PPM1D Mutations Silence NAPRT Gene Expression and Confer NAMPT Inhibitor Sensitivity in Glioma
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ABSTRACT:
PROVIDER: PRJNA554122 | ENA |
REPOSITORIES: ENA
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Project description:Genome wide DNA methylation profiling of wild type and PPM1D mutant astrocytes and patient-derived DIPG cell lines. The Illumina Human EPIC Bead Array (850k) platform was used to obtain methylation beta values of over 850k CpG sites throughout the genome across a panel of DIPG cell lines.
2019-07-12 | GSE134165 | GEO
Project description:Pediatric high-grade gliomas are among the deadliest of childhood cancers due to limited knowledge of early driving events in their gliomagenesis and the lack of effective therapies available. In this study, we investigate the oncogenic role of PPM1D, a protein phosphatase often found truncated in pediatric gliomas such as DIPG, and uncover a synthetic lethal interaction between PPM1D mutations and nicotinamide phosphoribosyltransferase (NAMPT) inhibition. Specifically, we show that mutant PPM1D drives hypermethylation of CpG islands throughout the genome and promotes epigenetic silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key gene involved in NAD biosynthesis. Notably, PPM1D mutant cells are shown to be sensitive to NAMPT inhibitors in vitro and in vivo, within both engineered isogenic astrocytes and primary patient-derived model systems, suggesting the possible application of NAMPT inhibitors for the treatment of pediatric gliomas. Overall, our results reveal a promising approach for the targeting of PPM1D mutant tumors, and define a critical link between oncogenic driver mutations and NAD metabolism, which can be exploited for tumor-specific cell killing.
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Project description:The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genomes sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of DMGs and are enriched in primary pontine tumors. Through the development of novel DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D activity is required for in vivo oncogenesis. Finally, we applied integrative phosphoproteomic and functional genomics assays and found that the oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition. Together, these findings highlight PPM1D mutations to represent a targetable driver of pediatric gliomas.
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Project description:Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.
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Project description:The majority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms. Our study show that frequent gain of the p53-regulating gene PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. A PPM1D transgenic mouse model develops cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1- and Pten-mutations, adenocarcinomas and PHOX2B-expressing neuroblastomas, therey establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice, proposing WIP1 as a therapeutic target in neural childhood tumors.
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Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. RNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. RNA sequencing libraries were prepared using KAPA RNA HyperPrep Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >120 for RNA samples, respectively. RNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129). All parts of RNAseq data analysis were conducted in R, version 4.3.2. and RNAseq read counts were normalized using R package DESeq2. Fold change (FC) and log2FC were calculated from normalized reads, nontransformed RPE-PPM1D-T1 cells were considered a reference. Significance of differential expression for each gene was evaluated by Fisher's t-test with simulated p-values and Holm's p-value correction for multiple comparisons.
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