PROJECT SUMMARY-ABSTRACT Background: Breast cancer is the number one killer of women between the ages of 44 and 55 in this country. It is estimated that more than 276,480 women will be newly diagnosed with breast cancer in 2020 and about 42,170 women are expected to die of breast cancer (BC) this year. Although ER+ tumors can be effectively treated, there are no effective therapies available for ER- and specially triple negative breast cancers (TNBC). TNBC accounts for 30% of all BC, is more aggressive and less responsive to treatment thus has highest mortality rates. Currently, there are no molecular therapies approved for TNBC which could be combined with radio- or immunotherapies. Objective: Mechanisms of radioresistance are poorly understood, and immunotherapy has been largely disappointing, but improvement of either could significantly impact outcomes for women with TNBC. Thus, identifying drivers of radioresistance and immune regulation that can be effectively targeted represents a critical unmet clinical need. The objective of this proposal is to establish BUB1 as a viable molecular target for radiosensitization and immune priming as well as for predicting therapeutic outcome in TNBC. Hypothesis: In an effort to identify actionable molecular targets for radiation- and immune- sensitization of TNBC, we have identified BUB1 expression as a strong prognostic correlate of aggressive disease and poor clinical outcome. We hypothesize that BUB1 mediates radioresistance and that BUB1 inhibition will sensitize resistant TNBC to ionizing radiation. Furthermore, we hypothesize that BUB1 regulates TGFβ signaling in tumor microenvironment leading to an increase in myeloid derived suppressor cells (MDSCs) population, decrease in cross-priming and immunotherapy resistance. We propose that genomic depletion or inhibition of BUB1 will reduce TGFβ and MDSCs, and will ascertain differentiation back to antigen presenting cells (APC) which will restore innate immunity and make tumors sensitive to immune checkpoint blockade. Impact: The proposed studies will validate BUB1 as a molecular target for TNBC therapy. This study will provide rationale for targeting BUB1 in combination with radio- or immune-therapy and will provide conclusive pre-clinical data for future clinical translation. We are certain that our approach of targeting BUB1 in combination with radio- and immunotherapy is fundamentally better than the current therapies as it exploits the multiple functions of BUB1 including cell-cycle, DNA- damage response and TGFβ signaling. We envision that the successful completion of the proposed study will lead to new treatment paradigms that go beyond minor improvements and will have major impact on breast cancer treatment, especially on TNBC.