HGG-02. Epigenetic dysregulation of PRC1 in paediatric high-grade glioma

Abstract Paediatric high-grade gliomas (pHGGs) are tumours of glial origin that include Diffuse Midline Glioma (DMG). pHGGs can be categorised according to regional location and mutational status; with many pHGGs displaying mutations in Histone 3 (H3) protein, commonly H3-K27M or H3-G34R/V mutations. DMGs (the majority of which are H3-K27M mutant) occur within the brainstem, where their diffuse nature means surgery is not often possible. Additionally, no available chemotherapies have a significant effect on survival. The H3 mutations cause epigenetic dysregulation, with H3-K27M causing disruption of the Polycomb Repressor Complex 1 (PRC1) responsible for ubiquitination of histone H2A to maintain epigenetic repression of target genes during development. H3-K27M can dysregulate PRC activity leading to re-distribution of repression that contributes to tumour maintenance and progression. BMI1, a member of PRC1, is frequently upregulated in pHGG, particularly in H3-K27M DMG. To understand the interplay between H3-K27M and BMI1, shRNA was used to stably knockdown BMI1 in primary H3-K27M lines and their edited H3-K27M knock out syngeneic cell lines, as well as H3-G34R and H3-WT primary lines. BMI1 binding sites, presence of selected histone marks and chromatin accessibility were assessed using ChIP-seq, CUT&Tag and ATAC-seq. We then used ChromHMM, a multivariate hidden Markov model, to annotate and identify changes in chromatin states that are dependent on BMI1 in the context of different H3 mutations. Interestingly, there were several chromatin states defined by repressive marks in a BMI1-high context (including PRC states) that transitioned to active state upon BMI1 knockdown specifically in the H3-K27M mutant cells. Importantly, we found that over 70% of genes presenting an activating chromatin switch upon BMI1 knockdown showed a concordant increase in expression levels. Therapeutic inhibitors of pathways enriched for within these transition states dependent on both BMI1 and K27M could help target aggressive pHGG subtypes.