PPM1D Expression Research Articles

MiR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia

Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.

Dual Targeting of HDM2 and PPM1D As a Novel Therapeutic Approach for Myelofibrosis

Dysregulation of TP53 in myeloproliferative neoplasms (MPNs) has been hypothesized to be a key driver of disease progression (Kubesova et al, Leukemia. 2018;32 (2):450-61). Targeting of double minute protein 2 (HDM2), a negative regulator of TP53, results in activation of the TP53 pathway which induces cell death in hematopoietic stem and progenitor cells (HSPCs). Nutlins, antagonists of HDM2, are currently being evaluated in multiple MPN clinical trials with promising results to date. Protein Phosphatase Mg2+/Mn2+ dependent 1D protein (PPM1D) is another key negative regulator of the TP53 pathway that dephosphorylates TP53 and stabilizes HDM2 (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). We and others have previously reported the presence of PPM1D mutations in MPNs that result in overexpression of the protein (Grinfeld et al, N Engl J Med. 2018 Oct 11;379 (15):1416-30). We have also reported overexpression of PPM1D in the mononuclear cells (MNC) of a subset of MPN patients not harboring PPM1D mutations. We hypothesize that inhibition of PPM1D in myelofibrosis (MF) would target the MF HSPC and sensitize MF HSPCs to HDM2 inhibitors resulting in further depletion of MF HSPCs. To further characterize PPM1D expression in the MF HSPC compartment, we utilized quantitative real time-PCR, to assess PPM1D transcript levels in CD34+ cells from 28 MF patients and compared these transcript levels to levels in CD34+ normal control cells (n=6). Forty-three percent (12/28) of the MF samples had overexpression of PPM1D (>1.5 fold increase) and the fold increase ranged from 1.8-59. To identify whether PPM1D is a viable therapeutic target for MF HSPCs, we utilized hematopoietic colony assays to test the activity of GSK2830371, an allosteric inhibitor of PPM1D (PPM1Di) alone and in combination with a low dose HDM2 inhibitor (HDM2i). Eight samples from distinct MF patients were tested under these conditions. There was not a significant reduction in the number of JAK2V617F+ colonies upon treatment with single agent HDM2i (50 nM) (p>0.05) or PPM1Di (1uM) (p>0.05). Combination of PPM1Di (1 uM) + HDM2i (50 nM) resulted in decreased JAK2V617F+ colony formation in all 8 samples (p<0.005) (Fig. 1). The reduction in JAK2V617F+ colony number was significant compared to either single agent treatment with HDM2i (p<0.001) or PPM1Di (p<0.05) (Fig. 1). Four samples had persistence of or an increase in JAK2 wild-type (WT) colony number with combination treatment. The MF samples utilized were from a heterogenous population of patients that all had WT TP53 and PPM1D but included patients with high risk myeloid mutations (in ASXL1, NRAS), complex cytogenetics and prior exposure to standard MF directed therapies. Additionally, to validate the on-target effect of PPM1Di, we showed that treatment of leukemia cell lines with PPM1Di led to increased protein levels of phosphorylated-p53 and p21 and resulted in decreased viability and increased apoptosis. To determine the effect of PPM1Di +/- HDM2i on distinct subclones we employed a multi-omics approach that allows for single cell mutation and epitope analysis of a clonally complex patient sample. We analyzed MNCs from a JAK2V617F, NRASG12D, TET2T1884A, ASXL1W960* MF patient treated in suspension culture with PPM1Di (1uM) alone, low dose HDM2i (50 nM) alone and PPM1Di (1uM) + low dose HDM2i (50 nM) in combination. Compared to vehicle alone or either drug alone, a marked effect was seen with the combination approach whereby the percent of WT cells increased by nearly 5 fold (4% to 19%) and 4/6 mutated clones were decreased in size, demonstrated by the fishplot (Fig. 2). These results show that the combination of the HDM2i + PPM1Di depletes multiple MF clones, while allowing for the emergence of more WT cells. Furthermore these studies demonstrate the utility of single cell genotyping to evaluate the effects of novel therapeutic strategies on MF clonal architecture. These studies indicate that PPM1Di sensitizes MF HSPCs to an HDM2i and is a promising strategy to treat MF patients. To this purpose, we are currently developing a novel clinical grade PPM1D targeting agent. Dual targeting of PPM1D and HDM2 has the potential to further deplete multiple MF HSPC clones while allowing the persistence of WT cells. Additional combinatorial mutation + epitope studies are underway to determine degree of MF HSPC subpopulation depletion utilizing this treatment strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular and Clinical Spectra of Protein Phosphatase, Mg2+/Mn2+-Dependent 1D (PPM1D)-Mutant Myeloid Neoplasms

