Abstract Bromodomain and Extra Terminal (BET) proteins are epigenetic “readers” that recognize acetylated histones and mark areas of the genome for transcription. BRD4, a BET family member protein, has been implicated in a number of types of cancer. It has been recently found to associate with super-enhancers, and elevated levels of BRD4 have been linked to increased expression of MYC as well as other oncogenes. BET protein inhibitors are currently being tested for their potential use in the treatment of HIV, heart failure, and cancer, all of which are diseases of aberrant transcription. However, little is known regarding their efficacy in triple-negative breast cancer (TNBC). We found JQ1, a prototypical BET inhibitor, impedes growth of seven TNBC cell lines in a dose-dependent manner within 72 hours of treatment. JQ1 also suppresses growth of three different TNBC xenografts, including a patient-derived model of basal-like breast cancer. Growth arrest, in vitro, is followed by either apoptosis or senescence. However, prior to the induction of these two terminal responses, prolonged treatment results in polyploidy in many of the cell lines, suggesting BETi disrupts mitosis/cytokinesis. Live-cell imaging revealed JQ1 significantly increased the duration of mitosis, and microarray analyses showed a significant JQ1-mediated downregulation of genes critical for cell cycle progression, mitosis, and cytokinesis. In all TNBC cell lines tested, Aurora kinases, proteins critical for proper progression through mitosis and cytokinesis, are suppressed in response to JQ1. Treatment with AZD1152, an Aurora kinase B inhibitor, elicits the same cellular responses as BET inhibition, indicating that the suppression of Aurora kinases plays a key role in the response of TNBC cells to BET inhibitors. These findings reveal that BET inhibitors block the growth of highly aggressive TNBC cells by inducing mitotic dysfunction and that these drugs are promising potential therapeutics for the treatment of TNBC. Citation Format: Jennifer M. Brancato, Sylvia Gayle, Kristen Weber-Bonk, Matthew Summers, Gurkan Bebek, Ruth Keri. BET protein inhibition blocks growth of triple-negative breast cancer by inducing mitotic and cytokinetic dysfunction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4647.