Abstract: Currently, therapeutic treatment with immune checkpoint inhibitors(ICI’s), such as α-CTLA4 and/or α-PD1, are increasing survival rates (>50% four-year survival), particularly in highly mutated tumors. Moreover, combining ICIs with therapies that target tumor oncogenes has been highly effective in some tumors. Our hypothesis is that response to ICI therapy in immunologically “cold” tumors can be enhanced by combining ICIs with therapies targeted to oncogenic drivers that also induce expression of key chemokines that recruit CD8+T cells and DCs to the TME and shift tumor associated macrophages to an anti-tumor phenotype, and reduce the infiltration by myeloid-derived suppressor cells. To test this hypothesis, we will utilize immune competent mouse models, humanized mouse models bearing patient-derived xenograft (PDX), and organotypic cultures of human tumor co- cultured with patient immune cells to determine if these specific precision medicine approaches to targeted therapies enhance response to ICIs, and improve survival in mice. We propose to advance therapeutic strategies for melanoma and breast cancer in four areas. There are two approaches we will develop for melanoma studies: 1) Using immune competent BRAFV600E/PTEN-/-, NRASQ61K,R/CDKN2a-/- or triple wild type mouse models, humanized PDX models and 3D organoids of human melanoma tumors co-cultured with immune cells isolated from the patient, we will determine whether treatment with either rigosertib (NRAS pathway inhibitor), CDK4/6 inhibitor +MDM2 antagonist combined with ICI therapy induces tumor regression. Mechanisms for alterations in anti-tumor immune response will be determined. 2). Using CyTOF and sc-NGS analysis we will characterize biopsy tissue and PBLs from melanoma patients who develop resistance to immune checkpoint inhibitors, evaluate the expression of other checkpoint proteins as well as activation of new driver mutations, and use these data to develop new strategies for treatment with other checkpoint inhibitors and targeted therapies using our organoid culture model. For our breast cancer studies: 1) We will treat tumors with AKT inhibitors plus paclitaxel in combination with ICI therapies or prior to ICI treatment using immune competent mouse models, humanized PDX models, and organotypic cultures of patient tumors co-cultured with patient immune cells; 2) We will examine the tumor growth in mice with targeted deletion of CXCR2 or CXCR4 in tumor tissue or in specific immune cell populations with or without targeted therapies and immune therapy. Where appropriate, CXCR2 and/or CXCR4 antagonists may be combined with the targeted therapies to enhance response to ICI therapy. Altogether these studies will provide key information for the design of new clinical trials I melanoma and breast cancer with our clinical collaborators and inform mechanisms of resistance to ongoing therapies. Melanoma and breast cancer can be major issues for our Veterans and development of imp...