Protein phosphatase Mg2+/Mn2+-dependent 1D (PPM1D) gene (17q23) cooperates with TP53 in regulating DNA damage response (DDR). PPM1DMT are frequently encountered in clonal hematopoiesis of indeterminate potential (CHIP) and in therapy-related myeloid neoplasms (t-MN). Most typically lesions lead to C-terminal loss leading to truncation with paradoxical gain-of-function amplification of PPM1D effects in terms of inactivation of TP53 and are thus functionally synergistic with TP53 deficiency. Here, we investigated the clonal architecture and clinical implications of PPM1DMT in the context of TP53MT MN. A cohort of 4479 patients (pts) screened for the presence of PPM1DMT and TP53MT was analyzed in terms of clinical phenotype, cytogenetics, clonal hierarchy and outcomes (see Table). Overall, 67 (1.5%) pts harbored PPM1DMT and 367 (8%) TP53MT. PPM1DMT (n=70) were truncating, of which 57% were frameshift and 43% were stop codons, distributed across exon 6. No biallelic PPM1DMT case was found. When we studied PPM1D expression in wild type, mRNA levels were elevated in AML (59%; P<.0001, but not in MDS), with high risk AML having higher expression PPM1D levels compared to favorable cases (59% vs. 46%, P=.08). PPM1DMT cases were older than those with TP53MT [median 74yrs (range, 39-93) vs. 71yrs (14-95), P=.02]. PPM1DMT were significantly enriched within pts with CH (including CHIP and CCUS) compared to TP53MT (27 vs. 1%, P=.0001) while TP53MT were more frequently encountered in MN (71 vs. 31%, P=.0001). Review of the medical records of PPM1DMT and TP53MT cases revealed history of malignancy was ascertained in 69% of PPM1DMTvs. 30% in TP53MT; P<.0001, of which 30% had hematological malignancies (HM) and 7.5% were breast cancer (BC) survivors. Of note is that 9% PPMD1MT cases had autoimmune disorders. Overall, 37% received only chemotherapy, 20% radiotherapy only, 22% both chemo/radiotherapy and 13% immunosuppression. A total of 14% of TP53MT carriers had a prior history of HM and 5% had BC history. When compared to PPM1DMT, a higher fraction (58%; P=.005) received chemotherapy, whereas similar rates were noted for radiotherapy (14%), chemo/radiotherapy combination (17%) and immunosuppression (11%). PPM1DMT were also more commonly found in t-MN than TP53MT (42 vs. 28%, P=.05). Overall, 37% of PPM1DMT showed a normal karyotype (NK) compared to 14% of TP53MT (P=.0003). Indeed, up to 61% of TP53MT had complex karyotypes (CK vs. 15% in PPM1DMT, P=.0001). Most common genes associated with PPM1DMTvs.TP53MT were DNMT3A (21 vs. 10%; P=.05), TET2 (19 vs. 11%; P=.2), ASXL1 (15 vs. 10%, P=.4), KRAS/NRAS (1 vs 7%, P=.06) and NF1 (1 vs. 7%, P=.03). Analysis of clonal architecture showed that PPM1DMT were either dominant / codominant (46%; n=33) including PPM1DMT as a sole lesion(n=19); in these cases TP53MT was the most common 2nd/codominant hit (25%) followed by DNMT3A (12.5%, P=.04) and RUNX1 (12.5%, P=.04). Cases with dominant PPM1DMT were encountered more in t-MN (46 vs. 36%, P=.3) and CH (30 vs. 24%, P=.4) compared to subclonal PPM1DMT (n=34, 51%), which were subclonal to TET2MT (21%) followed by JAK2 (12%), DNMT3A (9%, P=.02) and TP53 (9%, P=.02). Notably, all deaths in PPM1DMT CH cases were not due to progression to MN. No significant difference was found in OS between ancestral and subclonal PPM1DMT carriers. PPM1DMT were more likely encountered in older pts (OR=3.2; P=.001), MN-LR (OR=2.8, P<.0001), CH (OR=12.6, P<.0001), t-MN (OR=4.8, P<.0001), NK (OR=1.9, P=.01) and DNMT3AMT carriers (OR=1.86, P=.04) and less likely to occur in pts with MN-HR (OR=0.38, P=.002; see Figure) Survival analysis showed no significant difference between PPM1DMT and PPM1DWT and between CH and non-CH PPM1DMT cases while there was a significant difference between PPM1DMTvs. TP53MT (102 vs.12mo., P<.0001).Of note is that 12 pts had both PPM1DMT and TP53MT (50% t-MN, of which 25% had a prior history of lymphoma). In such cases, PPM1DMT were mostly ancestral compared to TP53MT (67 vs. 33%; P<.0001). Configurations of PPM1D in the clonal hierarchy and pairing with secondary or primary hits are consistent with their role in MN evolving from CH. Association with CH implies that some of the t-MN may be derived from pre-existing CH a theory which we are currently being testing in serial samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Functionalized Macrophage Exosomes with Panobinostat and PPM1D-siRNA for Diffuse Intrinsic Pontine Gliomas Therapy.

