Arun P. Wiita, Ph.D. - US grants

? DESCRIPTION (provided by applicant): This is a resubmission application for the K08 Mentored Clinical Scientist Research Career Development Award for Dr. Arun Wiita in the Dept. of Laboratory Medicine at the University of California, San Francisco. Dr. Wiita completed his MD/PhD at Columbia University, including highly successful graduate work in single molecule biophysics in the lab of Julio Fernandez, PhD. Since finishing his residency in Laboratory Medicine at UCSF he has been pursuing research in apoptosis in hematologic cancers with Jim Wells, PhD. Despite moving into a very different field, Dr. Wiita has made significant progress on two major projects, one published and the other submitted. His long-term goal is to understand in greater detail how cancer cells evolve and respond to therapies, eventually resulting in new diagnostic tools to assist cancer management. The K08 award will provide Dr. Wiita with the protected time and additional training in bioinformatics, proteomics, and cancer signaling networks critical to his development as a tenure-track physician-scientist primarily devoted to laboratory research. His mentor, Jim Wells, PhD, an expert on therapeutics and cell death, and his co-mentor, Kevin Shannon, MD, an expert on hematologic malignancies, have extremely strong records of mentorship. Additional advisory committee members include Jonathan Weissman, PhD, an expert on systems biology, Al Burlingame, PhD, a pioneer of biological mass spectrometry, and Scott Kogan, MD, a physician-scientist bridging clinical and basic research in Laboratory Medicine. Including coursework and participation in seminars and conferences, Dr. Wiita has drawn on the myriad resources of the UCSF scientific community to promote his career as an independent investigator. The research proposal is centered on using emerging, systems-level technologies to address pressing issues in management of multiple myeloma, an incurable, aggressive hematologic malignancy. In Aim 1 Dr. Wiita has begun to develop a unique pipeline combining mRNA deep sequencing, ribosome profiling, and quantitative proteomics to monitor bortezomib-induced cell death in myeloma cells. These studies have already revealed extensive biological insight into translational dynamics after bortezomib exposure. Here he will expand these studies with new proteomic and analysis methods to monitor the role of post-translational modifications and cellular signaling. In Aim 2 Dr. Wiita will use the first-of-its-kind data obtained in this pipeline to develop a new quantitatie model of protein translation and translational regulation. These processes are key determinants of cellular homeostasis and regulation and also play an important role in myeloma pathogenesis. In Aim 3 Dr. Wiita will use cell and molecular biology approaches to elucidate resistance and response markers as well as combination therapeutic targets in myeloma, driven by hypotheses based on systems-level data. These studies are deeply related to the missions of the NIH and NCI as they will greatly expand our understanding of therapeutic effects in myeloma and, in the future, potentially any malignancy.

