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…
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Evidence Hub reads this as a cohort study. Its evidence level is 3 of 5 (Moderate · trial / cohort / secondary analysis). Trials without a large randomized design, cohorts and secondary analyses give moderate evidence.
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