Diffuse intrinsic pontine glioma (DIPG) is a rare and fatal pediatric brain tumor. Mutation of p53‐induced protein phosphatase 1 (PPM1D) in DIPG cells promotes tumor cell proliferation, and inhibition of PPM1D expression in DIPG cells with PPM1D mutation effectively reduces the proliferation activity of tumor cells. Panobinostat effectively kills DIPG tumor cells, but its systemic toxicity and low blood–brain barrier (BBB) permeability limits its application. In this paper, a nano drug delivery system based on functionalized macrophage exosomes with panobinostat and PPM1D‐siRNA for targeted therapy of DIPG with PPM1D mutation is prepared. The nano drug delivery system has higher drug delivery efficiency and better therapeutic effect than free drugs. In vivo and in vitro experimental results show that the nano drug delivery system can deliver panobinostat and siRNA across the BBB and achieve a targeted killing effect of DIPG tumor cells, resulting in the prolonged survival of orthotopic DIPG mice. This study provides new ideas for the delivery of small molecule drugs and gene drugs for DIPG therapy.

PPM1D Is a Therapeutic Target in Childhood Neural Tumors.

Simple SummaryMedulloblastoma and neuroblastoma are childhood tumors of the central nervous system or the peripheral nervous system, respectively. These are the most common and deadly tumors of childhood. A common genetic feature of medulloblastoma and neuroblastoma is frequent segmental gain or amplification of chromosome 17q. Located on chromosome 17q23.2 is PPM1D which encodes WIP1, a phosphatase that acts as a regulator of p53 and DNA repair. Overexpression of WIP1 correlates with poor patient prognosis. We investigated the effects of genetic or pharmacologic inhibition of WIP1 activity and found that medulloblastoma and neuroblastoma cells were strongly dependent on WIP1 expression for survival. We also tested a number of small molecule inhibitors of WIP1 and show that SL-176 was the most effective compound suppressing the growth of medulloblastoma and neuroblastoma in vitro and in vivo.Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice.

Simple SummaryAberrant expression of the PPM1D gene which encodes a phosphatase called WIP1 is frequently observed in cancers of different origins. WIP1 is a negative regulator of the tumor suppressor p53. Improper inactivation of p53 results in genomic instability and can induce neoplastic transformation. We show that overexpression of PPM1D induces tumors in mice similar to cancers harboring p53 mutations. Our results suggest that PPM1D can act as an oncogenic driver by inducing genomic instability, impaired growth arrest, and apoptotic escape that can result in neoplastic transformation and malignant tumor development. PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a.

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.

Self-Assembled Plasmid Delivery System for PPM1D Knockout to Reverse Tumor Malignancy.

Gene delivery vectors possess critical roles in effective genome editing. In this study, a multiple functional vector for encapsulating CRISPR/Cas9 plasmid was designed to knock out PPM1D gene and prevent cancer malignancy. The plasmid was complexed with a KALA peptide with the capability of endosomal escape and histones for nuclear transportation and then decorated by hyaluronic acid (HA) and AS1411-incorporated hyaluronic acid (AHA) targeting CD44 and nucleolin overexpressed in cancer cells to form AHA/HA/KALA/histone/plasmid nanoparticles. The constructed multifunctional plasmid delivery system with the cancer targeting specificity can realize efficient genome editing for PPM1D knockout and thus dramatically downregulate PPM1D expression in targeted malignant cells. More importantly, PPM1D knockout results in upregulation of p21 and p-p38 as well as downregulation of cyclin D1, MMP9, CYR61, and vimentin. The edited cancer cells exhibit suppressed proliferation, migration, and invasion, indicating the successful reversal of tumor malignancy.

