DMG-46. Epigenomic Reprogramming by EZHIP Drives Tumorigenesis and Stem-like States in Diffuse Midline Gliomas.

Abstract EZHIP (EZH inhibitory protein) is a cancer-testis antigen (CTA) typically restricted to germline cells, where it inhibits Polycomb repressive complex 2 (PRC2)-mediated deposition of the repressive histone mark H3K27me3. Its aberrant expression in H3K27M mutation-negative diffuse midline gliomas (DMGs) and ependymomas leads to PRC2 inhibition and widespread epigenomic dysregulation. H3K27M-mutant gliomas are similarly characterized by global loss of H3K27me3 due to inhibition of PRC2 by the oncohistone H3K27M, positioning EZHIP as a functional epigenetic mimetic of this mutation.To explore its role in tumorigenesis, we overexpressed EZHIP in the H3K27 wild-type DMG cell lines that lack H3K27M mutations. EZHIP overexpression resulted in a significant reduction in H3K27me3 levels, indicating PRC2 inhibition. In a Nestin-TVA mouse model of DMG, the introduction of EZHIP into neural stem cells via the RCAS-EZHIP-P2A-PDGFβ vector led to the development of aggressive, high-grade diffuse gliomas. The tumorigenic potential of EZHIP was further supported by Western blot analysis of EZHIP-expressing neural stem cells, which revealed increased expression of stem cell markers, including Nanog, GFAP, and Nestin, consistent with a stem-like transcriptional state. RNA and ATAC sequencing of EZHIP-overexpressing DMG cell lines, compared to their isogenic wild-type counterparts, revealed a substantial increase in the number of upregulated genes typically repressed by PRC2, indicating widespread transcriptional activation associated with PRC2 inhibition. Comparative transcriptional analysis further showed that EZHIP-induced gene expression changes more closely resembled those in PRC2 knockout cells rather than in cells harboring the H3K27M mutation. Together, these findings highlight the complex role of EZHIP in disrupting PRC2-mediated gene silencing, promoting gliomagenesis, and altering the epigenomic landscape. While EZHIP and H3K27M mutations share some mechanistic similarities in inhibiting PRC2, key differences in their downstream effects suggest distinct contributions to tumor development. These findings advance our understanding of EZHIP-driven gliomas and reveal potential therapeutic vulnerabilities that could inform future treatments.