EPCO-10 - An oncogenic neurodevelopmental program in H3.3 K27M diffuse midline glioma invokes aberrant ASCL1-SMARCA4 epigenomic activity

The histone variant H3.3 K27M mutation is common in diffuse midline gliomas (DMG) and is associated with poor prognosis. While some insights into K27M epigenetic functions have emerged, much less is known about how K27M impacts chromatin structure and function, and the resulting transcriptomic consequences. Recently, we developed isogenic CRISPR-edited DMG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. ATAC-seq analysis of the matched sets of isogenic WT and H3.3K27M glioma cells pointed to unique K27M-associated accessible chromatin at regions corresponding to specific neurogenesis, NOTCH, and neuronal development pathways. These domains corresponded to genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell lines. Uniquely accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are important for tumorigenesis. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway including, in particular, ASCL1 and NEUROD1. CUT&RUN for ASCL1 defined novel target genes in DMG and BioID identified key epigenomic cofactors for ASCL1 including SMARCA4. Large-scale computational analysis of both our and other groups’ data including by chromHMM identified novel functional chromatin domains and potential looping events related to K27M as well. Overall, our findings indicate that H3.3K27M changes specific chromatin functions altering neurodevelopmental gene expression resulting in aberrant activity of an oncogenic epigenomic program. Encouragingly, this program appears at least partially reversible upon editing K27M back to wild-type, pointing to potential translational impact moving forward to targeting this pathway.