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. 2019 Mar 10;21(6):786–799. doi: 10.1093/neuonc/noz053

Inhibition of mutant PPM1D enhances DNA damage response and growth suppressive effects of ionizing radiation in diffuse intrinsic pontine glioma

Mwangala Precious Akamandisa 1,2,3,1, Kai Nie 2,3,1, Rita Nahta 4,5,6, Dolores Hambardzumyan 1,2,3,6, Robert Craig Castellino 2,3,6,✉
PMCID: PMC6556861  PMID: 30852603

Abstract

Background

Children with diffuse intrinsic pontine glioma (DIPG) succumb to disease within 2 years of diagnosis despite treatment with ionizing radiation (IR) and/or chemotherapy. Our aim was to determine the role of protein phosphatase, magnesium-dependent 1, delta (PPM1D) mutation, present in up to 25% of cases, in DIPG pathogenesis and treatment.

Methods

Using genetic and pharmacologic approaches, we assayed effects of PPM1D mutation on DIPG growth and murine survival. We assayed effects of targeting mutated PPM1D alone or with IR on signaling, cell cycle, proliferation, and apoptosis in patient-derived DIPG cells in vitro, in organotypic brain slices, and in vivo.

Results

PPM1D-mutated DIPG cell lines exhibited increased proliferation in vitro and in vivo, conferring reduced survival in orthotopically xenografted mice, through stabilization of truncated PPM1D protein and inactivation of DNA damage response (DDR) effectors p53 and H2A.X. PPM1D knockdown or treatment with PPM1D inhibitors suppressed growth of PPM1D-mutated DIPGs in vitro. Orthotopic xenografting of PPM1D short hairpin RNA–transduced or PPM1D inhibitor–treated, PPM1D-mutated DIPG cells into immunodeficient mice resulted in reduced tumor proliferation, increased apoptosis, and extended mouse survival. PPM1D inhibition had similar effects to IR alone on DIPG growth inhibition and augmented the anti-proliferative and pro-apoptotic effects of IR in PPM1D-mutated DIPG models.

Conclusions

PPM1D mutations inactivate DDR and promote DIPG growth. Treatment with PPM1D inhibitors activated DDR pathways and enhanced the anti-proliferative and pro-apoptotic effects of IR in DIPG models. Our results support continued development of PPM1D inhibitors for phase I/II trials in children with DIPG.

Keywords: DIPG, DNA damage response, GSK2830371, PPM1D, WIP1

Key Points.

  1. PPM1D mutant DIPGs exhibit increased growth and reduced survival in xenografts.

  2. PPM1D knockdown or inhibition extends survival of PPM1D-mutated DIPG xenografts.

  3. PPM1D inhibition augments radiation growth suppression in PPM1D-mutated DIPGs.

Importance of the Study.

DIPG is an aggressive brainstem glioma that is currently incurable. Recent studies identified novel mutations of PPM1D in up to 25% of DIPGs. We demonstrate that PPM1D mutation promotes DIPG through PPM1D protein stabilization and inactivation of DDR. Treatment with a PPM1D small-molecule inhibitor activates DDR pathways and enhances the anti-proliferative and pro-apoptotic effects of ionizing radiation in preclinical models of DIPG. Given that PPM1D mutations are linked to predisposition to breast and ovarian cancers, as well as some secondary leukemias, our findings have broader relevance than only to DIPG. Our findings also provide strong rationale for continued investigation of inhibition of mutant PPM1D, with the eventual goal of advancing a PPM1D inhibitor into phase I/II clinical trials in PPM1D-mutated malignancies, including DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brainstem glioma that is incurable by surgical resection or chemotherapy. Although ionizing radiation (IR) offers palliative benefit, most children succumb to disease within a year of diagnosis.1–3 Thus, DIPG carries the highest mortality rate of all pediatric brain tumors. Improved outcomes and survival demand an improved understanding of DIPG pathobiology, identification of novel molecular targets, and new treatment approaches.

Recent publications have identified somatic mutations, including those in TP53, ACVR1, PIK3CA, and protein phosphatase, magnesium-dependent 1, delta (PPM1D), hypothesized to drive DIPG tumorigenesis.4–8TP53 is mutated in 40–70% of DIPGs,4 and preclinical evidence suggests that TP53 mutation cooperates with other characteristic mutations, such as those in the histone gene H3F3A and/or PDGFRA, to drive DIPG tumor formation.9,10 Mechanisms whereby other mutations promote DIPG tumorigenesis are less clear.

