Search

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase toward the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we identified three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and promote p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38α reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a unique approach to achieving improved potency and specificity for therapeutic kinase inhibitors., Competing Interests: Competing interests statement:D.K. is co-founder of Relay Therapeutics and MOMA Therapeutics. N.B. and E.J.S. are the inventors on a pending patent on a new method for optimizing kinase inhibitors applied for by Brandeis University.

Mycobacterium tuberculosis (Mtb), the infectious agent of tuberculosis (TB), causes over 1.5 million deaths globally every year. Host-directed therapies (HDT) for TB are desirable for their potential to shorten treatment and reduce the development of antibiotic resistance. Previously, we described a modular biomimetic strategy to identify SMIP-30 , targeting PPM1A (IC 50 = 1.19 μM), a metal-dependent phosphatase exploited by Mtb to survive intracellularly. SMIP-30 restricted the survival of Mtb in macrophages and lungs of infected mice. Herein, we redesigned SMIP-30 to create SMIP-031 , which is a more potent inhibitor for PPM1A (IC 50 = 180 nM). SMIP-031 efficiently increased the level of phosphorylation of S403-p62 and the expression of LC3B-II to activate autophagy, resulting in the dose-dependent clearance of Mtb in infected macrophages. SMIP-031 possesses a good pharmacokinetic profile and oral bioavailability ( F = 74%). In vivo, SMIP-031 is well tolerated up to 50 mg/kg and significantly reduces the bacteria burden in the spleens of infected mice.

Metal-dependent protein phosphatases (PPMs) have essential roles in a variety of cellular processes, including inflammation, proliferation, differentiation, and stress responses, which are intensively investigated in cancer and metabolic diseases. Targeting PPMs to modulate host immunity in response to pathogens is an ambitious proposition. The feasibility of such a strategy is unproven because development of inhibitors against PPMs is challenging and suffers from poor selectivity. Combining a biomimetic modularization strategy with function-oriented synthesis, we design, synthesize and screen more than 500 pseudo-natural products, resulting in the discovery of a potent, selective, and non-cytotoxic small molecule inhibitor for PPM1A, SMIP-30. Inhibition of PPM1A with SMIP-30 or its genetic ablation (ΔPPM1A) activated autophagy through a mechanism dependent on phosphorylation of p62-SQSTM1, which restricted the intracellular survival of Mycobacterium tuberculosis in macrophages and in the lungs of infected mice. SMIP-30 provides proof of concept that PPMs are druggable and promising targets for the development of host-directed therapies against tuberculosis., Competing Interests: Declaration of interests Y.C.L., L.C., Z.X., W.Y., and J.S. are cited as inventors for a filed United States provisional patent related to this work. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D., (© 2022. The Author(s).)

Research Question: What role does wild-type p53-induced phosphatase 1 (WIP1) play in the regulation of primordial follicle development?, Design: WIP1 expression was detected in the ovaries of mice of different ages by western blotting and immunohistochemical staining. Three-day-old neonatal mouse ovaries were cultured in vitro with or without the WIP1 inhibitor GSK2830371 (10 μM) for 4 days. Ovarian morphology, follicle growth and follicle classification were analysed and the PI3K-AKT-mTOR signal pathway and the WIP1-p53-related mitochondrial apoptosis pathway evaluated., Results: WIP1 expression was downregulated with age. Primordial follicles were significantly decreased in the GSK2830371-treated group, without a significant increase in growing follicles. The ratio of growing follicles to primordial follicles was not significantly different between the control and GSK2830371 groups, and no significant variation was observed in the PI3K-AKT-mTOR signal pathway. The inhibition of WIP1 phosphatase accelerated primordial follicle atresia by activating the p53-BAX-caspase-3 pathway., Conclusions: These findings reveal that WIP1 participates in regulating primordial follicle development and that inhibiting WIP1 phosphatase leads to massive primordial follicle loss via interaction with the p53-BAX-caspase-3 pathway. This might also provide valuable information for understanding decreased ovarian reserve during ovarian ageing., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.

