Fig 2: PPM1D is a dependency in PPM1Dtr but not PPM1D-WT cell lines.A Fold change in proliferation compared to day 0 of PPM1D-mutant patient-derived cell line (PDCL) BT869 after transfection with PPM1D-KO sgRNAs, nontargeting (LacZ) sgRNA, or lethal (EXOSC8) sgRNA. Growth curves show mean ± SEM from three replicates and are representative of three independent experiments. P = 0.0021, 0.0011, and 0.0016 for PPM1D-KO #1, PPM1D-KO #2, and EXOSC8 respectively calculated using two-tailed t-test. B Growth of PPM1D-WT PDCL SU-DIPG-IV after transfection with indicated sgRNAs. Growth curves show mean ± SEM from three replicates and are representative of three independent experiments. P = 0.8963, 0.5524, and 0.0335 for PPM1D-KO #1, PPM1D-KO #2, and EXOSC8 respectively calculated using two-tailed t-test. C Probability of PPM1D dependency as determined by pooled CRISPR-cas9 assays across 558 cancer cell lines, subgrouped according to their TP53 mutation and PPM1D copy-number status. Bounds of the box represent the IQR, center represents the median, and the bounds of the whiskers represent 1.5 times IQR. P < 0.0001 for TP53 WT/PPM1D non-amplified cells vs both TP53-mutant/PPM1D-amplified and TP53-mutant/PPM1D non-amplified cells and P < 0.01 for TP53 WT/PPM1D non-amplified cells vs TP53 WT/PPM1D-amplified cells using Kruskal–Wallis test. D Growth of PPM1D-mutant colon cell line HCT116 after transfection with indicated sgRNAs. Growth curves show mean ± SEM from three replicates and are representative of three independent experiments. P = 0.0015, 0.0010, and 0.0059 for PPM1D-KO #1, PPM1D-KO #2, and EXOSC8 respectively calculated using two-tailed t-test. E–F Growth of PPM1D-mutant DMG cell lines BT869 (E) and SF7761 (F) after treatment with vehicle control (NT), 10 µM of GSK2830371 (PPM1Di), 2 Gy of ionizing radiation treatment (RT), or the combination of both (GSK + RT). Growth curves show mean ± SEM from at least three replicates and are representative of three independent experiments. BT869: P = 4.90083E-06, 2.18655E-07, 3.83657E-11, 2.8877E-11, and 1.1625E-06 for NT vs PPM1Di, NT vs RT, NT vs PPM1Di + RT, RT vs PPM1Di + RT, and PPM1Di vs PPM1D + RT respectively calculated using two-tailed t-test; SF7761: P = 0.003367278, 1.17616E-05, 5.59401E-08, 1.5694E-08, and 2.698E-05 for NT vs PPM1Di, NT vs RT, NT vs PPM1Di + RT, RT vs PPM1Di + RT, and PPM1Di vs PPM1D + RT respectively calculated using two-tailed t-test. Source data are provided as a Source Data file.

Fig 3: PPM1D suppresses apoptosis, DDR, and p53 pathways.Apoptosis and cell cycle responses were assessed in isogenic mouse neural stem cells (mNSCs) overexpressing H3F3A K27M (H3K27M) mutation plus either eGFP (vector control), full-length PPM1D (PPM1D FL), or C-terminus truncated PPM1D (PPM1Dtr) (A–B). A Cells were treated with 8 Gy of ionizing radiation (RT) or no treatment (NT) for 24 h, incubated with Annexin V-APC and Propidium Iodide (PI), and analyzed using flow cytometry. The total percentage of Annexin V positive cells representing both early and late apoptotic cells are shown. Data presented as mean ± SEM from three biological replicates. P = 0.027, 0.318, 0.044, 0.037 for GFP vs PPM1Dtr (NT), GFP vs PPM1D FL (NT), GFP vs PPM1Dtr (RT) and GFP vs PPM1D-FL (RT) conditions respectively calculated using two-tailed t-test. B Cells were treated with 8 Gy RT for 24 h and integration of BrdU was assessed to determine the percentage of cells in S-phase. Anti-BrdU APC and 7-AAD DNA staining were used to distinguish cells in each stage of the cell cycle. Data presented as mean ± SEM from three biological replicates. P = 0.276, 0.090 0.008, 0.005, 0.543, 0.848 for GFP vs PPM1D FL (G0/G1) GFP vs PPM1Dtr (G0/1), GFP vs PPM1D FL (S) GFP vs PPM1Dtr (S), GFP vs PPM1D FL (G2/M) GFP vs PPM1Dtr (G2/M) conditions respectively calculated using two-tailed t-test. C Cells were treated with 8 Gy of RT and lysates were collected at baseline (NT), 1 and 5 h post-RT respectively, and probed with the indicated antibodies. Three independent experiments were performed with similar results. D PPM1D-mutant patient-derived DMG cell lines BT869 and SF7761 were treated with 10 µM of GSK2830371 (PPM1Di) and/or 8 Gy of RT for 1 and 5 h, lysed and probed with indicated antibodies. Three independent experiments were performed with similar results. E Volcano plot showing differentially expressed genes in BT869 and SF7761 DMG cells (n = 3 per cell line per condition) treated with 10 µM of GSK2830371 (PPM1Di) for 5 h compared to vehicle treatment. Genes in the p53 signaling pathway are labeled. F GSEA enrichment plots of two p53 related pathways significantly enriched (FDR < 0.25) after inhibition of PPM1D in BT869 and SF7761 cells. Source data are provided as a Source Data file.

Fig 4: Integrative analysis of PPM1D-mutant DMG models.Schematic depicting the overall method and findings of the study. Top panel: an array of models used in the study including biopsy of DMG tumors, de novo mouse models of PPM1D driven DMG using in utero electroporation, and in vitro models including patient-derived cell lines. Middle panel: integrative approach employing whole-genome sequencing of patient tumors, gene expression analysis, proteomics, and phosphoproteomics screens, and CRISPR screens used to study the function and mechanism of PPM1D mutation in DMG. Bottom panel: convergence of the oncogenic function of PPM1Dtr on p53, cell cycle and DDR pathways using the integrative approach in the middle panel.

Fig 5: Reduced viability of glioblastoma cells was simultaneously observed with increased mRNA expression of PUMA, Bax, Apaf1 and p21 in MPT0B291-treated glioma cells. (A) Anti-tumor activities were evaluated using MTT assay comparing with SAHA and TMZ at 24, 48 and 72 h (See also Figure 2A). (B). The relative mRNA expression levels of caspase-3 in C6 cells with or without treatment are presented as mean ± SEM (n=3 in each group) (C) Representative images of Western blot analysis for PUMA, Bax and Apaf1. The relative protein levels are presented as mean ± SEM (n=4 in each group). The relative mRNA expression levels of PUMA (D), Bax (E), Apaf1 (F) and p21 (G) in vehicle- and MPT0B291-treated groups are presented as mean ± SEM (n=3 in each group) *p<0.05 versus the vehicle-treated group. (H) Schematic diagram of mechanistic studies. MPT0B291 inhibited HDAC activity leading to partially increased acetylation/activation of p53 as well as phosphorylation of p53, which in turn results in the induction of cell death, cell cycle arrest as well as a reduction in proliferation.