Overexpressed or amplified PPM1D, identified in breast and ovarian carcinomas, and subsequently in pediatric neuroblastoma and medulloblastoma, cooperates with other oncogenes to promote tumor growth.11–15 Dephosphorylation of activating serine or threonine residues on p53 itself, on ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase (CHK) 1 and 2 upstream of p53, and on murine double minute 2 (MDM2)/MDMX downstream of p53 are known PPM1D-associated mechanisms of tumorigenesis.16,17

PPM1D mutations exist in a variety of malignancies, including DIPG. Mosaic PPM1D mutations, resulting in protein-truncating variants, were first identified in breast and ovarian cancers. They conferred increased PPM1D activity, with suppressed phosphorylation of the PPM1D targets p53 and H2A.X in response to IR.18 A subsequent publication in osteosarcoma and colon cancer cells confirmed the hyperactive phenotype of PPM1D mutants and demonstrated increased stability of the mutant PPM1D.19 Recent studies identified PPM1D mutations in up to 25% of DIPGs.5,6,8,20 Most DIPGs that harbor a PPM1D mutation are distinct from TP53 mutant tumors.5 One publication suggested that the DIPG-characteristic mutation in the histone modifier, H3F3A, to H3K27M is usually followed by a mutation in TP53 or PPM1D.5PPM1D mutations have also been associated with chemoresistance in myeloid cancers.21 This suggests the importance of understanding mechanisms of PPM1D mutation in DIPG pathogenesis and treatment responsiveness.

We found that stable transduction of clinically relevant PPM1D mutations into murine and patient-derived DIPG cells increased proliferation and impaired survival of mice. PPM1D mutants inactivated DNA damage response effectors p53 and H2A.X, similar to prior reports in PPM1D-amplified medulloblastomas.12,22 Conversely, PPM1D knockdown suppressed proliferation and extended the survival of mice xenografted with PPM1D-mutated DIPG cells. PPM1D inhibitors suppressed the growth of PPM1D-mutated DIPGs in neurospheres, immunocompetent organotypic brain slices, and organotypic brain slices derived from symptomatic, orthotopically xenografted mice. PPM1D-mutated DIPGs treated with a PPM1D inhibitor exhibited reduced proliferation, increased apoptosis, and extended survival in vivo. PPM1D inhibition had similar effects on DIPG growth inhibition as IR alone, and augmented anti-proliferative and pro-apoptotic effects in combination with IR.

Materials and Methods

Cell Culture and Maintenance

SU-DIPG VI, HSJD-DIPG 007 (DIPG7), and CNMC-XD-625 cells were authenticated by short tandem repeat DNA profiling. Murine DIPG cells were derived from symptomatic Nestin/Tva; Ink4a/Arf−/−, RCAS-PDGFB–transduced mice. Cells were maintained in tumor stem medium. See Supplementary Methods for details.

Gene Sequencing

Sequencing was by Genewiz, analyzed using SnapGene v3.3.4 (GSL Biotech). See Supplementary Methods for details.

Lentiviral Particle Production and Infection

Green fluorescent protein (GFP)–PPM1D precision lentiORF expression constructs (ThermoFisher Scientific) were modified in the Emory Custom Cloning Core to encode a PPM1D mutation: L513*, T483, C478*, or S468*. Short hairpin (sh)PPM1D constructs have been described.23 Production and transduction with lentiviral particles are as described.24 See Supplementary Methods for details.

Immunoblotting

Protein analysis was previously described.24 Immunoblotting was performed using PPM1D (Santa Cruz Biotechnology), p53 (Santa Cruz), phospho-p53 Ser15 (Cell Signaling), H2A.X (Cell Signaling), phospho-H2AX Ser139 (Cell Signaling), β-actin (Sigma-Aldrich), and 14-3-3 (Cell Signaling). Secondary antibodies were Alexa Fluor 680 goat α-rabbit (ThermoFisher), or IRDye 800CW goat α-mouse (LI-COR), imaged with Odyssey scanner (LI-COR).

Viability Assays

CellTiter-Glo 2.0 (Promega) and GloMax-multi detection system (Promega) were used according to manufacturer’s specifications.

Cell Cycle Analysis

Cells were treated with EdU (ethynyl-labeled deoxyuridine; ThermoFisher) and stained using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Kit (ThermoFisher) per manufacturer’s protocol. Alternatively, cells were stained with propidium iodide (Sigma-Aldrich), analyzed on a CytoFLEX flow cytometer (Beckman Coulter). Analysis was performed in FlowJo. See Supplementary Methods for details.

Apoptosis Analysis

Cells stained with fluorescein isothiocyanate/annexin V (BioLegend) and 7-aminoactinomycin D (BioLegend) were analyzed on a CytoFLEX cytometer. Data were analyzed with FlowJo.

Colony Formation

Cells were plated in Vitrogel (TheWell Bioscience) per manufacturer’s specifications. Images were captured using a Leica MZ10F dissecting microscope with a DFC 365FX digital camera and the Leica Application Suite–Advanced Fluorescence software package. Images were analyzed using CellProfiler.