Aberrations in DNA damage response genes are recognized mediators of tumorigenesis and resistance to chemo- and radiotherapy. While protein phosphatase magnesium-dependent 1 δ (PPM1D), located on the long arm of chromosome 17 at 17q22-23, is a key regulator of cellular responses to DNA damage, amplification, overexpression, or mutation of this gene is important in a wide range of pathologic processes. In this review, we describe the physiologic function of PPM1D, as well as its role in diverse processes, including fertility, development, stemness, immunity, tumorigenesis, and treatment responsiveness. We highlight both the advances and limitations of current approaches to targeting malignant processes mediated by pathogenic alterations in PPM1D with the goal of providing rationale for continued research and development of clinically viable treatment approaches for PPM1D-associated diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein phosphatase 1B (PPM1B), a member of metal-dependent protein serine/threonine phosphatase family, is involved in the regulation of several signalling pathways. However, our understanding of its substrate interaction and physiological functions is still largely limited. There is no reported PPM1B inhibitor to date. In this study, we identified HN252, a p-terphenyl derivative, as a potent PPM1B inhibitor (K i  = 0.52 ± 0.06 µM). HN252 binding to PPM1B displayed remarkable and specific inhibition of PPM1B in both in vitro and ex vivo. With the aid of this small molecular inhibitor, we identified 30 proteins' serine/threonine phosphorylation as potential substrates of PPM1B, 5 of which were demonstrated by immunoprecipitation, including one known (CDK2) and 4 novel ones (AKT1, HSP90B, β-catenin and BRCA1). Furthermore, GO and KEGG analysis of dramatically phosphorylated proteins by PPM1B inhibition indicated that PPM1B plays roles in the regulation of multiple cellular processes and signalling pathways, such as gene transcription, inflammatory regulation, ageing and tumorigenesis. Our work provides novel insights into further investigation of molecular mechanisms of PPM1B., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Cancer can metastasize from early lesions without detectable tumors. Despite extensive studies on metastasis in cancer cells from patients with detectable primary tumors, mechanisms for early metastatic dissemination are poorly understood. Her2 promotes breast cancer early dissemination by inhibiting p38, but the downstream pathway in this process was unknown. Using early lesion breast cancer models, we demonstrate that the effect of p38 suppression by Her2 on early dissemination is mediated by MK2 and heat shock protein 27 (Hsp27). The early disseminating cells in the MMTV-Her2 breast cancer model are Her2 high p-p38 low p-MK2 low p-Hsp27 low , which also exist in human breast carcinoma tissues. Suppression of p38 and MK2 by Her2 reduces MK2-mediated Hsp27 phosphorylation, and unphosphorylated Hsp27 binds to β-catenin and enhances its phosphorylation by Src, leading to β-catenin activation and disseminating phenotypes in early lesion breast cancer cells. Pharmacological inhibition of MK2 promotes, while inhibition of a p38 phosphatase Wip1 suppresses, early dissemination in vivo. These findings identify Her2-mediated suppression of the p38-MK2-Hsp27 pathway as a novel mechanism for cancer early dissemination, and provide a basis for new therapies targeting early metastatic dissemination in Her2 + breast cancer.

Diffuse intrinsic pontine glioma (DIPG) is an invariably fatal brain tumor occurring predominantly in children. Up to 90% of pediatric DIPGs harbor a somatic heterozygous mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3.3 (H3F3A, 65%) or H3.1 (HIST1H3B, 25%). Several studies have also identified recurrent truncating mutations in the gene encoding protein phosphatase 1D, PPM1D , in 9%-23% of DIPGs. Here, we sought to investigate the therapeutic potential of targeting PPM1D, alone or in combination with inhibitors targeting specific components of DNA damage response pathways in patient-derived DIPG cell lines. We found that GSK2830371, an allosteric PPM1D inhibitor, suppressed the proliferation of PPM1D -mutant, but not PPM1D wild-type DIPG cells. We further observed that PPM1D inhibition sensitized PPM1D -mutant DIPG cells to PARP inhibitor (PARPi) treatment. Mechanistically, combined PPM1D and PARP inhibition show synergistic effects on suppressing a p53-dependent RAD51 expression and the formation of RAD51 nuclear foci, possibly leading to impaired homologous recombination (HR)-mediated DNA repair in PPM1D -mutant DIPG cells. Collectively, our findings reveal the potential role of the PPM1D-p53 signaling axis in the regulation of HR-mediated DNA repair and provide preclinical evidence demonstrating that combined inhibition of PPM1D and PARP1/2 may be a promising therapeutic combination for targeting PPM1D -mutant DIPG tumors. IMPLICATIONS: The findings support the use of PARPi in combination with PPM1D inhibition against PPM1D -mutant DIPGs., (©2020 American Association for Cancer Research.)

A greater understanding of factors causing cancer initiation, progression and evolution is of paramount importance. Among them, the serine/threonine phosphatase PPM1D, also referred to as wild-type p53-induced phosphatase 1 (Wip1) or protein phosphatase 2C delta (PP2Cδ), is emerging as an important oncoprotein due to its negative regulation on a number of crucial cancer suppressor pathways. Initially identified as a p53-regulated gene, PPM1D has been afterwards found amplified and more recently mutated in many human cancers such as breast cancer. The latest progress in this field further reveals that selective inhibition of PPM1D to delay tumor onset or reduce tumor burden represents a promising anti-cancer strategy. Here, we review the advances in the studies of the PPM1D activity and its relevance to various cancers, and recent progress in development of PPM1D inhibitors and discuss their potential application in cancer therapy. Consecutive research on PPM1D and its relationship with cancer is essential, as it ultimately contributes to the etiology and treatment of cancer., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

