The objective of this proposal is to identify viral and host factors that promote reovirus infection and cell killing of TNBC cells and identify small molecule inhibitors that can augment viral-mediated killing of cancer cells. In the U.S., breast cancer is the leading cause of cancer and a leading cause of cancer-related deaths in women. Mammalian orthoreovirus (reovirus) is a segmented, nonenveloped dsRNA virus that predominantly infects transformed cells and is in Phase I-III clinical trials to assess its efficacy as a viral oncolytic against several cancers. Preliminary data indicate that serotype 1 reoviruses induce more efficient TNBC cell death than serotype 3 reoviruses and that cell death occurs via a caspase 3-independent pathway. RNA sequencing showed that IL-24 is highly upregulated during reovirus infection of TNBC cells and high-throughput screening identified topoisomerase inhibitors (doxorubicin, epirubicin, etoposide, and topotecan) that when paired with reovirus promote efficacious TNBC cell death. The central hypothesis is that reovirus infection with serotype 1 reoviruses in the presence of topoisomerase inhibitors induces complementary signaling pathways that result in TNBC cell death. Two integrated specific aims are proposed. Specific Aim 1 will elucidate mechanisms of reovirus-induced cell death of triple-negative breast cancer cells. Specific Aim 2 will determine how topoisomerase inhibitors impact reovirus infection. These experiments will enhance an understanding of the mechanism viruses use to kill cancer cells, define how combinatorial virus and small molecule inhibitor regimens impact viral infection, and inform the development of personalized therapeutic options for patients afflicted with TNBC.