ID #448 Epigenomic erosion and enhancer dependence in H3.3K27M diffuse midline glioma

Abstract

Diffuse midline glioma, H3K27M-altered (DMG H3K27a), are the most agressive paediatric brain tumours, with a median survival of less than one year. In ∼60% of the cases, a somatzic mutation substitutes lysine 27 with methioninein the histone variant H3.3 (H3.3K27M). This mutation reducesrepressive mark H3K27me3 and increases active mark H3K27ac, but the impact of this increased acetylation on chromatin organisation and oncogenesis remains unclear, as global abundance (western blot) and local distribution (ChIP-seq) show discrepancies. Here, we aimed to define the role of hyperacetylated nucleosomes in DMG pathogenesis and explore their therapeutic relevance. We profiles the epigenome of three H3.3K27M+ DMG cell lines, mapping H3K27M, H3K27ac, H2AZac, H3K4me1, H3K4me3 and H3K27me3, distribution. Comparisons were made with normal astrocytes and isogenic h3K27M knockout (KO) models. Functional assaya assessed the impact of p300 inhibitionon DMG proliferationand colony formation. We identified a set of DMG-specific, highly accessible enhancers marked by H3K27M, H3K27ac and H2AZac that strongly predicted regulation of key oncogenic genes. In DMGs, acetylation profiles showed “epigenomic erosion”, with diffuse H3K27ac and H2AZacsignals extendingbeyond peak regions, validated by ATAC-seq. Treatment with p300 inhibitorssignificantly reduced proliferation in DMG cells and ex-vivo tumoroids, while sparing normal astrocytes. H3K27M+ cells were more sensitive to p300 inhibition than their KO counterparts. DMG cells rely on enhancer-driven transcription and a plasticchromatin statecharacterised by epigenomic erosion. Targeting p300 represents a promising therapeutic strategy to disrupt both enhancer activity abd chromatin plasticity in H3.3K27M DMG.