PROJECT SUMMARY / ABSTRACT Breast cancer is the most common cancer and the leading cause of cancer death in females. Triple negative breast cancer (TNBC) is a highly aggressive and lethal subtype of breast cancer with limited treatment options. A significant portion of TNBCs harbor hyperactivation of transcription factor MYC, which drives gene dysregulation and promote the elevated growth and proliferation of TNBCs. On the other hand, it has also been shown that transcriptional amplification in cancers, including those driven by MYC hyperactivation, places specific burdens on molecular machines involved in RNA metabolism. As one of such examples, we have shown that inhibiting molecules involved in RNA splicing can lead to significant cell stress and death of MYC+ TNBCs. However, while groundbreaking research has focused on dysregulation of RNA synthesis and processing in cancer, far less is known about how oncogenes such as MYC deregulate RNA decay pathways to drive aberrant gene regulation. Exoribonuclease-mediated RNA decay is a critical component of RNA metabolism. Although defects in exoribonucleases present in various disease settings have been linked to the cellular accumulation of misprocessed RNA transcripts, such relevance in cancer is far from understood. Through a prior genome-wide forward genetic screen, we identified that silencing of mitochondrial 3’ to 5’ exoribonuclease PNPT1 is lethal specifically in the context of MYC hyperactivation. Subsequent analysis of Cancer Dependency Map (DepMap) database further revealed that the function of PNPT1 is critical across models of TNBCs. Recent evidence demonstrated that defects in PNPT1 can lead to the accumulation of intracellular double-stranded RNA (dsRNA) and the induction of dsRNA-mediated antiviral-like immune and apoptotic responses in cancer cells. Similarly, we have confirmed that silencing of PNPT1 in SUM159, a MYC-dependent human TNBC line, also leads to the accumulation of intracellular dsRNA. These preliminary findings lead us to hypothesize that PNPT1 plays a critical role in the removal of aberrant RNA transcripts and prevents the induction of dsRNA-mediated cell stresses, thereby conferring survival benefits to TNBCs. Herein, we propose to leverage cutting-edge chemical- genetic (PROTAC) tools, RNA sequencing, and proteomic analysis approaches to elucidate the role of PNPT1 in TNBC survival and progression. We will 1) Assess the effects of PNPT1 perturbation on induction of dsRNA- mediated antiviral responses in TNBCs; 2) Examine the effects of PNPT1 perturbation on overall RNA metabolism in TNBCs; and 3) Determine the effects of PNPT1 perturbation on TNBC survival and progression. Successful completion of the proposed project will provide novel insights into how PNPT1-mediated RNA decay helps sustain TNBC growth and survival. Further, the study will serve as a foundation for the development of novel therapeutic approaches targeting PNPT1 for the potential treatment of TNBCs...