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.

Narrative The goals of this study are to understand how inhibiting a protein called DNA-PK helps makes tumors more detectable by the immune system and how this protein contributes to enhancing the ability for immune cells to remain an anticancer state longer. These studies can potentially develop more effective cancer immunotherapies.