ERBB2 amplification is a prognostic marker for aggressive tumors and a predictive marker for prolonged survival following treatment with HER2 inhibitors. We attempt to sub-group HER2+ tumors based on amplicon structures and co-amplified genes. We examined five HER2+ cell lines, three HER2+ xenographs and 57 HER2+ tumor tissues. ERBB2 amplification was analyzed using digital droplet PCR and low coverage whole genome sequencing. In some HER2+ tumors PPM1D, that encodes WIP1, is co-amplified. Cell lines were treated with HER2 and WIP1 inhibitors. We find that inverted duplication is the amplicon structure in the majority of HER2+ tumors. In patients suffering from an early stage disease the ERBB2 amplicon is composed of a single segment while in patients suffering from advanced cancer the amplicon is composed of several different segments. We find robust WIP1 inhibition in some HER2+ PPM1D amplified cell lines. Sub-grouping HER2+ tumors using low coverage whole genome sequencing identifies inverted duplications as the main amplicon structure and based on the number of segments, differentiates between local and advanced tumors. In addition, we found that we could determine if a tumor is a recurrent tumor or second primary tumor and identify co-amplified oncogenes that may serve as targets for therapy.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, P i We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens., (© 2019 Clausse et al.)

Objectives: Vascular smooth muscle cell (VSMC) proliferation is a crucial cause of vascular neointima hyperplasia and restenosis, thus limiting the long-term efficacy of percutaneous vascular intervention. We explored the role of wild-type p53-induced phosphatase 1 (Wip1), a potent regulator of tumorigenesis and atherosclerosis, in VSMC proliferation and neointima hyperplasia., Methods and Results: Animal model of vascular restenosis was established in wild type C57BL/6J and VSMC-specific Tuberous Sclerosis 1 (TSC1)-knockdown mice by wire injury. We observed increased protein levels of Wip1, phospho (p)-S6 Ribosomal Protein (S6), p-4EBP1 but decreased p-adenosine 5'-monophosphate-activated protein kinase (AMPK)α both in carotid artery at day 28 after injury and in VSMCs after 48 h of platelet derived growth factor-BB (PDGF-BB) treatment. By using hematoxylin-eosin staining, Ki-67 immunohistochemical staining, cell counting kit-8 assay and Ki-67 immunofluorescence staining, we found Wip1 antagonist GSK2830371 (GSK) or mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin both obviously reversed the neointima formation and VSMC proliferation induced by wire injury and PDGF-BB, respectively. GSK also reversed the increase in mRNA level of Collagen I after wire injury. However, GSK had no obvious effects on VSMC migration induced by PDGF-BB. Simultaneously, TSC1 knockdown as well as AMPK inhibition by Compound C abolished the vascular protective and anti-proliferative effects of Wip1 inhibition. Additionally, suppression of AMPK also reversed the declined mTORC1 activity by GSK., Conclusion: Wip1 promotes VSMC proliferation and neointima hyperplasia after wire injury via affecting AMPK/mTORC1 pathway.

Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.

Purpose: Accumulating reports have shown the oncogenic properties of PPMD1 (protein phosphatase, Mg2+/Mn2+ dependent 1D) in different cancer types. This study was undertaken to explore the role and therapeutic potential of PPM1D in colon cancer., Methods: HT-29 colon cancer cell line was used in this study. Expression analysis of PPMD1 was performed by qRT-PCR. Cell viability was determined by CCK-8 assay. DAPI, acridin orange/ethidium bromide (AO/EB) and propidium iodide (PI)staining assays were used for apoptosis detection. Cell cycle analysis was performed by flow cytometry. Protein expression was determined by western blot analysis., Results: The results showed that the expression of PPMD1 was significantly upregulated in colon cancer by 3.2 to 4.8 fold. Silencing of PPMD1 caused significant decline in the proliferation rate of the HT-29 colon cancer cells that was due to induction of apoptosis as evidenced by DAPI, AO/EB and PI staining. Annexin V/PI showed a significant increase in the percentage of apoptotic of HT-29 cells upon silencing of PPMD1. The induction of apoptosis was also accompanied by increase in Bax and decrease in Bcl-2 expression. PPMD1 silencing also resulted in arrest of the HT-29 cells in the G2/M phase of the cell cycle which was also associated with upsurge of p21 and p53 and depletion of cyclin B1 expression levels. PPMD1 silencing also caused decrease in the viability of the HT-29 cells which was concomitant with suppression of MMP-2 and MMP-9 expression., Conclusion: These findings suggest that PPMD1 has oncogenic properties in colon cancer and exhibit therapeutic implications in colon cancer treatment.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Epidemiologic evidence indicates that diabetes may increase risk of breast cancer (BC) and mortality in patients with cancer. The pathophysiological relationships between diabetes and cancer are not fully understood, and personalized treatments for diabetes-associated BC are urgently needed. Results: We observed that high glucose (HG), via activation of nuclear phosphatase PP2Cδ, suppresses p53 function, and consequently promotes BC cell proliferation, migration, and invasion. PP2Cδ expression is higher in tumor tissues from BC patients with hyperglycemia than those with normoglycemia. The mechanisms underlying HG stimulation of PP2Cδ involve classical/novel protein kinase-C (PKC) activation and GSK3β phosphorylation. Reactive oxygen species (ROS)/NF-κB pathway also mediates HG induction of PP2Cδ. Furthermore, we identified a 1,5-diheteroarylpenta-1,4-dien-3-one (Compound 23 , or C23) as a novel potent PP2Cδ inhibitor with a striking cytotoxicity on MCF-7 cells through cell-based screening assay for growth inhibition and activity of a group of curcumin mimics. Beside directly inhibiting PP2Cδ activity, C23 blocks HG induction of PP2Cδ expression via heat shock protein 27 (HSP27) induction and subsequent ablation of ROS/NF-κB activation. C23 can thus significantly block HG-triggered inhibition of p53 activity, leading to the inhibition of cancer cell proliferation, migration, and invasion. In addition, hyperglycemia promotes BC development in diabetic nude mice, and C23 inhibits the xenografted BC tumor growth. Conclusions and Innovation: Our findings elucidate mechanisms that may have contributed to diabetes-associated BC progression, and provide the first evidence to support the possible alternative therapeutic approach to BC patients with diabetes. Antioxid. Redox Signal . 30, 1983-1998.

