Abstract B018: PPM1D- versus TP53-altered H3K27M-altered diffuse midline gliomas define distinct tumor states with isogenic modeling of Ppm1d-driven replication stress

Abstract

H3K27M-altered diffuse midline glioma (DMG) is an aggressive pediatric brain tumor for which radiation is standard of care but only transiently delays progression. The oncogenic phosphatase PPM1D and the tumor suppressor TP53 both regulate DNA-damage-response (DDR) circuitry and are frequently mutated in H3K27M-altered DMG in a mutually exclusive manner, yet whether PPM1D- and TP53-altered tumors represent distinct states is incompletely defined. We analyzed newly diagnosed OpenPedCan H3K27M-altered DMGs by RNA-seq (n=112): PPM1D-altered (truncating PPM1D alterations after residue 370; n=16), TP53-altered (coding TP53 alterations; n=73), and neither (no qualifying alteration; n=23). Differential expression used DESeq2 Wald tests; Hallmark GSEA used DESeq2 Wald-statistic ranks. WGS single-base (SBS) and indel (ID) mutational signatures were evaluated in the same groups (n=89; 14/57/18) using COSMIC/SigProfiler. As an isogenic model, mouse embryonic fibroblasts (MEFs) expressing DMG-derived truncated versus wildtype Ppm1d (endogenous conditional flex-6 allele; SV40-immortalized, AdCre-induced; n=3/group) were analyzed by RNA-seq and S9.6-RPA proximity ligation assay (PLA). DESeq2 identified 699, 57, and 1,211 differentially expressed genes (FDR<0.05) in the PPM1D-vs-TP53, PPM1D-vs-neither, and TP53-vs-neither contrasts, respectively. Hallmark GSEA across all three contrasts distinguished group-specific from comparator-driven effects: proliferation/cell cycle was the distinct program enriched in PPM1D-altered tumors relative to both other groups (G2M checkpoint, E2F targets), whereas an interferon/inflammatory program was uniquely elevated in TP53-altered tumors (interferon-alpha/gamma, TNFA/NF-kB, IL6/JAK/STAT3). Leading-edge analysis supported this separation, with PPM1D-side cell-cycle/DDR-associated genes (H2AX, BRCA2, CDK1, CHEK1) and TP53-side interferon/cytokine-associated genes (STAT1, IL6, IRF1, CXCL9/10, TNFAIP3). In the direct PPM1D-vs-TP53 comparison, PPM1D-altered tumors were additionally enriched for OXPHOS, MYC-target, and DNA-repair pathways, whereas TP53-altered tumors showed EMT and inflammatory/TNFA-NF-kB enrichment. After modality-specific reconstruction (observed-vs-COSMIC cosine ≥0.90), SBS signatures were retained for 89/89 WGS tumors and ID signatures for 80/89 tumors; APOBEC-associated SBS2/SBS13 were notably TP53-skewed relative to PPM1D-altered and neither tumors. Isogenic Ppm1d truncation in MEFs recapitulated the human PPM1D proliferation/MYC axis (MYC, OXPHOS, E2F/G2M cell-cycle) and significantly increased S9.6-RPA PLA foci per nucleus versus controls, consistent with R-loop-associated replication stress. Together, PPM1D- and TP53-altered H3K27M-altered DMGs occupy distinct transcriptomic states within shared p53/DDR circuitry: a PPM1D-associated proliferative/cell-cycle state and a TP53-associated interferon/inflammatory state, with Ppm1d truncation sufficient to induce the MYC/proliferative program and a replication-stress phenotype in the MEF model, which may guide genotype-specific therapeutic vulnerabilities.