Silencing of PPM1D inhibits cell proliferation and invasion through the p38�MAPK and p53 signaling pathway in papillary thyroid carcinoma

Endeavors towards identifying key molecular markers for early diagnosis and treatment are driving the clinical study of papillary thyroid carcinoma (PTC). Recent studies have indicated that protein phosphatase, Mg2+/Mn2+ dependent, 1D (PPM1D) exerts an oncogenic function by increasing cell proliferation, migration and invasion in various cancer types. In addition, PPM1D has a high frequency of genetic alterations and has been proposed as a tumor driver in thyroid cancer, making PPM1D an attractive potential oncotarget for cancer treatment. The aims of the present study were to investigate the downstream targets of PPM1D and the potential molecular mechanisms of its oncogenic activities, as well as its clinical significance in PTC. As anticipated, PPM1D overexpression was confirmed in PTC clinical specimens. Furthermore, knockdown of PPM1D in thyroid cancer cell lines significantly suppressed the proliferation, migration and invasion but facilitated cell apoptosis. The protein levels of phosphorylated p38 mitogen-activated protein kinase (MAPK), p53 and Bax were increased in PPM1D-knockdown cells, while inhibition of p38 phosphorylation restored cell migration, proliferation and cell apoptosis. In addition, silencing of PPM1D expression induced nuclear translocation of p53 in K-1 and TPC-1 cells. The present results demonstrated that PPM1D regulated p38 MAPK and p53 signaling pathways to promote thyroid cancer progression. Collectively with the clinical results, these data qualified PPM1D as a potential diagnostic biomarker and therapeutic target in human thyroid cancer.

Significance of Abnormalities of PPM1D in Myeloproliferative Neoplasms

The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (&gt;1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

Sensitivity of Cells with Various Levels of Ppm1d Expression to Classical Chemotherapeutic Drugs for Colorectal Cancer Treatment

Mutations and amplifications of the Ppm1d gene encoding Wip1 phosphatase have been found in various tumors. Recent studies have shown that the presence of Wip1 stable mutant after chemotherapy suggests that Wip1 is involved in the resistance of malignant cells to chemotherapeutic drugs. In the present work, we investigated the role of Wip1 in the response of colon cancer cells to the antitumor drugs 5-fluorouracil and oxaliplatin. Cell lines with an increased level of Ppm1d expression were obtained with acquired using lentiviral transduction. It has been shown that Wip1 overexpression maintains the cell viability of cells exposed to oxaliplatinum, while deletion of the Ppm1d gene decreases the viability and clonogenicity both under the combined and single exposure to these drugs. These findings suggest that an increased Wip1 level in cancer cells during chemotherapy may contribute to the development of cancer cell resistance to antitumor therapy. Methods aimed at reducing the Wip1 level will increase the efficiency of colon cancer treatment.

APPBP2 enhances non-small cell lung cancer proliferation and invasiveness through regulating PPM1D and SPOP

BackgroundThe influence of amyloid protein-binding protein 2 (APPBP2) on lung cancer is unknown. MethodsThe function and mechanisms of APPBP2 were investigated in the NSCLC cell lines A549 and H1299. The ectopic expression of APPBP2, PPM1D and SPOP in NSCLS were examined in samples collected from ten pairs of human lung adenocarcinoma cancer tissues and adjacent normal lung tissues. shRNA vector was used for APPBP2 knockdown. Quantitative PCR and western blot assays quantified the mRNA and protein level of APPBP2, PPM1D, and SPOP. Cell proliferation was measured with BrdU, MTT, colony formation assays, and xenograft tumour growth experiments. Cell migration and invasion were analysed with transwell and wound healing assays. Co-Immunoprecipitation assay detected protein–protein interactions. FindingsAPPBP2 was upregulated in NSCLC tissues. Silencing APPBP2 in A549 and H1299 cells resulted in the inhibition of cell proliferation, migration, and invasion, enhancement of apoptosis, and a significant decrease in the expression of PPM1D and SPOP. Overexpression of PPM1D and SPOP attenuated the APPBP2-knockdown inhibition of NSCLC cells. Co-IP assay showed that PPM1D interacted with APPBP2. InterpretationThe expression level of APPBP2 positively correlates with NSCLC cell proliferation, migration, and invasiveness. APPBP2 contributes to NSCLC progression through regulating the PPM1D and SPOP signalling pathway. This novel molecular mechanism, underlying NSCLC oncogenesis, suggests APPBP2 is a potential target for diagnosis and therapeutic intervention in NSCLC. FundKey Program of Natural Science Research of Higher Education of Anhui Province (No. KJ2017A241), the National Natural Science Foundation of China (No. 81772493).

PPM1D Functions as Oncogene and is Associated with Poor Prognosis in Esophageal Squamous Cell Carcinoma.

