PROJECT SUMMARY/ABSTRACT Multiple myeloma is an incurable malignancy of plasma cells for which new treatments and precision guided approaches are acutely needed. Current treatments target the plasma cell biology thought to be common to all myeloma. However, response to these treatments is highly variable pointing to an underlying heterogeneity in biology. One recently identified source of heterogeneity with important therapeutic implications involves dependence on the Bcl-2 family of proteins, which includes Bcl-2, Bcl-xL, and Mcl-1. These proteins regulate cell survival and are the targets of a new class of drugs being studied for use in myeloma. Although both normal plasma cells and myeloma are typically dependent on Mcl-1 for survival, a subset of myeloma with a B cell-like phenotype is Bcl-2 dependent and sensitive to venetoclax, a Bcl-2 specific inhibitor. This B cell phenotype is notable because B cells are also Bcl-2 dependent and B cell malignancies such as CLL are highly sensitive to venetoclax. The objective of this proposal is to test the hypothesis that in a subset of myeloma, B cell transcriptional and signaling networks remain active and contribute to Bcl-2 dependence and venetoclax sensitivity as well as resistance to conventional plasma cell directed therapy. The studies proposed will define the mechanisms of Bcl-2 family dependence in myeloma and identify biomarkers capable of selecting myeloma patients most likely to respond to Bcl-2 inhibitors such as venetoclax. Aim 1 will characterize the mechanisms of BATF3 activity, an AP-1 transcription factor that increases Bcl-2 dependence and sensitizes myeloma to venetoclax, using genomic and epigenomic analyses on cells with CRISPR mediated BATF3 overexpression or knockdown. Aim 2 will directly interrogate the origins of Bcl-2 dependence and intrinsic resistance to venetoclax in myeloma using CRISPR screens targeting genes that are differentially expressed in venetoclax sensitive and resistant cells. Finally, Aim 3 will utilize state of the art mass cytometry on ex vivo tested myeloma patient samples to develop a novel biomarker that could be used for future precision medicine trials to predict response to inhibitors of Bcl-2, Bcl-xL, and Mcl-1. These studies will be conducted by Dr. Vikas Gupta, whose long-term career goal is to become an independently funded physician-scientist studying the molecular pathogenesis of multiple myeloma. He is seeking an NIH K08 career development award to acquire the additional skills necessary for independence. With the help of a multi-disciplinary group of experts serving as mentors and advisors, he will receive hands on training in advanced research techniques such as targeted CRISPR screens, RNA sequencing, bioinformatics, and mass cytometry as well as mentoring in manuscript and grant writing, clinical trial development, laboratory management, and leadership skills. Completion of the work proposed here will lay the foundation for future R01 applic...