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. 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. PPM1D is overexpressed in tumor-infiltrating neutrophils, both in humans and mice, and its genetic deletion or chemical inhibition in murine myeloid cells increased their anti-tumor phenotypes and suppressed tumorigenesis. These findings, published in Nature Communications, 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 to guide structure-activity investigations of inhibitors or activators identified in HTS studies. To that end, we have continued optimizing the expression of recombinant Wip1, identifying aggregation-prone residues, and screening for crystallization conditions in collaboration with Drs. Fred Dyda and Dalibor Kosek, Laboratory of Molecular Biology, NIDDK. We anticipate that these systematic efforts will provide the first structure of Wip1 to help better understand the specificity and potential for inhibition. 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 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 plan to assess the bioavailability of the lead molecules. Currently, there are no available Wip1 inhibitors which are potent and show favorable pharmacokinetics. We aim to discover a new Wip1 inhibitor scaffold that 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.