JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Ettore Appella
Title:	Principal Investigator
Address:	37 Convent Dr., Room 2140 Bethesda, MD 20892
Telephone:	301-402-4177
Fax:	301-435-8188
Email:	appellae@pop.nci.nih.gov
Sponsoring Laboratory/Branch:	Laboratory of Cell Biology

Project Title:	Identification and Biological Characterization of
Target(s) of Interest:	Wip1 phosphatase (PPM1D)

Project Synopsis:
The wild-type p53-induced phosphatase Wip1 (PP2Cδ 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 the p53 tumor suppressor after exposure of cells to DNA damage-inducing agents, such as ionizing radiation or UV light. The Wip1 protein is overexpressed and the PPM1D gene is amplified in several human cancers, such as breast, neuroblastoma, pancreatic adenocarcinoma, medulloblastoma, and ovarian and gastric carcinoma. In addition, Wip1 has been suggested to function as an oncogene, as the PPM1D gene cooperates with the oncogenes Ras, Myc, and Neu in the transformation of primary mouse embryo fibroblasts. When H-ras and ErbB2 trangenic mice were crossed with Wip1-deficient mice, a delay in the onset of mammary gland tumors was observed, while the accelerated onset of tumors was observed in mice overexpressing Wip1 and ErbB2 (Bulavin 2004 Nat Genet 36, 343-50 and Demidov 2007 Oncogene 16:2502-6). Finally, Wip1 has recently been shown to be important for tumorigenesis in mouse models of intestinal polyposis, B-cell lymphomas, and development of spontaneous tumors (Nannenga 2006 Mol Carincog 45:594-604; Demidov 2007 Cell Stem Cell 1:180-90; Shreeram 2006 J Exp Med 203:2793-9). Wip1 is similar in structure to a number of known type 2C phosphatases that are associated with stress response, sexual differentiation and cell cycle control in a range of organisms. The first target of Wip1 phosphatase activity to be identified was p38 mitogen-activated protein kinase (MAPK), which it specifically dephosphorylates and inactivates in the nucleus. The p38 MAPK pathway acts as a negative regulator of cell cycle progression, so it is likely that Wip1 also is important for cell cycle progression. p38 MAPK can phosphorylate and stimulate induction of apoptosis through p53 in response to UV and chemotherapeutic agents, suggesting that Wip1 may act in a p38 MAPK-p53 feedback loop. Interestingly, most tumors that have amplified Wip1 express wild type p53, suggesting that inhibition of Wip1 could be useful for the activation of p53 and, consequently, the induction of cell cycle arrest or apoptosis. Although inhibition of Wip1 activity is likely to have therapeutic value, no specific inhibitors are currently available. Our group has synthesized phosphopeptide inhibitors for Wip1 based upon its preferred substrates. These inhibitors are specific for Wip1, as two serine/threonine phosphatases, PP2Cα and PP2A, are not significantly inhibited by a phosphopeptide inhibitor containing a nonhydrolyzable phosphoserine mimetic moiety. A homology model of Wip1 bound to a cyclic phosphopeptide and site-directed mutagenesis helped to identify residues important for Wip1 inhibitor selectivity that are unique to the Wip1 active site (Yamaguchi 2006 Biochemistry 45:13193-202; Bang 2008 Chem Med Chem 3:230-2). These results have provided the first proof of concept of a specific inhibitor of the catalytic site of Wip1 and should be useful for developing potential anti-cancer drugs. In addition to the phosphopeptide inhibitors, we are also exploring small molecule inhibitors of Wip1; these compounds have been designed to mimic the structure of the phosphopeptide inhibitor obtained by NMR spectroscopy. The project will aim to characterize the cellular activity of Wip1 inhibitors made by our laboratory and that of our collaborator. To accomplish this, the fellow will make isogenic cell lines in which Wip1 is either stably knocked-down or expressed under the control of an inducible promoter. These cell lines will be used for screening and characterization of the biological activity and selectivity of the Wip1 inhibitors. Additionally, targeted libraries are being produced for screening through this cellular model system. The screening will identify the most active and specific compounds for further characterization. Synergy of the lead compounds with already known anti-cancer therapeutics will be also be explored.
Fellow Research Plan:
The fellow will first be trained in the purification of recombinant Wip1 and the in vitro enzymatic assays used to test inhibitors and substrate activities. The understanding of this method, already in use in the laboratory, will form the baseline for the study of the cellular activity of the compounds. The fellow will next prepare and characterize isogenic cell lines containing a stable knock-down of Wip1 using shRNA constructs already available in the laboratory. The fellow will similarly prepare and characterize isogenic cell lines with inducible expression of Wip1 using constructs being prepared in the laboratory. It is expected that the preparation of these cell lines will be straightforward and readily completed by the fellow. In these aims, the fellow will learn basic cell culture techniques for the manipulation of cell lines. Upon preparation of the cell lines, the fellow will use them to characterize the cellular activity of Wip1 inhibitors developed in our laboratory. The characterization will include evaluation of the ability of the compounds to enter the cells and cytotoxicity. The fellow will use Western blotting to look for changes in phosphorylation of Wip1 substrates and activation of p53. Finally, as p53 activation leads to apoptosis, the fellow will use cell sorting methods to quantify the extent of apoptosis induced by treatment with the inhibitors. As we have identified a number of lead compounds from in vitro studies, we anticipate that some of them will have good cellular activity. From the cellular study, we will select a cadre of lead compounds to be tested for inhibition of tumorigenesis in the APC-min/+ mouse model already shown to have great similarity to human cancers. The cell lines will also be used to screen selected inhibitors from both our targeted library and the NCI library of compounds. We have developed a targeted library of compounds based upon our model of the binding of the phosphopeptide inhibitor to Wip1. These compounds are all designed to bind the active site of Wip1, thereby inhibiting its catalytic activity. The NCI library will also be screened to identify inhibitors, which may bind to the active site or to an allosteric site on the protein. The fellow will use a similar methodology as described above to characterize the activity and specificity of compounds from the libraries. As the targeted library is based on our experimental studies and modeling, we predict that we will be able to identify good inhibitors from the targeted library. The NCI library screen is more general, but it is highly possible that we will identify something that has activity against Wip1. It remains to be seen, however, if we will be able to identify a specific inhibitor from the NCI library.