Mouse Xenografting and Drug Treatment

DIPG7 cells suspended in tumor stem medium containing negative control (shNC) versus PPM1D shRNA (shPPM1D), or dimethyl sulfoxide versus 5 μM GSK2830371 were xenografted into the brainstem of post-natal day 0–2 (P0-2) NSG mice (The Jackson Laboratory). Mice were housed in a facility accredited by the American Association for Laboratory Animal Care and maintained per guidelines of the National Institutes of Health. Animal care and experiments were approved by Emory’s Institutional Animal Care and Use Committee (PROTO201800212).

Brain Tissue Handling, Immunofluorescence, and Quantitation

Following injection with bromodeoxyuridine (BrdU; BD Biosciences), mice were perfused with 4% paraformaldehyde (Santa Cruz). Following de-paraffinization, tissues were incubated with Ki67 (Cell Signaling), BrdU (BD Biosciences), and/or GFP (Cell Signaling), followed by α-rabbit Alexa Fluor 488 or goat α-mouse Alexa Fluor 594.

DIPG cells were seeded on coverslips, coated with Poly-D-Lysine (Sigma-Aldrich) and Geltrex (ThermoFisher). Following treatment, cells were fixed and incubated with α-Ki67, followed by Alexa Fluor 594 goat α-rabbit secondary. Coverslips were mounted with Vectashield with 4′,6′-diamidino-2-phenylindole (DAPI). Imaging was as above. See Supplementary Methods for details.

Tissue Slice Culture, Immunofluorescence, Confocal Imaging, and Quantification

Whole brains from P14 CD1 (Charles River Laboratories; Strain Code: 022) or symptomatic DIPG-xenografted NSG mice were embedded, sectioned using a VT1200 vibratome (Leica), and placed onto membrane inserts (ThermoFisher).

GFP-expressing DIPG7 neurospheres were implanted onto P14 CD1 organotypic brain tissue slices. GFP+ fluorescent area was imaged for 120 hours, as above.

Organotypic brain tissue slices from symptomatic DIPG-xenografted NSG mice were treated and incubated with GFP (Abcam) and Ki67, before secondary, as above. Imaging was as above. See Supplementary Methods for details.

Statistical Analysis

Results were analyzed using a 2-tailed Student’s t-test or 2-way ANOVA in GraphPad Prism 7.3. P < 0.05 was considered statistically significant.

Results

Mutant PPM1D Increases Viability of Murine and Human Patient-Derived DIPG

Using Sanger sequencing, we verified TP53, but not PPM1D mutations in DIPG VI cells. Sequencing primers spanned PPM1D exons 5–6, where most PPM1D mutations are localized.18 Conversely, both DIPG7 and CNMC-XD-675 cells were TP53 wild type, but harbored homozygous and heterozygous mutations, respectively in PPM1D (Fig. 1A, B).

Fig. 1.

Fig. 1

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Mutant PPM1D increases viability of DIPG cells. (A) Clinical characteristics and mutational status of frequently altered genes in patient-derived DIPGs. (B) Sequencing identified PPM1D mutation in DIPG7 (red arrow; yellow arrow, corresponding amino acid alteration), but not DIPG VI cells. (C) Murine DIPG cells from RCAS-PDGFB transduced Nestin/Tva; Ink4a/Arf−/− mice (left panel) or DIPG VI cells (right panel) stably transduced with control (EV) or PPM1D-mutated lentivirus and assayed for viability in 7 days using CellTiter-Glo. Y-axis, relative luminescence (n = 4 replicates/construct/experiment). Experiments repeated 3 times. Error bars, standard error of the mean (SEM). (D) Kaplan–Meier survival for NSG mice orthotopically xenografted at P0-2 with murine DIPG cells transduced with EV or PPM1D-L513* (left panel), or human DIPG cells (right panel). (E) Brainstems of symptomatic mice (D) were immunostained (left panels) for GFP (green), BrdU (red), and DAPI (blue). BrdU+/high-power field (HPF) was quantified (right panel; n = 3 mice/xenograft, 6 non-overlapping fields/tumor). Horizontal line, mean; whiskers, standard deviation (SD). Scale bar, 200 μm. ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001.

Since PPM1D mutations usually function in a dominant negative fashion to drive cancer cell growth,25 we stably transduced murine and human DIPG VI cells with lentiviral constructs expressing PPM1D mutations identified in human DIPG patient tissues: S468*, C478*, T483, or L513*. Transduction with PPM1D-mutant constructs increased viability of murine and patient-derived DIPG cells (Fig. 1C).

Carboxy-terminal-truncated PPM1D typically demonstrates increased protein stability and intact phosphatase activity in adult cancer cells.19 We found that cycloheximide treatment reduced expression of full-length PPM1D in DIPG VI cells, but not of truncated, mutated PPM1D in DIPG7 cells. In DIPG VI-PPM1D-L513* cells stably transduced with mutant PPM1D, truncated PPM1D exhibited increased stability compared with endogenous, full-length PPM1D (Supplementary Figure 1). This suggests that increased protein stability is one mechanism whereby PPM1D mutation promotes DIPG tumorigenesis.

