Wip1, the product of the PPM1D gene, is a PP2C serine/threonine protein phosphatase that was first identified in my laboratory as a gene whose induction following DNA damage required wild-type p53. In humans and mice, Wip1 is expressed at low levels in most adult tissues, but is expressed at higher levels in proliferating tissues. Wip1 overexpression, either reflecting gene amplification or other mechanisms, is found in a substantial fraction of cases of several human cancers, including breast cancers, ovarian clear cell carcinomas and neuroblastoma, and is generally associated with a worse prognosis. To better understand the connections between Wip1 activity and the disregulation of cellular proliferation that characterizes cancer, we are investigating the regulation of Wip1 expression and activity, identifying targets of Wip1 phosphatase activity, and developing inhibitors of Wip1 phosphatase activity. Regulation of Wip1 expression and activity. In cells containing wild-type p53, the levels of Wip1 mRNA and protein transiently increase following exposure to genotoxic stress. Several recent papers have demonstrated that Wip1 plays a role in the down-regulation of DNA damage response signaling by dephosphorylating Ser1981 of ATM and Ser139 of gamma-H2AX in several cell lines. In primary fibroblasts from Xeroderma pigmentosum (XP) patients, exposure to UV results in elevated levels of p53 and sustained phosphorylation of many proteins involved in the DNA damage response, including ATM, H2AX, NBS1 and p53. To examine the connection between Wip1 induction and the persistence of H2AX phosphorylation in fibroblasts, we used a monoclonal antibody to detect endogenous Wip1 protein in normal and XP-B cells. Exposure of normal fibroblasts to UV resulted in elevation of Wip1 at 6hr and 24 hr, with concomitant decreased gamma-H2AX levels. In marked contrast, the level of endogenous Wip1 decreased at 6 h or 24 h after UV in the XP-B cells, with concomitant increased gamma-H2AX levels, suggesting that defective induction of Wip1 contributes to sustained H2AX phosphorylation in XP fibroblasts. To further understand the regulation of Wip1 following exposure to genotoxic stress, we have initiated studies into post-translational regulation of Wip1 protein levels after ionizing radiation. We examined the protein half-life and investigated the mechanism of degradation in unstressed cells and after cellular stress. We have determined that Wip1 undergoes proteosome-dependent degradation, but, interestingly, is not detectably ubiquitinated. We have also found that exposure to ionizing radiation results in increased stability of the Wip1 protein. These studies provide important insights into how the Wip1 mRNA and protein levels are regulated and how the increased stability of Wip1 following exposure to ionizing radiation may affect cell survival. In addition to the p53 response elements, the PPM1D promoter also contains several binding sites for cell cycle-related transcription factors, suggesting that in non-stressed cells, PPM1D mRNA and protein levels may be regulated in a cell cycle-dependent manner. To investigate the importance of regulation of Wip1 expression during the cell cycle, we have analyzed the changes in PPM1D mRNA and protein levels in different stages of the cell cycle in primary human cells and in tumor-derived cell lines. We have determined that the cell cycle-dependent regulation of Wip1 expression is frequently altered in tumor-derived cell lines. In addition, these findings suggest post-translational regulation of Wip1 protein levels may contribute to the association between high Wip1 protein levels and highly proliferative cells. Targets of Wip1 phosphatase activity. We have recently characterized the substrate specificity of Wip1 using biochemical studies. The substrates of Wip1 that have been characterized to date display pTXpY or pTQ/pSQ motifs. The former motif is exemplified by p38MAPK and UNG2, the nuclear isoform of the uracil DNA glycosylase. Many of the substrates that display the latter motif are targets of ATM kinase activity and function in the DNA damage response (DDR) pathway, including Chk1, Chk2, p53, H2AX and ATM itself. To identify potential novel substrates outside of the DDR pathway, we searched for motifs compatible with Wip1 specificity among known ATM targets. Among these, we identified Ser112 of 4E-BP1, an inhibitor of EIF4E-dependent mRNA export and cap-dependent translation. Wip1 is able to dephosphorylate this residue in vitro using as a substrate either a purified phosphopeptide or a recombinant protein phosphorylated in vitro. In addition, co-immunoprecipitation experiments showed that Wip1 interacted with 4E-BP1. Modulation of Wip1 levels resulted in changes in EIF4E activity consistent with dephosphorylation of