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 on 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. These structural studies have allowed us to better understand substrate binding in this family of phosphatases and to 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 observed that overexpression of Ppm1d in the blood cells of mice accelerated the growth of solid tumors. 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 by the presence of neutrophils. Genetic depletion of Ppm1d in neutrophils transformed them into efficient activators of CD8 + cytotoxic lymphocytes, in part, through p53-dependent induction of lymphocyte costimulating ligands 4-1BBL and OX-40L. Moreover, the depletion of Ppm1d in myeloid cells potentiated both anticancer chemotherapy and immunotherapy. Therefore, combining immune checkpoint therapy with genetic or chemical inhibition of Wip1 could be a valuable strategy to increase the efficiency of current anticancer immunotherapies. These findings demonstrate that Wip1 is a promising target for increasing the efficiency of anti-cancer immunotherapy. 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. We anticipate that these systematic efforts will provide the first structure of Wip1 to help better understand the specificity and potential for 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 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 more than 100,000 compounds from the NCATS Genesis library using physiologically relevant substrates to identify Wip1 modulators. This library comprises novel chemotypes with a combination of diverse chemical scaffolds as well as well-characterized and targeted compounds possessing new properties for further therapeutic development. Several hundred compounds were considered active after the primary screen and plated in dose response format in order to refine the selection. We implemented orthogonal readouts to reduce the prevalence of interference compounds, and follow-up counter assays eliminated those with non-specific activity. The most promising compounds were assessed to confirm their biophysical interaction with Wip1. Using a cell-based assay we optimized to determine Wip1 activity by measuring gammaH2AX phosphorylation following ionizing radiation we assessed the bioavailability of the lead molecules. Our biophysical, biochemical and cell-based studies of confirmed hits revealed two inhibitors, one competitive and one uncompetitive, and one activator. These new scaffolds are prime candidates for further optimization studies. We hope that the new Wip1 inhibitor scaffolds might be amenable to optimization for better pharmacokinetic properties. Also, very little work has been done on the activation of Wip1 in p53-negative tumors and no activator of this phosphatase has been described. Characterizing new Wip1 inhibitor and activator scaffolds represents a means to control the phosphatase activity using a precision medicine approach for cancer treatment.