PROJECT SUMMARY Triple-negative breast cancer (TNBC) has remained a considerable clinical challenge due to the lack of efficacious genetic targets. We need unique effective therapies and accurate biomarkers that can be used to predict patient responses in TNBC. We find that the ARF tumor suppressor is lost alongside p53 mutation in 60% of TNBC. Potentially stemming from the dual loss of ARF and p53, we have observed that type I IFN signaling is elevated in TNBC. We show that this IFN production is being kept in check by the ADAR1 enzyme. Notably, we discovered that ADAR1 is a novel binding partner for ARF. The central premise of this proposal is that the novel ARF-ADAR1 interaction provides key insights into how these two proteins function in the etiology of TNBC. The research application focuses on the role of this interaction in regulating the type I interferon response and sensitizing TNBC cells to cell death and immune recognition. The overarching hypothesis of the proposed research is that loss of ARF and p53 results in elevated type I IFN signaling and sensitizes cells to ADAR1 depletion. In Aim 1, we will define the functional interaction of ARF and ADAR1. In the absence of functional p53, ARF protein expression is induced. In this setting, we find that ARF can fully titrate all the cellular ADAR1 into ARF complexes. We will test the hypothesis that ARF mechanistically traps ADAR1 in the nucleolus to prevent ADAR1 from repressing the type I interferon pathway. In Aim 2, we present data that TNBC cells are sensitive to ADAR1 depletion. Importantly, this sensitivity is dependent on type I interferon signaling. We will test the hypothesis that activation of IFN production and signaling will combine with ADAR1 depletion to produce synthetic lethality in vitro and in vivo. We will utilize both agonists of the IFN pathway and synthetic ARF peptide mimics. In Aim 3, we will assess how the ARF-ADAR1 interaction influences the tumor microenvironment. While type I IFN release is a major component of the anti-viral response, chronic IFN- release by tumor cells can both alter the local immune environment and expression of PD-L1 on tumor cells. We will test the hypothesis that hyperactivation of the type I IFN pathway by ADAR depletion will result in gains in tumor infiltrating lymphocytes and elicit an anti-tumor immune response that will prevent metastasis. These studies are paramount to informing new approaches in treating TNBC through activation of IFN signaling in the tumor microenvironment.