A Team Science Approach Using Bioinformatics, Chemical Genetics and Proteomics to Dissect MAP3K1 Associated Biology in Cancer - Administrative Supplement for INBRE Parent

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PROJECT SUMMARY This application from the Nebraska Research Network in Functional Genomics (NE-INBRE) network responds to NIGMS call (NOT-GM-23-034) for supporting the development of team science projects. The proposal brings together four investigators with diverse training backgrounds from Nebraska to work on a team science project using bioinformatics, chemical genetics and proteomics to dissect MAP3K1 associated biology in cancer. MAP3K1, a mitogen activated protein kinase, is the only kinase that contains both a kinase domain and an E3-ubiquitin ligase domain. The presence of these two enzymatic domains uniquely positions MAP3K1 at the interface between several phosphorylation and ubiquitin-mediated signaling pathways involved in cellular signaling. This project addresses one of the main themes of the NE-INBRE which is cell signaling to understand the disease mechanisms using a diverse set of functional genomics tools that require a team science approach. Goals of the Parent Award: The mission of the Nebraska IDeA Networks of Biomedical Research Excellence (NE-INBRE) is to stimulate and develop biomedical research capacity at institutions of higher education in Nebraska. The NE-INBRE is structured around two major components: primary undergraduate institutions (PUIs) and PhD granting research institutions (RIs). Support for each PUI consists of: 1) campus research capacity development through support for faculty research and infrastructure enhancement, and 2) development of the undergraduate research pipeline of students through the NE-INBRE Scholars Program. Two features are associated with expanding research capacity at the RIs: 1) providing significant support to multi-user core facilities in order to allow investigators from PUIs and RIs access to cutting-edge technology, and 2) providing support in the form of first-year graduate assistantships for NE-INBRE scholars who matriculate into PhD programs at the RI campuses. The three participating RIs in the research network include two campuses of the University of Nebraska system and one privately supported medical center. Cutting edge multiuser core facilities include cores in genomics, bioinformatics, structural biology and imaging. The three themes reflect the scientific foci of the NE-INBRE, cell signaling and neuroscience, infectious disease, and structural biology and molecular biophysics. These themes serve to link faculty and students at the separate institutions into productive networks based on their areas of expertise and interest. Research Question to be addressed in the Supplement Proposal: The major limitation in developing drugs that block MAP3K1 is the failure of in vitro assays with recombinant MAP3K1 to recapitulate the native cellular state, where protein complexes associated with the full-length post-translationally modified MAP3K1 are preserved. The primary goal of this project is to develop genetic and chemical tools to perturb MAP3K1 in its native state. These are critical for evaluating the effect of MAP3K1 inhibition on NFκB signaling in a panel of cancer cell lines. To achieve this goal, we built a team of four accomplished investigators with expertise in molecular cell biology, chemical genetics, proteomics and bioinformatics. The overall project is divided into four sub-projects with four specific aims, each led by a co-Project Leader. In Aim 1, we will develop a panel of dox-inducible MAP3K1 wild type (WT) and MAP3K1 point mutant cell lines that will be tested by MAP3K1 inhibitors in Aim 2, and the interactomes of different mutants will be characterized by computational approaches in Aim 3 and mass-spectrometry based proteomics in Aim 4. The combined effort will not only validate the signaling defined by the reported knockdown and overexpression data but also unravel the signaling mechanisms as MAP3K1 is perturbed in its native state expressed at endogenous levels. Benefit of the Team Science Effort: Each sub-project in this proposal uses cutting-edge tools and relatively independent of each other, but the overall goal of developing orthogonal tools to dissect MAP3K1 signaling in its native state is achievable only by combining the complementary expertise of the four co-Project Leaders. For example, the MAP3K1 mutant cell lines generated by Dr. Ghosal in Aim 1, can be utilized for drug synthesis and testing by Dr. Natarajan in Aim 2 and analyzed using the proteomics approaches to determine their individual mechanistic defects by Dr. Woods in Aim 4. Dr. Woods also collaborate with Dr. Natarajan to characterize the function of the newly developed MAP3K1 inhibitors using proteomics-based methods optimized on IKAM-1. Similarly, both Drs. Woods and Guda will develop orthogonal approaches to map the MAP3K1 interactome using the mass-spectrometry proteomic and computational approaches, respectively, and use the data generated in each approach to validate their results and refine the protein-protein interaction network models. Together, the complementary aspects of our team allow for meaningful integration of approaches and data to comprehensively characterize MAP3K1 associated biology in cancer and to generate new tools and inhibitors. This project falls well within the scope of the parent NE-INBRE award, and the research questions proposed here do not duplicate any part of the work funded under the parent award.