The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Obesity is a worldwide public health problem, which is associated with various severe diseases including diabetes, hypertension, atherosclerosis, and cancer. Recent studies have revealed that combination treatment of several different compounds using low doses is effective to reduce side effects. Thus, there is a need to develop an efficient inhibitor for reducing lipid droplets with a divergent target/pathway. Ser/Thr protein phosphatase PPM1D is involved in cellular metabolic processes and is a promising target for anti-obesity treatment. We have previously developed a potent and specific PPM1D inhibitor, SL-176. In this study, we demonstrated that significant reduction of lipid droplet formation in adipocytes by the PPM1D specific inhibitor, SL-176. Using Oil-red O staining and fluorescent imaging of lipid droplet, we found that treatment of SL-176 significantly suppressed lipid droplet formation of 3T3-L1 cells both in amount and in size. SL-176 also repressed mRNA and protein expression of PPARγ and C/EBPα, adipogenic markers, at nontoxic conditions. Thus, SL-176 is a unique and potent inhibitor of lipid droplet formation that acts via PPM1D, a novel target toward inhibiting adipocyte differentiation., Competing Interests: The authors have declared that no competing interests exist.

Background: Excessive cardiac fibrosis due to maladaptive remodeling leads to progression of cardiac dysfunction and is modulated by TGF-β1-activated intracellular phospho-SMAD signaling effectors and transcription regulators. SMAD2/3 phosphorylation, regulated by protein-phosphatases, has been studied in different cell types, but its role in human ventricular fibroblasts (hVFs) is not defined as a target to reduce cytokine-mediated excessive fibrotic response and adverse cardiac remodeling. Statins are a class of drugs reported to reduce cardiac fibrosis, although underlying mechanisms are not completely understood. We aimed to assess whether simvastatin-mediated reduction in TGF-β1-augmented profibrotic response involves reduction in phospho-SMAD2/3 owing to activation of protein-phosphatase in hVFs., Methods and Results: Cultures of hVFs were used. Effect of simvastatin on TGF-β1-treated hVF proliferation, cytotoxicity, myofibroblast differentiation/activation, profibrotic gene expression and protein-phosphatase activity was assessed. Simvastatin (1 μM) reduced effect of TGF-β1 (5 ng/mL) on hVF proliferation, myofibroblast differentiation (reduced α-smooth muscle actin [α-SMA-expression]) and activation (decreased procollagen-peptide release). Simvastatin also reduced TGF-β1-stimulated time-dependent increases in SMAD2/3 phosphorylation and nuclear translocation, mediated through catalytic activation of protein-phosphatases PPM1A and PP2A, which physically interact with SMAD2/3, thereby promoting their dephosphorylation. Effect of simvastatin on TGF-β1-induced fibroblast activation was annulled by okadaic acid, an inhibitor of protein-phosphatase., Conclusions: This proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-β1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Maternal DNA damage during meiosis causes genetic abnormalities that can lead to infertility, birth defects, and abortion. While DNA damage can rapidly halt cell cycle progression and promote DNA repair in somatic cells, mammalian oocytes are unable to mount a robust G2/prophase arrest in response to DNA damage unless damage levels are severe. Here, we show that inhibition of WIP1 phosphatase enhances the ability of oocytes to respond to DNA damage. We found that WIP1 was expressed constantly during meiotic maturation, and that inhibition of WIP1 activity did not impair meiotic maturation. However, oocytes in G2/prophase were sensitized to DNA damage following WIP1 inhibition, not only increasing γ-H2AX level and ATM phosphorylation, but also decreasing entry into meiosis. Moreover, WIP1 inhibition significantly promoted the repair of damaged DNA during G2/prophase arrest, suggesting that WIP1 suppresses DNA repair in oocytes. Therefore, our results suggest that WIP1 is a key suppressor of the DNA damage response during G2/prophase arrest in mouse oocytes.

