transcriptional activity of EWSR1::FLI1-induced enhancers. KDM6A had a demethylase-independent role in recruiting BRG1 at EWSR1::FLI1-primed enhancers containing single GGAA motifs, which was critical for Ewing sarcoma tumor growth. Conversely, KDM6B demethylated H3K27me3 at specific EWSR1::FLI1-active enhancers, colocalizing with BRG1 at GGAA repeats. Loss of KDM6B impaired the growth of Ewing sarcoma tumor xenografts. These results highlight KDM6 demethylases as EWSR1::FLI1 functional partners with potential as targets for treating Ewing sarcoma.

KDM6A and KDM6B mediate critical mechanisms behind EWSR1::FLI1 transcriptional activation program involving demethylase-independent and dependent functions, respectively, supporting the development of therapeutic strategies targeting these demethylases in Ewing sarcoma.