Impact of Ppm1d Truncation on the Transcriptome of Mouse Embryonic Fibroblasts

Diffuse midline gliomas (DMGs) are incurable brain tumors with an urgent need for new therapeutic strategies. Approximately 20% of DMGs harbor truncating mutations in exon 6 of PPM1D, which stabilize the protein and deregulate p53 signaling. However, the consequences of these mutations for tumor initiation, progression, and therapeutic response remain poorly defined. We developed a conditional Ppm1d-loxP-exon6-loxP-exon6-E518X-tag allele (Ppm1d-flex-6) that enables lineage-, spatial- and temporal-specific expression of a DMG- derived truncated Ppm1d protein from the endogenous locus in the presence of Cre recombinase. Ubiquitous embryonic expression was modeled using the Meox2-Cre driver, and primary gliomas were modeled using the RCAS/tv-a retrovirus system to introduce Cre and PDGFB co- drivers into Nestin-positive neural stem cells. Complementary studies were performed in mouse embryonic fibroblasts (MEFs) expressing truncated Ppm1d following Cre recombination. While Meox2-Cre–driven ubiquitous expression produced muted phenotypes, Ppm1d- flex-6 accelerated gliomagenesis in combination with PDGF co drivers. Its oncogenic effect was weaker than complete p53 loss, and it did not enhance tumorigenesis further in p53-null tumors. Single-cell RNA-sequencing revealed that Ppm1d-flex-6 gliomas adopt more progenitor-like transcriptional states and upregulate p53- and cell cycle–associated pathways. In MEFs, Ppm1d- flex-6 enhanced proliferation and shifted transcriptomic programs toward MAPK, PI3K-Akt, and Rap1 signaling, while impairing DNA damage responses, including reduced γ-H2AX induction after irradiation. These defects sensitized cells to radiation and decreased clonogenic survival after IR or PARP inhibition, with the strongest effects in combination. We established a versatile, genetically faithful Ppm1d-flex-6 allele to model DMG-derived Ppm1d mutations in time, lineage, and space. Our findings indicate that Ppm1d mutations confer intermediate suppression of the p53 pathway, consistent with the clinical features of Ppm1d-mutant DMGs and are associated with radiosensitivity and PARP inhibitor vulnerability. This system provides new insights into PPM1D biology, transcriptomic and tumor hierarchy perturbations, and therapeutic opportunities.