Truncating mutations in the terminal exon of protein phosphatase Mg2 + /Mn2 + 1D ( PPM1D ) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D -mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D -mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D- mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D -mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D -mutant cells and the treatment of PPM1D -mutant disease., (© 2018 by The American Society of Hematology.)

Chlamydiae are obligate intracellular Gram-negative bacterial pathogens that undergo an essential, but poorly understood, biphasic developmental cycle transitioning between the infectious elementary body and the replicative reticulate body. Ser/Thr/Tyr phosphorylation has been increasingly recognized for its role in regulating bacterial physiology. Chlamydia spp. encode two Hanks'-type kinases in addition to a type 2C protein phosphatase (PP2C; CppA) and appears capable of global protein phosphorylation. While these findings substantiate the importance of protein phosphorylation in Chlamydia , the physiological impact of protein phosphorylation remains enigmatic. In this study, we investigated the in vivo role of CppA by using recombinant protein point mutants and small-molecule inhibitors. Recombinant CppA (rCppA) amino acid point mutants based upon missense mutations identified in growth-deficient Chlamydia trachomatis strains exhibited reduced, but not a complete loss of, phosphatase activity toward p -nitrophenyl phosphate (pNPP) and phosphopeptides. To more directly explore the importance of CppA in chlamydial development, we implemented a chemical "knockout" approach using derivatives of 5,5'-methylenedisalicylic acid (MDSA). Several MDSA derivatives significantly reduced CppA activity in vitro and the growth of C. trachomatis L2, C. trachomatis D, and Chlamydia muridarum in a cell culture infection model. The inhibition of C. trachomatis L2 growth was more pronounced when treated at earlier infection time points, and the removal of the inhibitors after 12 h postinfection did not rescue progeny production. Our findings revealed that altered CppA activity reduces chlamydial growth and that CppA function is likely crucial for early differentiation events. Collectively, our findings further support the importance of the protein phosphorylation network in chlamydial development. IMPORTANCE Chlamydia is a significant cause of disease in humans, including sexually transmitted infections, the ocular infection trachoma, and pneumonia. Despite the critical roles of protein phosphatases in bacterial physiology, their function in pathogenesis is less clear. Our findings demonstrate that CppA, a broad-specificity type 2C protein phosphatase (PP2C), is critical for chlamydial development and further substantiate reversible phosphorylation as a key regulatory mechanism in Chlamydia Additionally, our work highlights the potential of CppA to serve as a novel target for future therapeutic strategies and supports the feasibility of designing more potent PP2C phosphatase inhibitors for Chlamydia and other pathogenic bacteria., (Copyright © 2018 American Society for Microbiology.)

The wild-type p53 induced phosphatase 1, Wip1 (PP2Cδ), is a protein phosphatase 2C (PP2C) family serine/threonine phosphatase that negatively regulates the function of the tumor suppressor p53 and several of its positive regulators such as ATM, Chk1, Chk2, Mdm2, and p38 MAPK. Wip1 dephosphorylates and inactivates its protein targets, which are critical for cellular stress responses. Additionally, Wip1 is frequently amplified and overexpressed in several human cancer types. Because of its negative role in regulating the function of tumor suppressor proteins, Wip1 has been identified as a potential therapeutic target in various types of cancers. Based on a recently reported Wip1 inhibitor (G-1), we performed an extensive structure-activity relationship (SAR) analysis. This led us to interesting findings in SAR trends and to the discovery of new chemical analogues with good specificity and bioavailability., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogation leads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition of ATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by the presence of ATR-dependent phosphorylation marks (gamma H2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further, such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby 'replication catastrophe'. PPM1D phosphatase, a nucleolar localized protein, relocates to chromatin during replication stress and reverts back to nucleolus following stress recovery, under the control of ATR kinase action. Inhibition of ATR kinase activity, specifically during post replication stress, triggers dislodging of the chromatin-bound PPM1D from nucleus to cytoplasm followed by its degradation, thereby leading to persistence of activated ATR marks in the nuclei. Chemical inhibition of PPM1D activity or SiRNA mediated depletion of the protein during post replication stress recovery 'phenocopies' ATR kinase inhibition by failing to attenuate ATR signalling. Collectively, our observations suggest a novel role of ATR kinase in mediating its own signal attenuation via PPM1D recruitment to chromatin as an essential mechanism for restarting the stalled forks, cell-cycle re-entry and cellular recovery from replication stress.

