The goals of this study are to understand how inhibiting DNA-PK activity 1) induces and increases tumor antigen/neoantigen expression in weakly immunogenic tumors and 2) contributes to maintaining dendritic cells and myeloid cells in a T cell-activating state. These goals are motivated by exciting clinical results which demonstrate that T cell-based immunotherapies can mediate tumor regression but also because durable responses to current therapies are observed in only a subset of patients and against a limited number of cancers. Favorable responses to immunotherapies correlate with levels of neoantigens and changes in the tumor-reactive TCR repertory. Cancers can evade T cell detection by downregulating major histocompatibility complex (MHC I) and antigen expression. We screened ~2,500 compounds for the ability to selectively regulate the expression of various tumor-associated antigens (TAAs) and increase MHC I expression. Among the most effective drugs were DNA-PK inhibitors. Our preliminary studies indicate that DNA-PK inhibition increases and diversifies the expression of various TAAs and neoantigens in melanoma at the transcriptional level leading to increased protein expression. Further, reduced DNA-PK levels or DNA-PK mutations in patient samples are associated with increased tumor infiltrating lymphocytes (TIL) and tumor mutation burden. The overarching hypothesis is that DNA-PK plays a novel role as a transcriptional regulator that modifies the expression of melanoma tumor antigens, including neoantigens, and thereby increases the diversity, frequency, and activity of tumor-reactive T cells. We further postulate that inhibiting DNA-PK activity in DCs and myeloid cells reduces their propensity to enter a T cell-suppressive state. These hypotheses will be addressed through the following aims. In Aim 1, we will determine the molecular mechanisms by which DNA- PKcs regulates tumor antigen expression by determining the role of kinase activity, identifying the DNA-PK substrate(s) that contribute to its repressive function, and to define the roles that the individual DNA-PK subunits play in transcriptional regulation. Aim 2 will ascertain the in vivo impact that DNA-PK inhibition has on increasing and diversifying the tumor-reactive T cell repertoire. We seek to demonstrate that DNA-PK inhibition treatment increases the number and activity of neoantigen-reactive T cells. Aim 3 will determine the impact that inhibiting DNA-PK has on inducing and sustaining stimulatory signals on tumor derived dendritic and myeloid cells. We will determine how DNA-PK inhibition prevents DCs and myeloid cells from a entering a T cell-suppressive state. The proposed studies are significant as they will offer molecular and cellular insights as to how DNA-PK activity contributes to tumor immunogenicity and exploit these insights to develop more reliable biomarkers and effective therapies against weakly immunogenic tumors.