Ser112 of 4E-BP1. Thus, our results suggest a mechanism in which modulation of EIF4E activity by Wip1 occurs at two levels: first by inhibiting ATM activity through dephosphorylation of Ser1981 and second by dephosphorylation of Ser112 of 4E-BP1. Wip1 Inhibitors. Many human tumors in which the PPM1D gene is amplified or overexpressed contain wild type p53. Our published results indicate that Wip1 phosphatase is a candidate proto-oncogene that promotes tumorigenesis through inactivation of wild-type p53. Phosphatases in general play critical physiological roles in the cell as the antagonists of kinase activity. Because of this, phosphatases represent important targets in a number of diseases, including cancer. Most of the chemical scaffolds for inhibitors of phosphatases have limited selectivity and cell permeability. Based upon our previous work, we have continued to optimize the structure and inhibitory activity of small molecules designed to inhibit specifically Wip1 phosphatase. Most of the efforts on small molecule inhibitors thus far have focused on improving the solution-phase synthesis to facilitate the production of a small library of compounds to screen for activity against Wip1 and selectivity using a closely-related phosphatase. We have developed an efficient synthetic route for production of penta-substituted pyrrole Wip1 inhibitors. The synthesis has a high degree of flexibility for the modification of all five side-chains of the penta-substituted pyrroles and has facilitated structure-activity relationship studies. We are also considering means of getting the compounds into cells for cell-based studies of these compounds. For example, we are currently pursuing a prodrug approach to mask the two phosphate groups on the inhibitors and deliver them to the cell. At the same time that we have developed the synthesis of the small molecule inhibitors, we have also used cyclic peptides as a template to determine which residues and positions are most critical for inhibitory activity. We have identified the positions on the cyclic peptide inhibitor that are most important for inhibition and identified residues on Wip1 that seem to interact with these sites. Our best cyclic peptides show IC50 of 170 nM. We will use the structure of the most active cyclic peptides to drive the design of new small molecules that we anticipate will have similar to better inhibitory activity. Finally, we are pursuing a co-crystal structure of Wip1 with a lead cyclic peptide inhibitor to facilitate further optimization of both the peptide and small molecule inhibitors. The best compounds will be analyzed in cell-based assays and in vivo animal models for their activity. The animal models, in turn, will allow us to examine the efficacy of the inhibitors as a chemotherapeutic for a wide array of tumors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011197-02
Application #
8157690
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2010
Total Cost
$355,956
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Giardino Torchia, Maria Letizia; Dutta, Debjani; Mittelstadt, Paul R et al. (2018) Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome. Proc Natl Acad Sci U S A 115:2174-2179
Debnath, Subrata; Kosek, Dalibor; Tagad, Harichandra D et al. (2018) A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity. J Biol Chem 293:7993-8008
Alam, Muhammad S; Gaida, Matthias M; Debnath, Subrata et al. (2018) Unique properties of TCR-activated p38 are necessary for NFAT-dependent T-cell activation. PLoS Biol 16:e2004111
Tagad, Harichandra D; Debnath, Subrata; Clausse, Victor et al. (2018) Chemical Features Important for Activity in a Class of Inhibitors Targeting the Wip1 Flap Subdomain. ChemMedChem 13:894-901
Sakai, Hiroyasu; Fujigaki, Hidetsugu; Mazur, Sharlyn J et al. (2014) Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication. Cell Cycle 13:1015-29
Tanoue, Kan; Miller Jenkins, Lisa M; Durell, Stewart R et al. (2013) Binding of a third metal ion by the human phosphatases PP2C? and Wip1 is required for phosphatase activity. Biochemistry 52:5830-43
Dudgeon, Crissy; Shreeram, Sathyavageeswaran; Tanoue, Kan et al. (2013) Genetic variants and mutations of PPM1D control the response to DNA damage. Cell Cycle 12:2656-64
Goloudina, Anastasia R; Tanoue, Kan; Hammann, Arlette et al. (2012) Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents. Proc Natl Acad Sci U S A 109:E68-75
Lowe, Julie; Cha, Hyukjin; Lee, Mi-Ok et al. (2012) Regulation of the Wip1 phosphatase and its effects on the stress response. Front Biosci (Landmark Ed) 17:1480-98
Goloudina, Anastasia R; Mazur, Sharlyn J; Appella, Ettore et al. (2012) Wip1 sensitizes p53-negative tumors to apoptosis by regulating the Bax/Bcl-xL ratio. Cell Cycle 11:1883-7

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