In vivo, murine DIPG cells stably transduced with a PPM1D-mutated construct and orthotopically xenografted into neonatal NSG mice exhibited reduced survival (median, 108 ± 5 days) compared with empty vector-transduced cells (Fig. 1D, left panel). Similarly, patient-derived, PPM1D-mutated DIPG7 cells readily generated tumors (median survival, 53 ± 13 days). The median survival of DIPG VI–xenografted mice was significantly longer, 253 ± 37 days. Interestingly, when DIPG VI-PPM1D-L513* or DIPG VI-PPM1D-T483 stable cells were orthotopically xenografted into the brainstem at P0-2, mice became symptomatic at median 60 ± 17 and 89 ± 14 days, respectively (Fig. 1D, right panel). BrdU incorporation demonstrated increased proliferation of DIPG VI-PPM1D-L513* and DIPG VI-PPM1D-T483 xenografts compared with parental DIPG VI xenografts (Fig. 1E).

PPM1D Knockdown Significantly Inhibits Growth of PPM1D-Mutated DIPG

To verify the growth-promoting effects of PPM1D mutation, we transduced human DIPG cells harboring a de novo mutation or stably transduced with a mutated PPM1D construct with 2 unique PPM1D-targeting shRNAs. Lentiviral transduction reduced PPM1D protein expression by >60% (Supplementary Figure 2A) compared with negative control–transduced cells. PPM1D knockdown significantly reduced the viability of de novo PPM1D-mutated DIPG7 and CNMC-XD-625 cells. Interestingly, PPM1D knockdown also resulted in increased suppression of viability of DIPG VI-PPM1D-L513* cells versus parental DIPG VI cells (Supplementary Figure 2B).

In vivo, PPM1D knockdown suppressed tumor proliferation and improved the survival of NSG mice orthotopically xenografted with DIPG7 cells. Kaplan–Meier analysis identified a clear difference in survival of NSG mice xenografted with shPPM1D- versus shNC-transduced DIPG7 cells (Fig. 2A). Analysis of the brainstem of symptomatic mice demonstrated significant suppression of Ki67 and BrdU expression in shPPM1D- compared with shNC-transduced DIPG7 cells (Fig. 2B, C).

Fig. 2.

Fig. 2

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PPM1D knockdown suppresses proliferation of PPM1D-mutated DIPG cells in vivo. (A) DIPG7 cells stably transduced with a GFP-expressing construct transduced with negative control (shNC) or PPM1D-targeting shRNA (shPPM1D) were orthotopically xenografted into the brainstem of P0-2 NSG mice. Shown: Kaplan‒Meier survival. Immunostaining (left panels) for (B) Ki67 (red), and DAPI (blue), or (C) BrdU (red), and DAPI (blue) in brainstem of symptomatic mice. Ki67+/DAPI+ or BrdU+/DAPI+ nuclei were quantified, respectively (right panels). Experiments repeated twice. Error bars, SD. Scale bars, 50 μm. ****P < 0.0001.

PPM1D-Mutated DIPGs Exhibit Increased Sensitivity to PPM1D Inhibitors

Prior reports have shown that small-molecule inhibitors that target the phosphatase or flap domains of PPM1D suppress PPM1D function and tumor growth in cell culture and in vivo, including in subgroups of PPM1D-overexpressing medulloblastoma.22,26–29 A recent study demonstrated increased chemosensitivity of an acute myeloid leukemia cell line stably expressing mutant PPM1D in combination with a PPM1D inhibitor.21 We found that the PPM1D inhibitors CCT007093 and GSK2830371 suppressed PPM1D-mutated DIPG7 growth with half-maximal inhibitory concentrations of 19 μM and 4.6 μM, respectively (Supplementary Figure 3A, left panel). In comparison, the half-maximal inhibitory concentration for CCT007093 or GSK2830371 was >200 μM in PPM1D wild-type DIPG VI cells (Supplementary Figure 3A, right panel). In DIPG7 cells, GSK2830371 treatment increased phosphorylation at activating serines on the PPM1D targets p53 and H2A.X within 6 hours (Supplementary Figure 3B). GSK2830371 did not alter phosphorylation of p53 or H2A.X in DIPG VI cells (Supplementary Figure 3C). However, within 24 hours of treatment, GSK2830371 enhanced phosphorylation of H2A.X in DIPG VI-PPM1D-L513* cells (Supplementary Figure 3D). This suggests that PPM1D mutant protein sensitizes DIPG cells to the growth suppressive effects of PPM1D small-molecule inhibitors.

