Generation of Diffuse Midline Glioma-Derived Oncogenic Mutations in Primary Mouse Models

Abstract

Diffuse midline gliomas (DMGs) are aggressive, inoperable pediatric brain tumors that arise within the central nervous system. These tumors are defined by distinct genetic alterations that fundamentally reprogram DNA damage repair pathways. In an aim to better understand DMG biology and identify improved therapeutic strategies, we generated novel mouse models that faithfully recapitulate human DMG. DMGs are frequently characterized by p53-inactivating mutations and oncohistone mutations in the histone H3.3 that rewire the cellular epigenome to promote carcinogenesis. Using the RCAS/tv-a retroviral system, we engineered a mouse model to combine p53 inactivation with a H3.3K27M mutation. We then performed longitudinal survival studies in conjunction with pharmacologic inhibition, molecular profiling, and pathway-level analyses. Our results demonstrate that disruption of the ataxia-telangiectasia mutated (ATM) kinase, an important potential therapeutic target, enhances the efficacy of radiation therapy (RT) in both murine and patient-derived DMG models. These findings provide a strong rationale for therapeutic strategies in which radiation therapy is selectively optimized in p53-deficient DMGs through concurrent disruption of ATM signaling. DMGs are also known to harbor activating truncating mutations in the oncogenic phosphatase Ppm1d. We genetically engineered a mouse model to conditionally express a truncated Ppm1d allele (Ppm1d-flex-6) in a lineage-, spatial-, and temporal-specific manner. This Ppm1d-flex-6 allele was crossed with a Meox2-Cre driver to induce whole-body truncation of Ppm1d. Subsequently, the RCAS/tv-a system was used to deliver Cre recombinase and PDGFB into Nestin-positive neural stem cells, thereby restricting Ppm1d truncation to the brain. Complementary in vitro studies were conducted using mouse embryonic fibroblasts (MEFs) derived from Ppm1d-flex-6 mice to further elucidate the role of Ppm1d in tumorigenesis within a DMG-relevant context. Our findings reveal that truncated Ppm1d negatively regulates cellular proliferation, cell cycle progression, and the DNA damage response. Notably, PARP inhibition reduced colony formation in irradiated cells expressing truncated Ppm1d, highlighting a potential combinatorial therapeutic vulnerability. Collectively, these results establish Ppm1d as a critical modulator of DNA damage signaling in DMG and support the development of rational combination therapies targeting DNA repair pathways.