PROJECT SUMMARY Pediatric high-grade gliomas (pHGG), which include diffuse midline glioma (DMG), are among the most lethal of all pediatric cancers, with survival rarely exceeding two years. The most common mutations in pHGG are found in histone variant H3.3 (H3F3A), with nearly 50% of all pHGG exhibiting gain-of-function mutations at either lysine 27 (K27M) or glycine 34 (G34R/V). Both histone mutants result in widespread epigenetic impacts on transcriptional control and distinct clinical behavior. Additionally, roughly 30% of pHGG harbor loss-of- function mutations in the chromatin remodeling protein ATRX, which co-occur in about 20% of H3K27M tumors and 90% of those with H3.3G34R/V mutations. However, there is currently no known mechanism to explain the gap in co-mutation between the different H3F3A-mutant tumors. Studies have demonstrated the ability of H3K27M to inhibit the catalytic function of EZH2, an H3K27 methyltransferase, and ATRX has been shown to impact localization of PRC2, but no such association has been made for H3G34R/V mutations. EZH2, one of the catalytic subunits of the polycomb repressive complex 2 (PRC2), is responsible for maintaining repression of many early developmental genes, including those involved in early brain development. The overall objective of this proposal is to determine how ATRX loss impacts developmental gene expression in H3G34R/V mutant tumor cells. The central hypothesis states that co-mutation of H3G34R/V and ATRX will result in (i) inappropriate PRC2 localization and H3K27me3 placement, leading to (ii) expression of PRC2- repressed neurodevelopmental genes and a subsequent increase in tumor cell growth. The preliminary data outlined in this proposal suggests that the same set of PRC2 target genes are upregulated both by H3K27M alone and ATRX loss/H3G34R together. The first aim assesses the role of concurrent H3F3A and ATRX mutations on PRC2 dysfunction, which will look at both localization and function of PRC2 at known targets and throughout the genome. The second aim will look into how H3F3A and ATRX co-mutation affects expression of NPC marker genes and how this impacts cell proliferation and rates of tumor growth. Successful completion of this proposal is expected to result in a clearer understanding of why ATRX loss is preferentially selected for in H3G34R/V-mutant gliomas over H3K27M-mutant gliomas. This knowledge will result in the ability to design and utilize more specific therapies for H3F3A-mutant gliomas, which could prolong survival in upwards of 50% of pHGG patients.