PROJECT SUMMARY Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has a very poor prognosis due to its tendency to metastasize and relapse. Immune checkpoint blockade (immunotherapy) plus chemotherapy is the first-line treatment for metastatic TNBC, but the tumor responses are limited and not durable. Additionally, the lack of robust predictive biomarkers of response remains a limiting factor in maximizing the efficacy of immunotherapy. Therefore, there is an urgent need to develop a more effective immunotherapy combination and establish novel biomarkers to identify TNBC patients who will benefit from this treatment. Because the tumor microenvironment (TME) critically influences TNBC response to immunotherapy, we set out to identify mechanisms that broadly mediate the immune response in TNBC. Our preliminary studies showed that the c-Jun N-terminal kinase (JNK) pathway acts as a TME master switch in promoting a persistent immunosuppressive TME in TNBC. Building on this evidence, our central hypothesis is that JNK inhibitors (JNKi) synergize with immunotherapy by converting the TNBC TME from an immunosuppressive to an immunoactive state. Our hypothesis will be tested through 3 specific aims: Aim 1) Determine how JNK regulates the immunosuppressive TME and aggressiveness in TNBC; Aim 2) Establish JNK signaling-related biomarkers of the immunosuppressive status of the TNBC TME; and Aim 3) Develop an optimal JNKi-immunotherapy combination for TNBC. Completing these aims will provide a robust scientific framework for developing effective therapeutic strategies for TNBC. The experimental approach will be as follows: In Aim 1, we will investigate how JNK promotes TNBC aggressiveness by immunologically modulating the TME using clinically relevant immunocompetent syngeneic TNBC mouse models with well-defined immune TMEs. We will also identify molecules responsible for JNK’s immunological modulation of the TME. In Aim 2, we will establish novel JNK signaling-related biomarkers reflecting the immunosuppressive status of the TNBC TME using patient samples. In Aim 3, we will test whether JNKi synergize with immunotherapy in TNBC by promoting an immunoactive TME, using clinically relevant immunocompetent syngeneic TNBC mouse models and our established patient-derived xenografts of TNBC molecular subtypes (sensitive or resistant to immunotherapy) in humanized mouse models. We expect to 1) generate sufficient preclinical data to support the development of an effective JNKi-immunotherapy combination for patients with TNBC and 2) establish biomarkers of the immunosuppressive status of the TNBC TME. The proposed research is significant because it will fundamentally advance our understanding of mechanisms by which cancers promote suppression of the response to immunotherapy and may lead to the development of a novel combination immunotherapy that improves the survival of TNBC patients.