Mentor Name: Zachary Reitman

Diffuse midline glioma (DMG) is a leading cause of pediatric brain tumor mortality. Currently, radiation therapy remains a standard treatment option. However, it only offers a transient delay in disease progression. Up to 25% of DMGs harbor truncating mutations in Mn2+/Mg2+-dependent protein phosphatase 1D (PPM1D), yielding a hyperactive protein that suppresses DNA damage response (DDR). Notably, these mutations co-occur with the defining H3K27M oncohistone. Further, PPM1D mutations are mutually exclusive with tumor suppressor p53 mutations. This pattern suggests a reliance on weakening p53-mediated checkpoint control but also on H3K27M-driven chromatin and transcriptional reprogramming for tumorigenesis. However, how these mutations interact remains unknown. This summer, I will investigate how H3K27M-driven chromatin dysregulation interacts with PPM1D truncation across defined p53 states to determine whether they act cooperatively or redundantly in DMG formation and treatment response. I hypothesize that H3K27M and PPM1D mutations affect replication stress and cell cycle checkpoint signaling in DMG, thereby promoting increased proliferation and tumorigenic activity in a p53-dependent manner. To test this hypothesis, I will utilize the Ppm1d-flex6 mouse model, which allows for the conditional expression of C-terminally truncated Ppm1d in mice. By interbreeding these with H3f3a-LSL-K27M mice, I will generate a model system that recapitulates the co-occurrence of these mutations in DMG. Following genotyping and phenotypic characterization, I will perform histological and immunohistochemical analyses on collected mouse brain tumor tissue. By comparing tumor-free survival across genotypes, I will determine if Ppm1d truncation alters the tumor phenotype within the H3.3K27M background. In parallel, I will perform complementary in vitro studies using mouse embryonic fibroblasts derived from Ppm1d-flex6 mice and isogenic human DMG cell lines (truncated PPM1D ± H3K27M). I will quantify cell proliferation and DDR marker expression (via immunoblotting) at baseline and following treatment with ionizing radiation or DDR inhibitors. Ultimately, this project will determine if the PPM1D-H3K27M axis synergistically drives replication stress and DDR suppression, yielding distinct patterns of DDR pathway dependence, radiosensitivity, and response to DDR inhibitors that are not seen with either lesion alone. These results will guide rational combinations of radiation and DDR-targeted therapies, clarify whether PPM1D/H3K27M status supports genotype-directed precision trials versus broader DDR-based approaches in DMG, and inform stratification of PPM1D tumors.