Regulation and Function of WIP1 Phosphatase and its Role in Tumor Cells

Division Of Basic Sciences - Nci

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Abstract

The wild-type p53-induced phosphatase Wip1 (PP2Cdelta or PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. The Wip1 protein is frequently overexpressed or the PPM1D gene is amplified in several human cancers, and this increased expression is associated with worse outcomes. Studies of human cells have shown that overexpression of Wip1 compromises tumor suppressor functions, and studies of mice that lack Wip1 show that they are resistant to tumorigenesis. The current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens (HTS) of small molecule libraries to identify specific modulators of Wip1 phosphatase activity. Recently, we characterized the effects of the binding of the labile metal ion and the phospho-peptide substrate on the conformation of human PPM1A, a family member of Wip1, by both hydrogen/deuterium exchange mass spectrometry and x-ray crystallography. Together, these structural studies have allowed us to better understand substrate binding in this family of phosphatases and characterize the labile third metal ion that is essential for catalytic activity, both critical aspects that could be abrogated by the binding of a specific inhibitor. In collaboration with Dr. Oleg Demidov (University of Burgundy, Dijon, France), we have used syngeneic tumor models to investigate the effects of ablating Wip1 in the immune system on tumor progression. We found that myeloid-specific deletion of Wip1 delayed the growth of both B10 melanoma tumors and LLC1 lung cancer tumors, confirming an important role of Wip1-deficient innate immune cells in anti-tumor immunity. The loss of Wip1 expression significantly increased the infiltration of solid tumors by myeloid granulocytes and neutrophils. Genetic depletion of Ppm1d in neutrophils transformed them into efficient activators of CD8 + cytotoxic lymphocytes, in part, through p53-dependent induction of lymphocyte co-stimulating ligands 4-1BBL and OX-40L. Recently, we have used RNA Seq and found that tumor-infiltrated neutrophils induce profound phenotypic changes resulting in an anti-tumor phenotype characterized by increased expression of genes involved in antigen presentation and a metabolic switch. Determination of a high-resolution structure of the Wip1 catalytic domain that includes the conformation of the B-loop would greatly aid further development of specific inhibitors of Wip1 phosphatase activity. Additionally, high-resolution structural information for the Wip1 catalytic site would be useful for further optimization of known inhibitors and activators and to guide structure-activity investigations. To that end, we have continued optimizing the expression of recombinant Wip1, and screening for crystallization conditions. These systematic efforts have provided the first structure of Wip1 that will help to better understand the specificity and potential for Wip1 inhibition and activation. As Wip1 is amplified or overexpressed in numerous human cancers including breast cancer, ovarian clear cell carcinoma, gastric cancer, pancreatic adenocarcinoma, medulloblastoma, and neuroblastoma, developing inhibitors of Wip1 activity may be beneficial in the treatment of several human cancers. Wip1, though, can function as a tumor suppressor in cancer cells bearing inactive mutated p53. Therefore, developing activators of Wip1 is as important as characterizing inhibitors of this phosphatase. We have developed and validated two orthogonal plate-based Wip1 activity assays for high-throughput screens (HTS). The two assays have high sensitivity and broad dynamic range enabled by either fluorescence detection or mass spectrometry and are suitable for screening compound libraries for modulators of Wip1 activity. In collaboration with the National Center for Advancing Translational Sciences (NCATS), we have used the above-mentioned HTS method to screen 102,277 compounds at a single concentration from the NCATS Genesis library using physiologically relevant substrates to identify Wip1 modulators. Hits were evaluated over a range of 11 concentrations with both the Rapid-Fire MS assay and the orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a Wip1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class Wip1 activator. Characterizing new Wip1 inhibitor and activator scaffolds represents a means to control the phosphatase activity using a precision medicine approach for cancer treatment. We have also identified a series of alkyl-substituted N-methylaryl-N'-aryl-4-aminobenzamides and tested their Wip1 inhibitory activity. A straightforward synthetic route was developed to synthesize the target compounds from commercially available starting materials. Three different portions of the core scaffold were extensively modified to examine structure-activity relationships. This study revealed interesting trends about a new molecular scaffold to inhibit Wip1. Additional work may lead to more potent inhibitors that interact with a larger portion of the Wip1 binding site and increase its specificity.