PROJECT SUMMARY/ABSTRACT Multiple myeloma is an aggressive hematologic malignancy that remains incurable despite recent progress. This disease of malignant plasma cells is fundamentally associated with aberrant protein homeostasis, defined by an extremely high burden of immunoglobulin synthesis. Proteasome inhibitors (PIs), a widely-used first-line therapy in myeloma, are thought to directly take advantage of this aberrancy by increasing unfolded protein stress leading to cell death. However, this mechanism is not fully proven, and further insight into PI-induced cell death may lead to more effective combination strategies. In addition, PI resistance is a major clinical problem in myeloma, and new strategies are needed to overcome this condition. Here, we hypothesize that the remodeling of the plasma cell proteome after therapy is central to both PI response and resistance. We specifically propose that proteome remodeling is mediated through rewiring of proteostasis pathways involving chaperones, the VCP/p97 complex, and the ubiquitin-proteasome system, as well as through changes to the alternative splicing landscape, as mediated by post-translational modification of the splicing machinery. To explore this hypothesis we will take advantage of novel pharmacologic and genetic perturbation tools, cellular and biochemical assays, in vivo models, clinical trial genomic data, primary sample analysis, RNA sequencing, and mass spectrometry approaches. The overall goals of this proposal are 1) develop new therapy strategies either in combination with PIs or in the PI-refractory setting, and 2) describe a new, systematic approach to probe the architecture of proteostasis networks. Importantly, our preliminary results challenge existing paradigms related to PI efficacy. In Aim 1, we address paradoxical findings relating the unfolded protein response, the interaction between the p97 degradation machinery and PIs, and the relevance of inducible HSP-family chaperones. We will take advantage of novel pharmacology available to us, including active site and allosteric inhibitors of p97 and allosteric inhibitors of HSP70, in combination with functional genetics by CRISPR interference, to define the role of central protein homeostasis nodes defining PI response and resistance. Furthermore, we will use our unique expertise in pulsed-SILAC proteomics to determine specific substrates of the p97 machinery and the proteasome in the presence of clinically-relevant resistance modifications. Toward Aim 2, our preliminary studies using unbiased mass spectrometry have revealed significant phosphorylation of the spliceosome after PI treatment. We first aim to characterize the relationship between specific alternative splicing events and proteome remodeling after PIs. We then aim to extend our promising preliminary data demonstrating the efficacy of splicing inhibitors as a new anti-myeloma therapy. Overall, the studies here will have a direct impact on delineating the surprisingly broad range of PI-mediated effects in plasma cells, validate the novel therapeutic strategy of splicing inhibition, and reveal new mechanistic approaches to dissect proteostasis networks and alternative splicing that could extend far beyond myeloma.

PROJECT SUMMARY/ABSTRACT Engineered cellular therapies, such as CAR-T cells, hold great promise as ?living drugs?. However, the application of this approach beyond B-cell origin malignancies has been hampered by a lack of tumor-specific cell surface antigen targets. Current methods to identify new targets rely largely on expression patterns from RNA-seq data. However, the limitations of this transcriptome-based strategy have rapidly become apparent. We were struck by the recent serendipitous discovery of a tumor- specific surface antigen in the blood cancer multiple myeloma, amenable to CAR-T targeting, defined not by its expression but by its structural conformation compared to normal hematopoietic cells. The central hypothesis of this proposal is that many additional conformation-specific tumor antigens likely exist, across cancers, but currently there is no technology yet available to detect them. Here we aim to develop such a technology, combining our expertise in cell surface proteomics with crosslinking mass spectrometry, which we call ?structural surfaceomics?. While this approach could be applied to any cancer, here we first explore acute myeloid leukemia (AML), a hematologic malignancy with poor clinical outcomes and a lack of highly-specific cell surface targets. In preliminary data, utilizing an initial version of the structural surfaceomics technology, we have already identified a novel conformation-specific antigen in AML that may be a promising therapeutic target. Here, we propose two Specific Aims: 1) Further development of the structural surfaceomics approach. Our preliminary protocol is restricted to highly-expressed surface antigens with sufficient lysine crosslinks to report on structural changes. To broaden applicability, we first aim to implement recently-described XL-MS strategies that can achieve much higher proteomic coverage. We will further assess the performance of our method using biochemical control of specific surface antigens, as well as profile additional AML lines for novel target discovery. 2) Development of novel CAR-T's targeting a conformation-specific AML antigen. We will generate CAR-T compatible binders both using a standard scFv approach, based on an existing murine antibody, as well as fully in vitro-selected nanobodies via yeast display. Our lab has recently demonstrated the latter strategy as a promising approach for cellular therapy in acute leukemia (Nix et al., in revision for Cancer Discovery). We will perform initial in vitro and in vivo validation of these cellular therapies. Overall, we anticipate developing an approach to identify an entirely new class of immunotherapy targets. Furthermore, we aim to demonstrate that our approach can nominate a promising cellular therapeutic candidate specific for AML. In future work, beyond this pilot funding, we anticipate applying structural surfaceomics to broader profiling of malignancies, as well as more complete preclinical validation of our AML conformation-targeting CAR-T's.