PPM1D Inhibition Suppresses Growth of PPM1D-Mutated DIPG Cells in Organotypic Brain Slice Cultures

Due to increasing recognition of important interactions between cancer cells and their microenvironment,30–32 we generated organotypic brain slice (OBS) cultures from immunocompetent CD1 and symptomatic NSG mice. Studies suggest similarities, such as the presence of stemlike cells, between the ventral brainstem of 6- to 7-year-old children, peak ages for DIPG presentation, and the ventral brainstem of P14-21 mice.33 Thus, we implanted GFP-stable DIPG7 neurospheres into the dorsal and ventral pons of OBS from P14 CD1 mice. The GFP+ area expanded significantly by 5 days following DIPG7 implantation in the dorsal and ventral pons (Fig. 3A), suggesting that murine immunocompetent OBS are capable of supporting human DIPG cell growth. However, while DIPGs may originate in the ventral pons,33 both the dorsal and ventral pons supported growth of established DIPG cells.

Fig. 3.

Fig. 3

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A small-molecule PPM1D inhibitor suppresses proliferation of PPM1D-mutated DIPG cells in OBS cultures. (A) Mid-line sagittal tissue section (left panel) of P14 CD1 brainstem. DIPG7-GFP neurospheres implanted into the dorsal (D) or ventral (V) brainstem (BS) of P14 CD1 OBS. Representative image (middle panel). CBL, cerebellum. GFP+ neurospheres imaged on day of implantation (day 0), and days 3 and 5 following implantation. GFP+ area quantified versus GFP+ on day 0 (right panel) (n = 6 replicates/location on brain slices/day). Scale bar, 300 μm. (B) DIPG7 neurosphere-implanted P14 CD1 OBS treated with 5 μM GSK2830371 for 48 hours and incubated with Ki67 antibody (left panels). Shown: percentage of Ki67+ (red)/nuclear DAPI+ (blue) cells/HPF (right panel) (n = 4 replicates/condition/experiment). Scale bar, 50 μm. (C, D) OBS from symptomatic NSG mice xenografted with DIPG7-GFP (C) cells or DIPG VI PPM1D-L513*-GFP stably transduced cells (DIPG VI-L513*-GFP) (D) were treated with vehicle (Vh) or 5 μM GSK2830371 (GSKi) for 48 hours and immunostained for GFP (green), Ki67 (red), and DAPI (blue) (left panels). Shown: Ki67+ cells/HPF (right panel) (n = 6 non-overlapping HPFs/slice, 3 slices/condition). Experiments repeated 3 times. Boxes, range; middle line, mean; error bars, SD. Scale bars, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

By Ki67 immunofluorescence (IF), PPM1D inhibition with GSK2830371 significantly reduced proliferation of DIPG7 cells embedded into P14 CD1 OBS (Fig. 3B). GSK2830371 also caused a dose-dependent reduction in proliferation of DIPG7 and DIPG VI-PPM1D-L513* cells in ex vivo OBS from symptomatic NSG mice (Fig. 3C, D).

PPM1D Inhibition Extends Survival of Mice Bearing PPM1D-Mutated DIPG Orthotopic Xenografts

In vivo efficacy of a PPM1D inhibitor has been demonstrated against subcutaneous flank xenografts of PPM1D-overexpressing cancers.27 However, no studies to date have demonstrated in vivo efficacy of PPM1D small-molecule inhibitors against mutant PPM1D-containing malignancies. We identified a clear difference in survival of NSG mice orthotopically xenografted with DIPG7 cells that were treated with GSK2830371 immediately before injection into the brainstem at P0-2 compared with vehicle-treated, xenografted controls (Fig. 4A). Analysis of the brainstem of symptomatic mice showed that GSK2830371 suppressed Ki67 and BrdU expression (Fig. 4B, C) and increased apoptosis (Fig. 4D) of GSK2830371-treated, DIPG7-xenografted cells.

Fig. 4.

Fig. 4

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PPM1D inhibition suppresses proliferation of PPM1D-mutated DIPG cells in vivo. (A) DIPG7-GFP cells treated with vehicle or 5 μM GSK2830371 were xenografted into the brainstem of P0-2 NSG mice. Shown: Kaplan–Meier survival. Immunostaining (left panels) for (B) GFP (green), Ki67 (red), and DAPI (blue), or (C) GFP (green), BrdU (red), and DAPI (blue) in the brainstem of symptomatic mice. Ki67+/DAPI+ or BrdU+/DAPI+ nuclei, respectively, were quantified (right panels). (D) The brainstem of vehicle and GSK2830371-treated mice was immunostained (left panels) for GFP (green), TUNEL (red), and DAPI (blue). TUNEL+/HPF was quantified (right panel) (n = 6 non-overlapping HPFs/tumor, 3 tumors/condition). Experiments repeated twice. Error bars, SD. Scale bars, 50 μm. ***P < 0.001, ****P < 0.0001.

Since PPM1D mutations are rare in TP53-mutated DIPG,5 one concern regarding PPM1D inhibition is that GSK2830371-treated DIPG cells may select for TP53 mutation. We did not sequence the TP53 locus in xenografts. However, since strong nuclear p53 expression is used as a surrogate for TP53 mutation, we used IF to assay TP53 status. IF showed extensive nuclear p53 expression in TP53-mutated DIPG VI, but sparse p53 expression in untreated DIPG7 orthotopic, xenografted DIPGs (Supplementary Figure 4A). IF did not show a difference in total or nuclear p53 expression in GSK2830371-treated versus vehicle-treated DIPG7 xenografts (Supplementary Figure 4B). This suggests that PPM1D inhibition does not promote acquisition of a TP53 mutation and is a viable treatment for PPM1D-mutated DIPGs.