Mounting evidence has demonstrated that PPM1D participates in the development and progression of a wide variety of tumors. However, its precise roles in esophageal squamous cell carcinoma (ESCC) remain under investigation. Here, UALCAN, an interactive web-portal to perform the expression analyses of PPM1D using TCGA gene expression data, and PPM1D high expression was exhibited in primary esophageal cancer. Further investigation revealed that PPM1D expression was obviously higher in ESCC tissues than in normal tissues (P < 0.01), which was consistent with the results from real-time qPCR and Western blotting in ESCC tissues and paired normal esophageal tissues. Besides, PPM1D expression was closely correlated with TNM staging, tumor differentiation and lymph node metastasis (P < 0.01), but not related to the patients' gender and age (P > 0.05). Notably, PPM1D expression in metastatic ESCC patients was markedly higher than that in non-metastatic ESCC patients (P < 0.01), and the ESCC patients with high PPM1D expression predicted poor prognosis. Multivariate assay demonstrated that PPM1D and lymph node metastasis were considered as independent prognostic factors for the ESCC patients. These findings suggest PPM1D plays a pivotal important role in onset and progression of ESCC, and may be a new biomarker for metastasis and prognosis of the ESCC patients.

Abstract B05: Mechanisms of PPM1D-mediated tumorigenesis in diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG), the most common pediatric brainstem tumor, is associated with a dismal prognosis with almost no survivors within two years of diagnosis. It is unamenable to surgical resection, is not responsive to conventional chemotherapy, and responds only temporarily to radiation therapy (XRT). Recent publications identified carboxy (C)-terminal truncating mutations of PPM1D, which encodes the protein WIP1, in up to 25% of DIPGs. WIP1 is a serine/threonine protein phosphatase that plays an important role in DNA damage response (DDR). Prior publications show that most, but not all, PPM1D C-terminal mutants function in a dominant negative fashion to promote tumor growth in cancer models. We hypothesized that PPM1D mutation promotes DIPG growth, and WIP1 inhibition suppresses tumor proliferation. We further hypothesized that, due to its key role in DDR, WIP1 inhibition enhances the antitumor effects of XRT. Using Sanger sequencing, we identified de novo mutation of PPM1D in the C-terminus of multiple patient-derived DIPG cell lines. We also stably introduced clinically relevant PPM1D mutations, which we identified in DIPGs resected from patients, into a PPM1D wild-type DIPG cell line, DIPG VI, as well as into DIPG cells derived from a DIPG mouse model. Using trypan blue exclusion, bioluminescence, and immunofluorescence detection of markers of proliferation and cell death, in the presence or absence of PPM1D knockdown or pharmacologic WIP1 inhibition, and/or following XRT exposure, we measured proliferation of DIPG VI, DIPG VI L513X, a cell line with stable expression of a mutant PPM1D, and DIPG7, a cell line with a de novo PPM1D mutation. We also assayed proliferation in organotypic brain slice cultures derived from symptomatic immunocompromised mice orthotopically xenografted with DIPG cells and of DIPG cells embedded within the brainstem of organotypic brain slices derived from immunocompetent mice. Finally, we orthotopically xenografted DIPG cells into the early post-natal (P0-2) brainstem of immunodeficient mice, treated mice with vehicle or with a small-molecule WIP1 inhibitor, and measured effects on survival, as well as proliferation in tumor cells post-necropsy. Under cell culture conditions, expression of PPM1D mutants promoted tumor cell proliferation in wild-type PPM1D murine and human DIPG cells. Conversely, PPM1D knockdown significantly suppressed DIPG cell proliferation, especially in PPM1D mutated DIPG cells. Similarly, treatment with a small-molecule WIP1 inhibitor suppressed the growth of PPM1D-mutated, but not PPM1D wild-type DIPG cells, in neurosphere cultures, on immunocompetent organotypic brain slices, and on organotypic brain slices derived from symptomatic, orthotopic, xenografted mice. WIP1 inhibition had similar effects on growth inhibition to XRT alone and augmented the antiproliferative and proapoptotic effects of XRT in vitro. Mice bearing an orthotopic xenograft of PPM1D-mutated human DIPG cells, DIPG7, died of disease earlier than mice xenografted with PPM1D wild-type human DIPG cells, DIPG VI. Treatment of mice bearing orthotopically, xenografted DIPG7 cells with a WIP1 inhibitor via a continuous 14-day infusion into the lateral ventricles resulted in a significant reduction in proliferation of tumor cells. In summary, our results show that C-terminal mutated PPM1D is oncogenic in DIPG, promoting proliferation and reduced survival in vivo. WIP1 inhibition suppresses growth and increases the radiosensitivity of PPM1D-mutated DIPG. These results suggest that WIP1 is a druggable target alone and in combination with XRT in DIPG. Further studies will examine the PPM1D-mediated mechanisms of tumorigenesis and treatment responsiveness in DIPG in order to develop clinically viable treatment paradigms that target PPM1D-mutated tumors. Citation Format: Mwangala P. Akamandisa, Kai Nie, Jing Wen, Rita Nahta, Dolores Hambardzumyan, Robert C. Castellino. Mechanisms of PPM1D-mediated tumorigenesis in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B05.

