CSIG-28. ATRX LOSS LEADS TO H3K4ME3-DRIVEN EPIGENETIC DYSREGULATION OF OLIGODENDROCYTE DIFFERENTIATION AND CELL CYCLE CONTROL IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMA (DMG)

Abstract Diffuse midline gliomas (DMGs) are lethal pediatric cancers. Histone H3.3 (H3-3A) is the most commonly mutated gene in DMG; up to 80% exhibit a gain-of-function mutation at lysine 27 (K27M). K27M mutations often co-occur with loss of ATRX, a chromatin remodeler. We generated isogenic mouse and human cell lines to characterize the interaction between H3.3K27M and ATRX co-mutations. Interestingly, CUT&RUN-seq demonstrates that ATRX loss drives global loss of H3K4me3 at promoters in murine K27M cells when compared to H3.3WT or H3.3G34R models. Integration of RNA- and ATAC-seq shows that on a K27M background, ATRX loss downregulates differentiation-related gene sets, consistent with the role of ATRX in healthy brain differentiation. Gene loci for oligodendrocyte precursor genes OLIG2 and CSPG4 exhibit both H3K4me3 loss and decreased chromatin accessibility with ATRX loss. Separately, RNA- and ATAC-seq integration show ATRX loss leads to dysregulation of cell cycle genes in H3K27M cells. p16, a critical G1/S checkpoint regulator which has been shown to be decreased in H3K27M cells, is further decreased with ATRX loss. ATRX deficiency leads to loss of H3K4me3 binding and further decrease in chromatin accessibility at the p16 promoter uniquely in H3K27M cells. This leads to: (1) absent p16 expression, (2) inappropriate release of cell cycle checkpoints after radiation, and (3) heightened sensitivity to CDK4/6 inhibitors in ATRX-H3K27M co-mutant cells. Ongoing work includes determining the impact of ATRX on H3K4 demethylase function and association of H3K4me3 distribution with established K27M “poised” promoters. We are additionally pursuing in vivo CDK4/6 inhibitor studies by H3 and ATRX status, as well as assembling anecdotal use of abemaciclib in human DMG patients with H3.3/ATRX mutation. In summary, ATRX loss leads to a unique epigenetic landscape in H3K27M tumor cells, with global H3K4me3 loss at promoters, resulting in pathways that may be therapeutically targetable.