Protein Phosphatase 2C (PP2C) is an important phosphatase-like protein in eukaryotic organisms that can negatively regulate protein kinase cascade abscisic acid (ABA) signal system through phosphorylation and carry out vital roles in various cell processes. The previous study indicated that the accumulation of reactive oxygen species (ROS) is a part of mechanism of glucohexaose-induced resistance in cucumber cotyledons, and CsPP2C80s might play a crucial role in processes related to ROS produce and signal transduction. To identify the mechanism of CsPP2C80s involved in glucohexaose and ABA signaling regulating cell redox status, the effects of glucohexaose and ROS inhibitor pretreatment on endogenous ABA content and ABA signaling genes expression levels of cucumber seedlings were analysed. These results suggest that cucumber CsPP2C80s are involved in ROS accumulation and ABA signal transduction pathway induced by glucohexaose, CsPP2C80s play a positive regulatory role in process of ABA combined with ABA receptors (PYLs) to activate SNF1-related protein kinases 2 (SnRK2s) and regulate NADPH oxidase to produce extracellular hydrogen peroxide (H2O2), providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in cell redox status induced by glucohexaose.

Background: Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2-p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells., Methods: A panel of three p53 WT (A375, WM35 and C8161) and three p53 MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT-PCR gene expression profiling and flow cytometry., Results: GSK2830371, at doses (⩽10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53 WT but not p53 MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis., Conclusions: GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.

The blood brain barrier (BBB) is a diffusion barrier that maintains the brain environment. Wip1 is a nuclear phosphatase induced by many factors and involved in various stresses, tumorigenesis, organismal aging, and neurogenesis. Wip1's role in BBB integrity has not been thoroughly investigated. The purpose of the present study was to investigate the effect and mechanism of Wip1 on lipopolysaccharide (LPS)-induced BBB dysfunction and inflammation in an in vitro BBB model. The in vitro BBB model was established by co-culturing human brain-microvascular endothelial cells and human astrocytes and then exposing them to 1μg/ml LPS for 6, 12, 18, 24, and 48h. Wip1 expression was significantly elevated by LPS treatment. Knockdown of Wip1 aggravated the increased permeability and decreased transepithelial electrical resistance, protein expression of ZO-1, and occludin induced by LPS. Wip1 silencing augmented the elevated inflammatory cytokines TNF-α, IL-1β, IL-12, and IL-6 of the BBB induced by LPS, whereas overexpression of Wip1 showed a contrary effect. Sonic hedgehog signaling (SHH) was activated by Wip1 overexpression and inhibited by Wip1 silencing. Additionally, activating or inhibiting the SHH pathway by purmorphamine or cyclopamine, respectively, abolished the Wip1-induced changes in transepithelial electrical resistance and permeability and inflammatory responses in the LPS-injured BBB model. Our results demonstrate that Wip1 may protect the BBB against LPS-induced integrity disruption and inflammatory response through the SHH signaling pathway., (Copyright © 2017. Published by Elsevier Ltd.)

Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy. It has been reported that PPM1D knockout mice exhibit neutrophilia in blood and show a defective immune response. Here, we found that inhibition of PPM1D induced neutrophil differentiation of human promyelocytic leukemia cell line HL-60. The combination of a PPM1D inhibitor and all-trans retinoic acid significantly increased their differentiation efficiency. The PPM1D inhibitor also induced G1 arrest in HL-60 cells. Our results suggest that PPM1D may be a potential therapeutic target for blood cell diseases including leukemia., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Aquaporin 2 (AQP2) trafficking is regulated by phosphorylation and dephosphorylation of serine residues in the AQP2 COOH terminus. Vasopressin (VP) binding to its receptor (V2R) leads to a cascade of events that result in phosphorylation of serine 256 (S256), S264, and S269, but dephosphorylation of S261. To identify which phosphatase is responsible for VP-induced S261 dephosphorylation, we pretreated cells with different phosphatase inhibitors before VP stimulation. Sanguinarine, a specific protein phosphatase (PP) 2C inhibitor, but not inhibitors of PP1, PP2A (okadaic acid), or PP2B (cyclosporine), abolished VP-induced S261 dephosphorylation. However, sanguinarine and VP significantly increased phosphorylation of ERK, a kinase that can phosphorylate S261; inhibition of ERK by PD98059 partially decreased baseline S261 phosphorylation. These data support a role of ERK in S261 phosphorylation but suggest that, upon VP treatment, increased phosphatase activity overcomes the increase in ERK activity, resulting in overall dephosphorylation of S261. We also found that sanguinarine abolished VP-induced S261 dephosphorylation in cells expressing mutated AQP2 S256A, suggesting that the phosphorylation state of S261 is independent of S256. Sanguinarine alone did not induce AQP2 membrane trafficking, nor did it inhibit VP-induced AQP2 membrane accumulation in cells and kidney tissues, suggesting that S261 does not play an observable role in acute AQP2 membrane accumulation. In conclusion, PP2C activity is required for S261 AQP2 dephosphorylation upon VP stimulation, which occurs independently of S256 phosphorylation. Understanding the pathways involved in modulating PP2C will help elucidate the role of S261 in cellular events involving AQP2., (Copyright © 2017 the American Physiological Society.)