PPM1D Inhibition Cooperates with Gamma Irradiation to Suppress Viability of DIPG

To date, the only effective treatment for DIPG is IR. Unfortunately, IR is not curative. We hypothesized that combining PPM1D inhibition with IR will increase antitumor effects. We treated DIPG VI and DIPG7 cells with the PPM1D inhibitors CCT007093, GSK2830371, and reactivation of p53 and induction of tumor cell apoptosis (RITA)34 ± IR. DIPG VI exhibited minimal responsiveness to PPM1D inhibition or IR, alone or in combination (Supplementary Figure 5A). Responsiveness to combination therapy with a PPM1D inhibitor and IR was most apparent 72 hours following exposure to combined treatments.

PPM1D-mutated DIPG7 was more sensitive to IR. As opposed to cancer cells that overexpress PPM1D, DIPG7 cells were relatively insensitive to CCT007093.22,24,28 At 72 hours, RITA alone resulted in greater growth suppression than IR alone in DIPG7 cells. Combined RITA and IR was similar in growth suppression to RITA alone. The growth suppressive effects on DIPG7 cells, at 72 hours, were similar for IR versus GSK2830371. However, combined GSK2830371 and IR significantly reduced growth of DIPG7 cells compared with either modality alone (Supplementary Figure 5B). We also observed a significant reduction in colony formation and cell viability when GSK2830371 was combined with IR (Supplementary Figure 5C).

To validate results from CellTiter-Glo assays, we treated DIPG cells suspended in basement membrane matrix with GSK2830371 ± IR. Treatment with either GSK2830371 or IR resulted in a similar level of suppression of proliferation of DIPG7 cells. Combined treatment with GSK2830371 and IR resulted in further reduction in DIPG7 proliferation, compared with either modality alone (Supplementary Figure 6A, B). In comparison, neither GSK2830371 nor IR significantly altered proliferation of DIPG VI cells (Supplementary Figure 6C, D). Analogous to our findings in DIPG7 cells, proliferation of DIPG VI-PPM1D-L513* cells was suppressed by either GSK2830371 or IR treatment alone. Combination therapy with GSK2830371 and IR further suppressed proliferation compared with IR alone (Supplementary Figure 6E, F).

Previous studies in PPM1D-amplified cancers demonstrated that PPM1D inhibition suppresses growth by enhancing phosphorylation and activation of PPM1D targets critical for DNA damage response (DDR), including p53 and H2A.X.16,17 DIPG7 cells treated with GSK2830371 and IR exhibited increased phosphorylation of p53 and H2A.X for up to 6 hours and 1 hour, respectively, compared with vehicle-treated, irradiated controls (Fig. 5A).

Fig. 5.

Fig. 5

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PPM1D inhibition and IR enhance PPM1D target activation in PPM1D-mutated DIPG cells. Expression of PPM1D targets p53 and H2A.X in (A) DIPG 7, (B) DIPG VI, and (C) DIPG VI-PPM1D-L513* cells treated with vehicle or 5 μM GSK2830371 (GSKi) and 10 Gy IR. Protein densitometry (right panels) shows expression, relative to loading control, β-actin, and vehicle-treated controls (n = 3 replicates/timepoint/condition). Experiments repeated 3 times. Error bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001.

GSK2830371 with IR did not alter phosphorylation of p53 or H2A.X in DIPG VI compared with controls (Fig. 5B). DIPG VI-PPM1D-L513* cells exhibited increased H2A.X phosphorylation by 1 hour after GSK2830371 and IR compared with controls (Fig. 5C). Thus, similar to PPM1D-amplified cancer cells, PPM1D-mutated DIPG cells demonstrate enhanced activation of DDR pathways in response to combined IR and PPM1D inhibition.

Combined PPM1D Inhibition and Gamma Irradiation Enhances Suppression of S Phase and Proliferation While Promoting Apoptosis of PPM1D-Mutated DIPG Cells

GSK2830371 caused an accumulation of DIPG7 cells in G0/G1 phase of the cell cycle and a reduction in the percentage of cells in S phase. Combination therapy with GSK2830371 and IR significantly suppressed the percentage of DIPG7 cells in S phase (Fig. 6A). Alone or in combination, neither GSK2830371 nor IR dramatically affected the cell cycle profile of DIPG VI cells (Fig. 6B). But GSK2830371 caused accumulation of DIPG VI-PPM1D-L513* cells in G0/G1 and reduced the percentage of DIPG VI-PPM1D-L513* cells in S phase compared with vehicle. Combined treatment with GSK2830371 and IR also resulted in a modest but statistically significant reduction in the percentage of DIPG VI-PPM1D-L513* cells in S phase compared with irradiated cells (Fig. 6C).

