Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is recalcitrant to molecular targeted therapies and accounts for about 200,000 new diagnoses per year worldwide. Immunomodulators, therapies that harness the immune system against cancer, have revolutionized the treatment of some cancers and have recently been approved for treatment of TNBC. Disappointingly, TNBC patient response to these therapies has been heterogeneous and significantly lower than other types of cancers. TNBCs are notoriously immunologically cold, with relatively low levels of baseline immune infiltration limiting the efficacy of immunomodulators that amplify productive antitumor immune response. Therefore, a great deal of effort has been focused on promoting immune engagement in these tumors. We have previously shown that oncogenic aberrations common in TNBC result in hypersensitivity to perturbation of the RNA splicing machinery. Importantly, multiple studies, including our own (Bowling, Wang, et al. 2021), have revealed that therapeutic efficacy of spliceosome-targeted therapies (STTs) is in part due to engaging the host immune system. While these discoveries hold great promise for clinical deployment of STTs, especially in combination with immunomodulators, several aspects of their role in activation of antitumor immunity require further understanding. Herein, we propose to interrogate the species of mis-spliced RNA that trigger viral mimicry and trigger tumor inflammation and antitumor immunity. Aim 1. Investigate combinatorial therapy approaches based on STT-induced antitumor immune response. Our preliminary data indicate that activation of tumor-intrinsic antiviral signaling by STT treatment modifies interaction with the tumor microenvironment. By leveraging a diverse cohort of immune-competent syngeneic models of TNBC, we will elucidate the STT-induced change in immune infiltrate and evaluate treatment in combination with immunomodulators to enhance the efficacy of STTs. Aim 2: Define cellular modulators of endogenous dsRNA-mediated antiviral signaling. Prominent oncogenic aberrations have been shown to modulate baseline and agonist-induced inflammatory signaling in tumor cells. Indeed, we have observed heterogeneity in antiviral transcriptional response across a panel of TNBC cell lines with STT-induced dsRNA accumulation. Thus, we will leverage an interferon reporter-based genetic screen to identify novel regulators of cell-intrinsic immune response to endogenous dsRNA accumulation. Aim 3: Evaluate features of STT-induced mis-spliced RNA that stimulate antiviral signaling. Preliminary studies indicate that distinct chemotypes of STTs being investigated in the clinical setting result in differential antiviral immune signaling. Using chemical genetic perturbation and novel RNA labeling and sequencing methods, we will profile immunogenic misprocessed RNA generated by perturbation of distinct spliceosome components encompassing the entire...