DNA damage response (DDR) pathway protects cells from genome instability and prevents cancer development. Tumor suppressor p53 is a key molecule that interconnects DDR, cell cycle checkpoints, and cell fate decisions in the presence of genotoxic stress. Inactivating mutations in TP53 and other genes implicated in DDR potentiate cancer development and also influence the sensitivity of cancer cells to treatment. Protein phosphatase 2C delta (referred to as WIP1) is a negative regulator of DDR and has been proposed as potential pharmaceutical target. Until recently, exploitation of WIP1 inhibition for suppression of cancer cell growth was compromised by the lack of selective small-molecule inhibitors effective at cellular and organismal levels. Here, we review recent advances in development of WIP1 inhibitors and discuss their potential use in cancer treatment.

The phytohormone abscisic acid (ABA) plays a crucial role in mediating plant growth and development by recruiting genetically redundant ABA receptors. To overcome its oxidation inactivation, we developed a novel ABA analog named 2',3'-benzo-iso-ABA (iso-PhABA) and studied its function and structural characterization with A. thaliana ABA receptors. The (+)-iso-PhABA form showed much higher ABA-like activities than (+)-ABA including inhibitory effects on the seed germination of lettuce and A. thaliana, wheat embryo germination and rice seedling elongation. The PP2C (protein phosphatases 2C) activity assay showed that (+)-iso-PhABA acted as a potent and selective ABA receptor agonist, which is preferred to PYL10. In some cases, (-)-iso-PhABA showed moderate to high activity for the PYL protein inhibiting PP2C activity, suggesting different mechanisms of action of iso-PhABA and ABA. The complex crystal structure of iso-PhABA with PYL10 was determined and elucidated successfully, revealing that (+)-iso-PhABA was better coordinated in the same binding pocket compared to (+)-ABA. Moreover, the detailed interaction network of iso-PhABA/PYL10 was disclosed and involves hydrogen bonds and multiple hydrophobic interactions that provide a robust framework for the design of novel ABA receptor agonists/antagonists.

Microcystins are a group of toxins produced by freshwater cyanobacteria. Uptake of microcystin-leucine arginine (MC-LR) by organic anion transporting polypeptide 1B2 in hepatocytes results in inhibition of protein phosphatase 1A and 2A, and subsequent cell death. Studies performed in primary rat hepatocytes demonstrate prototypical apoptosis after MC-LR exposure; however, no study has directly tested whether apoptosis is critically involved in vivo in the mouse, or in human hepatocytes. MC-LR (120 μg/kg) was administered to C57BL/6J mice and cell death was evaluated by alanine aminotransferase (ALT) release, caspase-3 activity in the liver, and histology. Mice exposed to MC-LR had increases in plasma ALT values, and hemorrhage in the liver, but no increase in capase-3 activity in the liver. Pre-treatment with the pan-caspase inhibitor z-VAD-fmk failed to protect against cell death measured by ALT, glutathione depletion, or hemorrhage. Administration of MC-LR to primary human hepatocytes resulted in significant toxicity at concentrations between 5 nM and 1 μM. There were no elevated caspase-3 activities and pretreatment with z-VAD-fmk failed to protect against cell death in human hepatocytes. MC-LR treated human hepatocytes stained positive for propidium iodide, indicating membrane instability, a marker of necrosis. Of note, both increases in PI positive cells, and increases in lactate dehydrogenase release, occurred before the onset of complete actin filament collapse. In conclusion, apoptosis does not contribute to MC-LR-induced cell death in the in vivo mouse model or in primary human hepatocytes in vitro. Thus, targeting necrotic cell death mechanisms will be critical for preventing microcystin-induced liver injury., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Temozolomide (TMZ) is approved for use as first-line treatment for glioblastoma multiforme (GBM). However, GBM shows chemoresistance shortly after the initiation of treatment. In order to detect whether silencing of human protein phosphatase 1D magnesium dependent (PPM1D) gene could increase the effects of TMZ in glioma cells, glioma cells U87-MG were infected with lentiviral shRNA vector targeting PPM1D silencing. After PPM1D silencing was established, cells were treated with TMZ. The multiple functions of human glioma cells after PPM1D silencing and TMZ chemotherapy were detected by flow cytometry and MTT assay. Significantly differentially expressed genes were distinguished by microarray-based gene expression profiling and analyzed by gene pathway enrichment analysis and ontology assessment. Western blotting was used to establish the protein expression of the core genes. PPM1D gene silencing improves TMZ induced cell proliferation and induces cell apoptosis and cell cycle arrest. When PPM1D gene silencing combined with TMZ was performed in glioma cells, 367 genes were upregulated and 444 genes were downregulated compared with negative control. The most significant differential expression pathway was pathway in cancer and IGFR1R, PIK3R1, MAPK8 and EP300 are core genes in the network. Western blotting showed that MAPK8 and PIK3R1 protein expression levels were upregulated and RB1 protein expression was decreased. It was consistent with that detected in gene expression profiling. In conclusion, PPM1D gene silencing combined with TMZ eradicates glioma cells through cell apoptosis and cell cycle arrest. PIK3R1/AKT pathway plays a role in the multiple functions of glioma cells after PPM1D silencing and TMZ chemotherapy.

