PROJECT SUMMARY The expression levels of protein arginine methyltransferases (PRMTs) are significantly elevated in cancer cells and are associated with poor clinical outcomes. This strongly suggests their potential as a novel class of therapeutic targets in oncology. Major pharmaceutical companies have developed over ten selective and potent PRMT inhibitors (PRMTis) that are currently in early-stage clinical evaluation. However, due to the understudied nature of arginine methylation as a post-translational modification, a comprehensive mechanistic understanding of PRMT functions in cancer is urgently needed to enable the clinical application of these inhibitors. Notably, recent clinical trial results indicate that the activity of PRMTis alone may be insufficient for effective cancer management in patients. Therefore, it is imperative to conduct mechanism-driven preclinical evaluations of combination strategies that target PRMTs alongside other therapeutic drugs to achieve clinical success. Both high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) share clinical and genomic characteristics such as poor prognosis, homologous recombination deficiency, and potential immunoreactivity. Given the urgent unmet medical need to develop effective therapeutic strategies for these diseases, our preliminary studies have merged large-scale drug combination screening and cancer genomic profiling to establish a strong rationale for applying PRMTis in treating HGSOC and TNBC. Our hypothesis is that because of uncontrolled proliferation, increased DNA damage, and activated oncogenes, cancer cells must sustain abnormal levels of transcription, splicing, and protein arginine methylation through enhanced PRMT activities (PRMT addiction). Treatment with PRMTis can disrupt the hyperactivated PRMT-regulome that enables cancer cells to survive. We've assembled a team of investigators with diverse expertise and resources to test this hypothesis through three specific aims: Specific Aim 1: Characterize molecular mechanisms of PRMTi mono- and combination therapies; Specific Aim 2: Evaluate therapeutic potentials of PRMTis in HGSOC and TNBC preclinical models; and Specific Aim 3: Investigate effects of PRMTi treatments on anti-tumor immune responses. The overarching objective of this application is to systematically explore the dynamic changes in the regulome induced by PRMTi treatments in cancer cells. This will offer fresh insights into their mechanisms of action and provide a rationale for the clinical development of PRMTi mono- and combination therapies in the field of oncology.