MicroRNA-499a-5p inhibits osteosarcoma cell proliferation and differentiation by targeting protein phosphatase 1D through protein kinase B/glycogen synthase kinase 3β signaling.

A number of studies have attempted to elucidate the association between mircoRNAs (miRNAs/miRs) and cancer-associated processes. The aim of the present study was to determine how miR-499a-5p intervenes in human osteosarcoma cell proliferation and differentiation. The cancerous tissues and adjacent non-cancerous tissues of 62 patients with osteosarcoma (OS) were collected. miRNA microarray analysis revealed that 29 miRNAs were upregulated while 26 were downregulated, among which miR-499a-5p expression was the most decreased. Western blot analysis and reverse transcription-quantitative polymerase chain reaction demonstrated that the mRNA and protein expression of miR-499a-5p was lower, while that of protein phosphatase 1D (PPM1D) was higher in OS tissues compared with expression levels in normal tissues. Furthermore, miR-499a-5p expression was markedly decreased in the metastatic tumors and in those at stage III+IV compared with the non-metastatic tumors and those at stage I, respectively. In addition, following transfection of the human OS MG-63 cell line with an miR-499a-5p mimic, the expression of miR-499a-5p was elevated while the protein and mRNA expression of PPM1D was decreased. When combining these findings with the information obtained from the Targetscan predictive software, it was confirmed that PPM1D was targeted by miR-499a-5p. In MG-63 cells transfected with an miR-499a-5p mimic, PPM1D-associated downstream proteins phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) were significantly downregulated compared with the negative control (NC) group, while the expression of p-Akt and p-GSK-3β were significantly elevated in the tumor tissues compared with the adjacent non-tumor tissues. Simultaneously, the growth and proliferation activity of MG-63 cells were notably reduced when transfected with the miR-499a-5p mimic, compared with the NC group. Therefore, it may be concluded that miR-499a-5p suppresses OS cell proliferation and differentiation by targeting PPM1D through modulation of Akt/GSK-3β signaling.

Protein Phosphatase Magnesium-Dependent 1δ (PPM1D) Expression as a Prognostic Marker in Adult Supratentorial Diffuse Astrocytic and Oligodendroglial Tumors.

BackgroundProtein phosphatase magnesium-dependent 1δ (PPM1D) is a p53-induced serine/threonine phosphatase, which is overexpressed in various human cancers. A recent study reported that a mutation in the PPM1D gene is associated with poor prognosis in brainstem gliomas. In this study, we evaluated the utility of PPM1D as a prognostic biomarker of adult supratentorial diffuse astrocytic and oligodendroglial tumors.MethodsTo investigate PPM1D protein expression, mRNA expression, and copy number changes, immunohistochemistry, RNAscope in situ hybridization, and fluorescence in situ hybridization were performed in 84 adult supratentorial diffuse gliomas. We further analyzed clinical characteristics and overall survival (OS) according to PPM1D protein expression, and examined its correlation with other glioma biomarkers such as isocitrate dehydrogenase (IDH) mutation, and p53 expression.ResultsForty-six cases (54.8%) were PPM1D-positive. PPM1D expression levels were significantly correlated with PPM1D transcript levels (p= .035), but marginally with PPM1D gene amplification (p=.079). Patients with high-grade gliomas showed a higher frequency of PPM1D expression than those with low-grade gliomas (p <.001). Multivariate analysis demonstrated that PPM1D expression (hazard ratio [HR], 2.58; p=.032), age over 60 years (HR, 2.55; p=.018), and IDH1 mutation (HR, 0.18; p=.002) were significantly independent prognostic factors; p53 expression had no prognostic significance (p=.986). The patients with tumor expressing PPM1D showed a shorter OS (p=.003). Moreover, patients with tumor harboring wild-type IDH1 and PPM1D expression had the worst OS (p<.001).ConclusionsOur data suggest that a subset of gliomas express PPM1D; PPM1D expression is a significant marker of poor prognosis in adult supratentorial diffuse astrocytic and oligodendroglial tumors.

De Novo Truncating Mutations in the Last and Penultimate Exons of PPM1D Cause an Intellectual Disability Syndrome

Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in ∼35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders.

