KDM6A and B-mediated transcriptional activation of EWSR1-FLI1 targets coordinates demethylase dependent and independent mechanisms in Ewing sarcoma

Ewing Sarcoma (EwS) 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 neo-enhancers 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 EwS, neo-enhancers are characterized by strong enrichment of the active H3K27ac mark and concomitant lack of the repressive H3K27me3 mark. These regions are typically decorated with high levels of H3K27me3 prior to EWSR1::FLI1-activation. Here, upon expression of EWSR1::FLI1 in human pediatric mesenchymal stem cells, considered the putative cell of origin of EwS, 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 EwS tumor growth. Conversely, KDM6B demethylated H3K27me3 at specific EWSR1::FLI1-active enhancers, co-localizing with BRG1 at GGAA repeats. Loss of KDM6B impaired the growth of EwS tumor xenografts. These results highlight KDM6 demethylases as EWSR1::FLI1 functional partners with potential as targets for treating EwS.