Abstract 1736: Elucidating the oncogenic mechanisms in diffuse midline glioma: Comparative analysis of h3k27m mutation and k27 methylation loss on gene expression profiles via RNA sequencing

Abstract Background: Diffuse Midline Glioma (DMG), a severe pediatric brain tumor, is characterized by its aggressive nature and poor prognosis. Common in brain midline structures like the thalamus and brainstem, DMGs often harbor H3K27M mutations, leading to reduced histone trimethylation at lysine 27 and loss of serine 31 phosphorylation. Building on these findings, our laboratory has undertaken a novel approach by engineering a knockout of the Polycomb Repressive Complex 2 (PRC2) in selected cell lines. This was achieved using CRISPR-Cas technology to target the EZH1 and EZH2 methyltransferases (PRC2KO), alongside the introduction or reversal of histone H3.3 mutations. The goal of this study is to investigate the effects of K27 Methylation loss and H3.3 mutations on gene expression in DMG to better understand DMG pathogenesis and potential therapeutic avenues. Methods: RNA and ATAC sequencing were performed on DMG cell lines including H3.3WT, H3.3 S31A, H3.3K27M, and PRC2KO. Utilizing 150 bp paired-end reads, 25 to 45 million unique reads were mapped to the human genome (hg38) using STAR. Differential gene expression was analyzed using DESeq2 in R, complemented by ATAC-sequencing to explore potential epigenetic changes. Results: The analysis identified 2,564; 2,946; 4,618; and 5,490 differentially expressed genes (DEGs) in the K27M, S31A, K27M-PRC2KO, and WT-PRC2KO cell lines, respectively, when compared to WT. A four-way Venn diagram analysis identified unique and shared gene cohorts among the different mutations. H3K27M cells showed 414 downregulated and 385 upregulated genes, mainly affecting cell migration. In contrast, PRC2KO cells, characterized by a complete loss of histone methylation, showed extensive changes with 1,302 genes downregulated and 1,779 upregulated, primarily impacting cell cycle and developmental processes. The S31A cells displayed a unique profile with 548 genes uniquely downregulated and 547 genes upregulated. The lack of enrichment in specific pathways for these genes suggests their involvement in a broader range of affected pathways. A significant discovery was the identification of a set of genes common to both S31 and K27M cells, but absent in PRC2 knockouts. This cohort, consisting of 350 downregulated and 389 upregulated genes, may represent a molecular link between serine phosphorylation and the K27M mutation. Additionally, ATAC-sequencing analysis was conducted to determine if the expression changes were a direct result of epigenetic dysregulation or an indirect effect. Conclusion: This study delineates the intricate gene regulation in DMG resulting from H3K27M mutations beyond loss of loss of K27 Methylation. It highlights unique and overlapping pathways influenced by, providing valuable insights into the molecular basis of DMG and guiding future therapeutic strategies. Citation Format: Suraj Bhattarai, Faruck L. Hakkim, Charles Day, Florina Grigore, Alyssa Langfald, Edward Hinchcliffe, James Robinson. Elucidating the oncogenic mechanisms in diffuse midline glioma: Comparative analysis of h3k27m mutation and k27 methylation loss on gene expression profiles via RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1736.