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Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer subset with poor outcomes. Since TNBC is resistant to hormone therapies, there are few effective therapies available for TNBC patients. One potential therapeutic strategy exists in targeting specific molecular components of an individual patient’s cancer. Prior work in our group has nominated monopolar spindle kinase I (TTK) as a gene upregulated in breast cancer patients. Specifically, TTK expression was found to be increased in cancerous breast tissue compared to healthy tissue and correlated with cancer recurrence following radiotherapy. Importantly, the implications of TTK inhibition and radiotherapy on the immune system is not well understood. In this study, we aimed to elucidate the role of combined TTK inhibition and radiotherapy in syngeneic murine mouse models. We hypothesize that TTK inhibition will radiosensitize murine TNBC models to radiotherapy both in vitro and in vivo and modulate the immune tumor microenvironment. Methods: Cell viability assays were implemented to determine the half-maximal inhibitory concentrations (IC50) of TTK inhibitor. Clonogenic survival assays were used to determine the radiation enhancement ratios (rERs) of TTK inhibition in vitro. Syngeneic murine mouse models were used to assess therapeutic effects of TTK inhibition and RT in vivo. 4T1 TNBC cells were injected bilaterally into the flanks of BALB/c mice and treated with combinations of radiotherapy and TTK inhibition. Tumor growth was monitored and, following the completion of the study, final tumor weights were recorded and tumor tissue was collected to perform immunofluorescent microscopy. Results: Single-agent TTK inhibition via treatment with the ATP-competitive inhibitor empesertib inhibits the growth of murine TNBC cell lines with IC50 values up to 30nM. Sub-IC50 values of TTK inhibitor induced radiosensitization in the murine TNBC cells 4T1 (rERs ≤ 2.4) and Py8119 (rERs ≤ 1.6). TTK knockdown also resulted in changes in radiosensitization in vitro. Furthermore, we also observed a similar phenotype in vivo. In our 4T1 model system, mice receiving combined treatment had significantly decreased tumor growth compared to mice that receiving single-agent therapies or vehicle control alone. Quantities of monocyte derived suppressor cells and CD8+ T cells were altered with radiotherapy and TTK inhibition. Conclusion: Our data suggests that TTK inhibition and radiotherapy is synergistic in murine TNBC and alters the tumor immune microenvironment. This combined therapy suggests that changes in the underlying immune mechanisms as a result of the synergistic treatment efficacy are important in TNBC. Future work will examine the underlying mechanisms of TTK inhibition and radiotherapy on systemic and tumoral immune changes. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline R. Bishop, Kalli R. Jungles, Cydnee Wilson, Meilan Liu, Ashley N. Pearson, Erin A. Holcomb, Ben Chandler, Jadyn James, Amanda Huber, Lori J. Pierce, Corey Speers, James M. Rae, Michael D. Green. Targeting monopolar spindle kinase I (TTK) as a radiosensitizing strategy in syngeneic murine models of triple negative breast cancer (TNBC) and its implications on the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2823.