The Pathophysiological Significance and Therapeutic Targeting of PPM1D-Mediated Signaling in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin lymphoma genetically characterized by balanced t(11:14) translocation and cyclin D1 overexpression. A great majority of patients with MCL remain incurable. The selection of chemoresistant subclones occurs during therapies, leading to relapse of MCL. Many chemotherapeutic agents induce DNA damage and activate the tumor suppressor p53. Recently, non-genotoxic p53 activators like MDM2 (master regulator of p53) inhibitors and XPO1 (nuclear exporter for p53) inhibitors have entered clinical trials. p53 stabilization, however, has not always led to p53-mediated transcriptional activation in MCL. PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. PPM1D has been thought to be an oncoprotein that inhibits p53. PPM1D overexpression or amplification has been reported in various cancers, including lung, breast, kidney and ovarian cancers. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 (GSK) (Nat Chem Biol 2014) in MCL.RESULTS Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart B-lymphocytes. PPM1D mRNA levels positively correlated with CCND1 (Cyclin D1) mRNA levels (r = 0.33, P = 0.0014; n = 92) and with proliferation signature averages (r = 0.54, P < 0.0001; n = 92) in a series of MCL samples. Increased PPM1D expression at diagnosis was itself associated with a poorer prognosis in MCL patients (median overall survival of 3.9 years and 1.4 years for cases in the lowest and highest PPM1D expression tertiles, respectively; P = 0.0047). PPM1D levels in MCL were as high as those in aggressive lymphomas including Burkitt’s lymphoma and diffuse large B-cell lymphoma, and were significantly higher than those in indolent lymphomas including chronic lymphocytic leukemia/small lymphocytic lymphoma (P = 0.0076) and follicular lymphoma (P = 0.011). Eight MCL (three p53 wild-type (WT) and five mutant (MUT)) cell lines were exposed to GSK. GSK inhibited the cell growth, being more prominent in p53 WT cells (48.3 ± 9.8% versus 14.8 ± 4.7% growth inhibition, P = 0.036). In sensitive cells, GSK caused a significant loss of mitochondrial membrane potential in addition to increased annexin V positivity, indicating apoptosis induction. Stable p53-specific shRNA-expressing cells were generated in p53 WT Z-138 and JVM-2 cells, with > 85% knockdown efficiency. p53 knockdown cells were less susceptible to GSK than control cells, indicating that GSK utilizes p53-mediated signaling to eradicate MCL cells. This idea is supported by the Western blot data that GSK treatment increased total and phosphorylated p53 levels and those of p53 targets p21 and PUMA. Basal and GSK-induced levels of PPM1D and its target proteins NFkB-p65, p38 MAPK, and histone H2A.X did not predict MCL cell sensitivity to GSK. GSK and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 WT MCL cells. Importantly, GSK sensitized MCL cells to bortezomib (BTZ) and doxorubicin (DOX) irrespective of p53 mutational status. BTZ and DOX are clinically active agents against MCL. In p53 WT cells, GSK/BTZ and GSK/DOX combinations potently activated p53-mediated apoptosis signaling. In p53 MUT cells, combination treatment did not activate p53 signaling. Instead, p38 signaling appeared to be actively involved in the GSK/BTZ lethality, as the selective p38 inhibitor SB203580 significantly attenuated bortezomib- and GSK/bortezomib-induced lethality. Activation of p38 MAPK has been found to cause MCL cell death. SB203580 did not protect MCL cell from doxorubicin- or GSK/doxorubicin-induced lethality.CONCLUSION PPM1D inhibition increases levels of phosphorylated p53 at Ser15 in a non-genotoxic manner, which enhances p53-mediated transcription in MCL cells. In addition, PPM1D inhibition shows anti-lymphoma effects in p53 MUT cells, partially through activation of p38 signaling. Although a single-agent activity of GSK was modest against MCL cells, especially against those with mutant p53, its p53-independent potentiation effect on BTZ and DOX may support the use of PPM1D inhibitors as part of a combination therapeutic strategy for MCL. Collectively, PPM1D inhibition may offer a novel therapeutic strategy for MCL. DisclosuresNo relevant conflicts of interest to declare.

PPM1D Truncating Mutations Confer Chemotherapy Resistance in Hematopoietic Stem Cells, Which Is Reversible By PPM1D Inhibition

