Abstract B041: Defining essential PPM1D-regulated DNA damage response phosphosites in diffuse midline gliomas

Abstract Diffuse midline gliomas (DMGs) are universally fatal and highly aggressive pediatric brain tumors. While radiation can extend survival, traditional chemotherapies and newer targeted therapies remain largely ineffective, and tumors ultimately progress. About 10% of pediatric high-grade gliomas harbor mutations in PPM1D (also known as WIP1) which encodes for a phosphatase that negatively regulates the DNA damage response (DDR) and cell cycle checkpoint pathways. In DMGs, truncating mutations in PPM1D stabilize the protein and enhance phosphatase activity, thereby promoting tumor growth. Specific inhibition of PPM1D with tool compound GSK2830371 induces cell death in glioma cells with PPM1D mutations. However, the potential dephosphorylation and downstream phosphorylation targets of PPM1D are numerous and occur on multiple sites across many proteins, making it difficult to identify which phosphosites are critical for DMG growth. Using mass spectrometry, our lab previously identified 797 phosphosites in a PPM1D-mutated patient-derived cell line (BT869) that were differentially phosphorylated following treatment with GSK283037. Yet, only a small number of these sites have been functionally evaluated. Consistent with earlier findings, enrichment analysis of post-translational modifications revealed that the top significantly altered phosphosites are associated with the DDR pathway (FDR 0. 05). Therefore, we hypothesize that PPM1D-dependent dephosphorylation of a limited number of DDR-related phosphosites is essential for DMG growth and treatment resistance by enabling cell cycle progression, inhibiting apoptosis, and increasing tolerance to replication stress. To address this hypothesis, we will leverage CRISPR-based homology- directed repair (HDR) technology to generate BT869 cell lines with specific phosphosite mutations that prevent phosphorylation at candidate DDR-related residues identified in our previous phosphoproteomic screen. We will assess the functional relevance of each candidate phosphosite in promoting cell proliferation and survival using live-cell imaging. Cells will be cultured in the presence and absence of GSK2830371 to determine whether the phosphosite mutation can rescue PPM1D inhibition. Effects on cell cycle progression, apoptosis, and replication stress will be evaluated using flow-cytometry, immunofluorescence, and western blotting. We will also determine the contribution of individual phosphosites to resistance against genotoxic therapies such as radiation and cisplatin using the same approaches. This work will enhance our understanding of the regulation of the DDR pathway and may reveal novel therapeutic vulnerabilities not only for DMGs with PPM1D mutations but also for other PPM1D-driven cancers. Citation Format: Ria Kedia, Timothy H. Chang, Rameen Beroukhim, Pratiti Bandopadhayay. Defining essential PPM1D-regulated DNA damage response phosphosites in diffuse midline gliomas abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr B041.