Fig. 6.

Fig. 6

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PPM1D inhibition and IR enhance suppression of S phase in PPM1D-mutated DIPG cells. (A) DIPG 7, (B) DIPG VI, and (C) DIPG VI-PPM1D-L513* cells were treated with vehicle or 5μM GSK2830371 (GSKi), mock irradiated or treated with 4Gy IR, and analyzed using Click-iT EdU, with propidium iodide (PI) and allophycocyanin (APC)-conjugated EdU. Shown: cell cycle analysis (right hand panels; n = 4 replicates/condition). Experiments repeated 3 times. Error bars, SD. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Changes in proliferation of PPM1D-mutated DIPG cells correlated inversely with changes in apoptosis. Treatment with GSK2830371 or IR resulted in a similar increase in apoptosis of DIPG7 cells. Combined treatment with GSK2830371 and IR resulted in an additional, significant increase in apoptosis of DIPG7 cells (Supplementary Figure 7A). Alone, neither GSK2830371 nor IR promoted apoptosis of DIPG VI cells (Supplementary Figure 7B). However, by flow cytometry, combined treatment with GSK2830371 and IR resulted in a modest, but statistically significant, increase in apoptosis of DIPG VI-PPM1D-L513* cells compared with treatment with either modality or with vehicle controls (Supplementary Figure 7C).

We also assayed immunostaining by TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) in OBS from symptomatic mice orthotopically xenografted with DIPG7 or DIPG VI-PPM1D-L513* cells. Analogous to our findings by flow, treatment with either GSK2830371 or IR increased the percentage of TUNEL+ DIPG7 cells in OBS. Combined treatment with GSK2830371 and IR resulted in an even greater increase in the percentage of apoptotic, TUNEL+ DIPG7 and DIPG VI-PPM1D-L513* cells (Supplementary Figure 8), which suggests a significant advantage to combined therapy with PPM1D inhibition and IR in PPM1D-mutated DIPGs.

Discussion

We have used patient-derived DIPG cells in vitro and in vivo to show that PPM1D mutation promoted PPM1D stabilization in DIPG cells, increased proliferation in culture, and increased tumor formation in the developing brainstem. Conversely, genetic ablation using PPM1D-targeting shRNAs impaired cell viability in cell lines that contain a de novo PPM1D mutation and in a DIPG cell line with stable expression of a DIPG-relevant mutant PPM1D. Zhang et al previously used homologous recombination to show that genetic conversion of mutant to wild-type PPM1D results in reduced proliferation of PPM1D mutated HCT116 colon cancer cells.8 But, our findings suggest that PPM1D mutation is an important driver in DIPG and support the findings of Filbin et al and Nikbakht et al, who used single cell RNA-seq and evolutionary reconstruction in a cohort of 134 DIPG tissues, respectively, to identify mutation of TP53 or PPM1D as driver mutations downstream of the characteristic DIPG H3F3A mutation.5,35

Of interest is our finding of increased proliferation in vitro and reduced survival of orthotopically xenografted DIPG VI stably transduced with PPM1D-mutated compared with parental DIPG VI cells. Prior studies have shown that overexpressed PPM1D promotes tumorigenesis by inactivating components of the DDR pathway, especially p53 itself and its targets. Our findings of increased tumorigenicity in DIPG VI cells with stable expression of mutant PPM1D, which also harbor inactivating TP53 mutations, suggest a p53-independent mechanism of tumorigenesis for PPM1D-mutated DIPGs. PPM1D is known to regulate mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase signaling through dephosphorylation of p38MAPK and nuclear factor-kappaB, as well as mammalian target of rapamycin complex 1. It is possible that these pathways contribute to the oncogenic function of mutant PPM1D.

We have further shown that some inhibitors with activity against PPM1D-overexpressed or -amplified malignancies also inhibit proliferation of PPM1D-mutated DIPGs. CCT007093, a cyclopentanone identified in a chemical screen against PPM1D-overexpressing cancer cells, induces apoptosis via activation of p38MAPK and works synergistically with paclitaxel in breast cancer cells.28 CCT007093 is hypothesized to inhibit the phosphatase domain of PPM1D.