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.

Cytosolic RNA sensing is a prerequisite for initiation of innate immune response against RNA viral pathogens. Signaling through RIG-I (retinoic acid-inducible gene I)-like receptors (RLRs) to TBK1 (Tank-binding kinase 1)/IKKε (IκB kinase ε) kinases is transduced by mitochondria-associated MAVS (mitochondrial antiviral signaling protein). However, the precise mechanism of how MAVS-mediated TBK1/IKKε activation is strictly controlled still remains obscure. We reported that protein phosphatase magnesium-dependent 1A (PPM1A; also known as PP2Cα), depending on its catalytic ability, dampened the RLR-IRF3 (interferon regulatory factor 3) axis to silence cytosolic RNA sensing signaling. We demonstrated that PPM1A was an inherent partner of the TBK1/IKKε complex, targeted both MAVS and TBK1/IKKε for dephosphorylation, and thus disrupted MAVS-driven formation of signaling complex. Conversely, a high level of MAVS can dissociate the TBK1/PPM1A complex to override PPM1A-mediated inhibition. Loss of PPM1A through gene ablation in human embryonic kidney 293 cells and mouse primary macrophages enabled robustly enhanced antiviral responses. Consequently, Ppm1a(-/-) mice resisted to RNA virus attack, and transgenic zebrafish expressing PPM1A displayed profoundly increased RNA virus vulnerability. These findings identify PPM1A as the first known phosphatase of MAVS and elucidate the physiological function of PPM1A in antiviral immunity on whole animals.

Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists., Competing Interests: The authors declare no conflict of interest.

PP2C family serine/threonine phosphatase WIP1 acts as a negative regulator of the tumor suppressor p53 and is implicated in silencing of cellular responses to genotoxic stress. Chromosomal locus 17q23 carrying the PPM1D (coding for WIP1) is commonly amplified in breast carcinomas and WIP1 was proposed as potential pharmacological target. Here we employed a cellular model with knocked out PPM1D to validate the specificity and efficiency of GSK2830371, novel small molecule inhibitor of WIP1. We have found that GSK2830371 increased activation of the DNA damage response pathway to a comparable level as the loss of PPM1D. In addition, GSK2830371 did not affect proliferation of cells lacking PPM1D but significantly supressed proliferation of breast cancer cells with amplified PPM1D. Over time cells treated with GSK2830371 accumulated in G1 and G2 phases of the cell cycle in a p21-dependent manner and were prone to induction of senescence by a low dose of MDM2 antagonist nutlin-3. In addition, combined treatment with GSK2830371 and doxorubicin or nutlin-3 potentiated cell death through a strong induction of p53 pathway and activation of caspase 9. We conclude that efficient inhibition of WIP1 by GSK2830371 sensitizes breast cancer cells with amplified PPM1D and wild type p53 to chemotherapy.

Sensitivity to MDM2 inhibitors is widely different among responsive TP53 wild-type cell lines and tumors. Understanding the determinants of MDM2 inhibitor sensitivity is pertinent for their optimal clinical application. Wild-type p53-inducible phosphatase-1 (WIP1) encoded by PPM1D, is activated, gained/amplified in a range of TP53 wild-type malignancies, and is involved in p53 stress response homeostasis. We investigated cellular growth/proliferation of TP53 wild-type and matched mutant/null cell line pairs, differing in PPM1D genetic status, in response to Nutlin-3/RG7388 ± a highly selective WIP1 inhibitor, GSK2830371. We also assessed the effects of GSK2830371 on MDM2 inhibitor-induced p53(Ser15) phosphorylation, p53-mediated global transcriptional activity, and apoptosis. The investigated cell line pairs were relatively insensitive to single-agent GSK2830371. However, a non-growth-inhibitory dose of GSK2830371 markedly potentiated the response to MDM2 inhibitors in TP53 wild-type cell lines, most notably in those harboring PPM1D-activating mutations or copy number gain (up to 5.8-fold decrease in GI50). Potentiation also correlated with significant increase in MDM2 inhibitor-induced cell death endpoints that were preceded by a marked increase in a WIP1 negatively regulated substrate, phosphorylated p53(Ser15), known to increase p53 transcriptional activity. Microarray-based gene expression analysis showed that the combination treatment increases the subset of early RG7388-induced p53 transcriptional target genes. These findings demonstrate that potent and selective WIP1 inhibition potentiates the response to MDM2 inhibitors in TP53 wild-type cells, particularly those with PPM1D activation or gain, while highlighting the mechanistic importance of p53(Ser15) and its potential use as a biomarker for response to this combination regimen., (©2016 American Association for Cancer Research.)