Project Abstract Breast cancer, the most prevalent cancer in women, is responsible for more than 15% of new cancer cases and about 6.9% of all cancer-related death in the US. A major cause of therapeutic failure in breast cancer is the development of resistance to chemotherapy, especially for triple- negative breast cancer (TNBC), a type of breast cancer considered to be more aggressive and have a poorer prognosis than other types of breast cancer. Therefore, how to overcome chemoresistance is the major challenge to improve the life expectancy of TNBC patients. Preliminary data demonstrate that TNBC cells surviving from the chronic treatment of chemotherapeutic drugs show significantly higher expression of a special protein kinase, dual serine/threonine and tyrosine protein kinase (DSTYK), than parental cells without treatment. Additionally, from both in vitro and in vivo results, we found that both the knockout of DSTYK and exosome-mediated DSTYK silence can lead to the apoptosis of chemoresistant cells upon drug treatment. This novel finding suggests that DSTYK exerts a previously unknown and important role in promoting chemoresistance. The objective of this proposal is to elucidate the novel role and the underpinning mechanism of DSTYK in regulating chemoresistance in TNBC cells and further explore the potential of exosome-mediated DSTYK silence in TNBC chemotherapy with preclinical models. We hypothesize that DSTYK is a novel target for chemotherapy to induce TNBC cell death. We will test our hypothesis with three specific aims. In Aim 1, we will investigate the mechanism by which DSTYK promotes chemoresistance in TNBC cells. In Aim 2, we will establish DSTYK as a prognostic biomarker in clinical TNBC. In Aim 3, we will evaluate the therapeutic benefit of DSTYK-siRNA-exosomes in chemotherapy. We engineered exosomes containing siRNA against DSTYK that can specifically target TNBC cells. We found these engineered exosomes can significantly promote tumor regression and inhibit metastasis in both cellular and orthotopic models treated with drugs. Successful completion of the proposed studies will provide fundamental insights into the role of DSTYK in chemoresistance in TNBC cells and will lay the essential foundation for the development of an exosome-mediated strategy targeting DSTYK to significantly improve clinical TNBC chemotherapy.