One of the adverse consequences of chemotherapy exposure is the development of therapy-related myeloid neoplasms (t-MNs). However, the cause and origin of most t-MNs are unknown, and the prognosis remains dismal. Novel work has shown that in addition to TP53, PPM1D is selectively mutated in 15% of therapy related MDS (Lindsley et al., ASH Abstract). Truncating mutations of PPM1D are also found to commonly occur in clonal hematopoiesis (Jaiswal et al., NEJM 2014; Genovese et al., NEJM 2014; Xie et al., Nat Med 2014), as well as in the blood of cancer patients, particularly after exposure to chemotherapy (Ruark et al., Nature 2013; Swisher et al., JAMA Oncol 2016). We hypothesized that PPM1D truncating mutations confer chemotherapy resistance, causing selective outgrowth of PPM1D mutant hematopoietic stem cells in states of genotoxic stress. In addition, since PPM1D mutations lead to a gain of function, we examined the potential of targeting PPM1D-mutant cells pharmacologically.The protein phosphatase PPM1Dis a direct regulator of TP53 activity and the DNA damage response pathway (Fiscella et al., PNAS 1997). Consequently, gain-of-function PPM1D mutations lead to decreased TP53 activity. To examine whether PPM1D mutations drive chemotherapy resistance through an abrogation of the TP53 dependent DNA damage response, we engineered PPM1D-mutant subclones using the CRISPR-Cas9 system in the TP53 wild-type AML cell line MOLM-13. PPM1D exon 6 truncation led to increased expression of PPM1D and resistance to DNA damaging agents, including cytarabine, cyclophosphamide and cisplatin. In addition, PPM1D-mutant cells exhibited a selective advantage over wild-type cells in the presence of chemotherapy, expanding 100-fold over a 24-day period (Fig. 1). While treatment with chemotherapy induced phosphorylation of Chk1 and p53, cell cycle arrest and apoptosis in wild-type cells, this response was abrogated in PPM1D-mutant cells due to gain of function of PPM1D. Using phosphoproteomic analysis, we further demonstrate decreased phosphorylation of known and novel targets in PPM1D-mutant compared to wild-type cells.We next investigated the effect of PPM1D mutation on normal marrow progenitors in response to chemotherapy treatment in vivo. We performed a competition experiment in which mouse bone marrow c-Kit+ cells expressing Cas9 were transduced with guide RNAs targeting exon 6 of PPM1D or a control guide, and then transplanted into mice in a 1:5 ratio. We observed a selective outgrowth of PPM1D-mutant myeloid cells in the peripheral blood of mice after exposure to chemotherapy. In addition, we found expansion of PPM1D-mutant cells in the lineage negative, c-Kit+ Sca-1+ population, which is enriched for hematopoietic stem cells and multipotent progenitors, indicating that PPM1D-mutant stem and progenitor cells have a competitive advantage over wild-type cells after exposure to genotoxic stress.The generation of a selective, allosteric inhibitor of PPM1D (Gilmartin et al., Nat Chem Biol 2014) allowed us to examine whether pharmacologic inhibition of PPM1D decreases the chemotherapy resistance or survival of PPM1D-mutant cells. We found that PPM1D-mutant cells have a significantly increased sensitivity to PPM1D inhibition when compared to wild-type controls. In addition, PPM1D inhibitor treatment was able to re-sensitize mutant cells to chemotherapy and abrogate the selective outgrowth of PPM1D-mutant cells during chemotherapy exposure. Lastly, we demonstrate that the proteome-wide phosphorylation profile characteristic of PPM1D-mutant cells can be reversed through treatment with the PPM1D inhibitor.In sum, these results demonstrate that PPM1D mutations confer a competitive advantage to hematopoietic stem cells undergoing genotoxic stress by abrogating the DNA damage response, and are likely to be the initiating mutation in a large proportion of t-MNs. Due to the gain-of-function nature of this mutation, PPM1D-mutant cells are differentially sensitive to treatment with a PPM1D inhibitor. PPM1D inhibition may therefore provide an opportunity for the prevention and targeted treatment of hematologic malignancies that harbor PPM1D mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The pathophysiological significance of PPM1D and therapeutic targeting of PPM1D-mediated signaling by GSK2830371 in mantle cell lymphoma.

PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 in mantle cell lymphoma (MCL). Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart cells. Higher PPM1D expression was associated with higher expression of the proliferation gene signature and poorer prognosis in patients. Eight MCL (three p53 wild-type and five mutant) cell lines were exposed to GSK2830371. GSK2830371 inhibited the cell growth, being prominent in p53 wild-type cells. GSK2830371 induced apoptosis in sensitive cells, as evidenced by induction of phosphatidylserine externalization and loss of mitochondrial membrane potential. p53 knockdown de-sensitized cell sensitivity. GSK2830371 increased the levels of total and Ser15-phosphorylated p53, and p53 targets p21 and PUMA. GSK2830371 and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 wild-type cells. Interestingly, GSK2830371 sensitized MCL cells to bortezomib and doxorubicin in p53 wild-type and mutant cells; p38 signaling appeared to be involved in the GSK2830371/bortezomib lethality. PPM1D inhibition may represent a novel therapeutic strategy for MCL, which can be exploited in combination therapeutic strategies for MCL.