EPCO-64. EXPLOITING EPIGENETIC CELL STATE HETEROGENEITY TOWARDS DIFFERENTIATION THERAPY IN H3K27M-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract The H3K27M mutation inhibits PRC2 activity resulting in a global hypomethylation and concomitant increase in acetylation of H3K27 wildtype histones. Such epigenetic alterations force diffuse midline glioma (DMG) cells to retain a highly proliferative stem-like cell state. However, single cell transcriptomics revealed that some cancer cells, despite harboring the H3K27M mutation, can exit the cell cycle and acquire features of mature glial-like cells, providing rationale for differentiation therapies. Here, we sought to dissect the epigenetic landscape of these distinct cancer cell states within DMG to better understand how tumor cell identity might be modulated for therapeutic benefit. To do so, we profiled the histone posttranslational modifications landscape and genome accessibility in both stem- as well as glial-like DMG cells. We observed that the more mature glial-like cells overturn the oncogenic activity of the histone mutation on H3K27 marks, and consequently carry higher levels of the repressive histone mark H3K27me3 and lower levels of the activating mark H3K27ac. Next, we questioned whether H3K27 acetylation loss would reciprocally drive DMG cell differentiation. Intriguingly, our epigenetically-focused CRISPR screen identified the histone acetyltransferases responsible for acetylating H3K27, EP300/CBP, as essential genes for the continuous proliferation of stem-like DMG cells. In agreement, DMG cells are selectively sensitive to EP300/CBP inhibition or degradation, unlike human astrocytes and H3G34R/V high-grade glioma cells. Interestingly, targeting EP300/CBP reverts the epigenetic reprograming caused by H3K27M by not only depleting H3K27ac but also rescuing H3K27me3 levels. Finally, ATAC and single cell RNA sequencing revealed that the epigenetic changes caused by EP300/CBP targeting shift DMG stem-like cells towards more differentiated glial-like cell states. In summary, we show that distinct DMG transcriptomic cell states reflect underlying epigenetic heterogeneity which can be exploited for treatment. In particular, we demonstrate that EP300/CBP are promising therapeutic targets for differentiation therapy in this universally fatal glioma.