RITA, a tricyclic thiophene derivative, inhibits the interaction of p53 and MDM2, its major regulator, and prevents p53 polyubiquitination, resulting in increased p53 stability and activity, cell cycle arrest, and apoptosis.36 In PPM1D-overexpressing neuroblastomas, RITA inhibits the expression of oncogenes, including N-myc, Aurora kinase, MDM2, MDMX, and PPM1D.37 The mechanism by which RITA inhibits PPM1D expression is unclear, but PPM1D repression results in activation of ATM and downregulation of human MDMX.34

GSK2830371, a thiophenecarboxamide derivative, is an orally active, allosteric, non-competitive PPM1D inhibitor that binds a structural flap domain which is unique to PPM1D, resulting in p53-dependent inhibition of tumor growth in vitro and in vivo.27 In a recent publication, GSK2830371 altered phosphorylation of key DDR proteins, including ATM, CHK1/2, MDM2/X, p53, and p21, preferentially killed PPM1D-mutated cells, and re-sensitized myeloid leukemia cells to conventional chemotherapy.21

In vitro, CCT007093 and GSK2830371 exhibited 10–100x increased efficacy against PPM1D-mutated DIPG7 cells compared with PPM1D wild-type DIPG VI cells. Additionally, RITA was as effective as IR in suppressing the growth of DIPG7, but not DIPG VI cells. Using OBS from immunocompetent and NSG mice containing either DIPG7 or DIPG VI-PPM1D-L513* cells, we also showed that PPM1D inhibition suppressed the growth of PPM1D-mutated DIPGs within the brainstem microenvironment. We verified PPM1D-targeted activity by GSK2830371 using western blotting in DIPG7 and DIPG VI-PPM1D-L513* cells, which showed increased activation of PPM1D targets p53 and H2A.X. Prior evidence that RITA and GSK2830371 suppress expression or activity of PPM1D, while simultaneously activating DDR proteins, including ATM and p53, likely helps explain their increased efficacy against PPM1D-mutated DIPG cells, alone and in combination with IR compared with CCT007093, which is primarily known to promote apoptosis by activating p38MAPK signaling in cancer cells that overexpress PPM1D.

Given our findings of efficacy of PPM1D inhibitors in PPM1D-mutated cells under cell culture conditions and in OBS, we examined in vivo efficacy by treating DIPG7 cells with vehicle, GSK2830371, or PPM1D shRNA and immediately xenografting cells into the pons of neonatal NSG mice. This allowed us to circumvent potential issues with drug delivery. PPM1D knockdown or GSK2830371 treatment resulted in a significant survival advantage, as well as both decreased proliferation and, in GSK2830371-treated mice, increased apoptosis. A limitation of our study is that these differences in survival might be due to reduced engraftment of PPM1D knocked-down or GSK2830371-treated DIPG7 cells. Thus, future studies will examine the response of established PPM1D-mutated murine and human DIPGs to PPM1D inhibition.

Prior publications have shown that chemotherapy selects for TP53 mutations and poor outcomes in some cancers, such as neuroblastoma.38 Given that TP53 and PPM1D mutations do not often coexist, one concern regarding PPM1D inhibition is that it may select for TP53 mutation in DIPG. We did not detect differences in total or nuclear p53 expression in GSK2830371-treated DIPG7 xenografts. Thus, results presented in this study suggest that PPM1D-targeted inhibition is a viable treatment for PPM1D-mutated DIPGs.

DIPGs are radio-responsive at diagnosis. Recent reports show that they also respond to re-irradiation following progression.39 Current trials are employing molecularly targeted or immune therapies to enhance the efficacy of IR and promote tumor regression. Our results show that RITA is as effective as IR, but only GSK2830371 increased the anti-DIPG efficacy of IR. Future studies will address the role of combined treatment with PPM1D inhibitors and IR in vivo. Nevertheless, our results provide strong rationale for continued development of clinically viable PPM1D inhibitors and eventual trials combining PPM1D inhibitors with IR in DIPG.

Funding

This study was supported in part by the Emory Integrated Genomics Core (EIGC), Georgia Clinical and Translational Science Alliance of the NIH (UL1TR002378), Emory University Integrated Cellular Imaging Microscopy Core, the Cancer Animal Models Shared Resource, NIH (CA172392), Peer Reviewed Cancer Research Program, Department of Defense (CA140056), CURE Childhood Cancer Foundation, and the Aflac Cancer and Blood Disorders Center.

Conflict of interest statement. The authors declare no potential conflicts of interest.

Authorship statement. Experimental design/implementation, data analysis, and manuscript preparation: MPA, KN, RN, DH, and RCC.

Supplementary Material

noz053_suppl_Supplementary_Figure_S1
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noz053_suppl_Supplementary_Materials
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Acknowledgments

We acknowledge technical and intellectual assistance from Oskar Laur, Zhihong Chen, Cameron Herting, Chris Jones (ICR, UK), Michelle Monje (Stanford), Angel Carcaboso (Barcelona), Javad Nazarian (Children’s National Medical Center), Tobey MacDonald and Anna Kenny (Emory University).

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Associated Data

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Supplementary Materials

noz053_suppl_Supplementary_Figure_S1
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noz053_suppl_Supplementary_Figure_S3
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noz053_suppl_Supplementary_Figure_S4
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noz053_suppl_Supplementary_Figure_S5
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noz053_suppl_Supplementary_Figure_S6
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noz053_suppl_Supplementary_Figure_S7
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noz053_suppl_Supplementary_Figure_S8
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