Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas

Abstract

Gliomas arising in the brainstem and thalamus are devastating tumors that are difficult to surgically resect. To determine the genetic and epigenetic landscape of these tumors, we performed exomic sequencing of 14 brainstem gliomas (BSGs) and 12 thalamic gliomas. We also performed targeted mutational analysis of an additional 24 such tumors and genome-wide methylation profiling of 45 gliomas. This study led to the discovery of tumor-specific mutations in PPM1D, encoding wild-type p53-induced protein phosphatase 1D (WIP1), in 37.5% of the BSGs that harbored hallmark H3F3A mutations encoding p.Lys27Met substitutions. PPM1D mutations were mutually exclusive with TP53 mutations in BSG and attenuated p53 activation in vitro. PPM1D mutations were truncating alterations in exon 6 that enhanced the ability of PPM1D to suppress the activation of the DNA damage response checkpoint protein CHK2. These results define PPM1D as a frequent target of somatic mutation and as a potential therapeutic target in brainstem gliomas.

Figure 1

Exome sequencing results for BSG and thalamic gliomas. (a) The frequency of mutations per case. A: Astrocytoma; O: Oligodendroglioma; OA: Oligoastrocytoma; GBM: Glioblastoma (b) The overview for mutation status of genes mutated in at least 2 cases. No deletions were detected in these genes. (c) The frequency of mutation per gene.

Figure 2

Mutation status of TP53, PPM1D, H3F3A, IDH1 in the extended series. Mutational status as determined by Sanger sequencing is shown for BSGs (n=33) and thalamic gliomas (n=17).

Figure 3

Landscape of PPM1D mutations and IHC staining of PPM1D in tumor samples. (a) All mutations occurred in the C-terminal portion of PPM1D, leaving the phosphatase domain untouched. All mutations were identified in BSGs, except E472X which was identified in a cerebral glioma. (b) Tumors with or without PPM1D mutations were selected for IHC staining with an N-terminal anti-PPM1D antibody. PPM1D-WT (case ID); PPM1D-mutant (case ID, mutation type). Scale bar, 100 µm.

Figure 4

Heatmap of variant methylation probes in 45 glioma samples, with genetic information. Forty-five glioma samples (columns) and top 2% variant probes (rows) are arranged by unsupervised average-linkage hierarchical clustering. IDH1 mutated, H3F3A (K27)-mutated, and WT samples clustered separately by unsupervised average-linkage hierarchical clustering. Within the IDH1 mutated subcluster, two hemizygous IDH1 mutation samples described previously were included (marked as light green in the status of IDH1), which showed distinct methylation patterns compared with other IDH1-mutated samples. Gene expression-based classification into proneural, neural, classical, and mesenchymal subtypes by TCGA classification is shown for samples for which material was available for gene expression analysis.

Figure 5

The effect of cancer-derived truncated PPM1D on Chk2, p53, and H2AX activation and cell growth. (a) HEK293T cells transfected with full length wild type, E472X truncated PPM1D, E540X truncated PPM1D, and D314A PPM1D were exposed to ionizing radiation (IR, 10Gy). Cells were collected before IR and 1 h and 5 h after IR. Whole cell lysates were analyzed for Chk2 Thr68 phosphorylation, p53 Ser15 phosphorylation, γ-H2AX, and GAPDH. Western blot against PPM1D showed successful transfection. (b) Gene targeting to repair a native PPM1D mutation. HCT116 cells contain a heterozygous frameshift deletion that results in a truncated PPM1D mutant (L450X). Homologous recombination was used to replace the mutated PPM1D allele with wild type PPM1D in two HCT116 sublines. Western blot demonstrates expression of full length and truncated PPM1D in the parental cell line, but only full length PPM1D expression in the two repaired cell lines. PPM1D-F: Full length PPM1D. PPM1D-T: Truncated PPM1D. (c) HCT116 parental (PPM1D mutant) and two isogenic repaired lines (PPM1D wild type) were exposed to ionizing radiation (IR, 10 Gy). Cells were collected before IR and 1 h and 5 h after IR. Whole cell lysates were analyzed for Chk2 Thr68 phosphorylation, p53 Ser15 phosphorylation, γ-H2AX, and GAPDH. (d) Proliferation assay for HCT116 parental cell line (PPM1D mutant) and isogenic repaired lines (PPM1D wild type). 1000 cells were seeded in each well (triplicates) of 96-well plates on day 0. Relative cell number was determined each day after seeding. Error bars represent mean ± SEM. (e) Colony formation by PPM1D-mutant HCT116 parental cell line and isogenic wild type PPM1D lines. Colony formation was assessed in the absence of IR, and under 2 Gy, or 4 Gy IR. P < 0.05 (*) between parental cells and isogenic repaired line 1 or 2 under 0 Gy, 2 Gy, and 4 Gy IR. n=4, error bars represent mean ± SEM.