PROJECT SUMMARY/ABSTRACT Since the discovery of the IL-12 cytokine, it has been considered a “master regulator” of the immune system. Primarily produced by dendritic cells, IL-12 is the key signaling molecule linking innate to adaptive immunity to drive IFN𝛾𝛾/Th1-mediated T cell responses against pathogens and cancer. Preclinical studies have long shown that enhancing IL-12 signaling improves vaccines. Recently, IL-12 in dendritic cells was shown to be required for anti-PD1 efficacy in mouse models of cancer. Despite the data nominating the IL-12 pathway as a therapeutic target in cancer, previous attempts to directly administer IL-12 in clinical trials have resulted in patient death from cytotoxic response or poor efficacy due to IL-10-mediated negative feedback. Subsequent efforts in cancer have largely focused on improving methods to deliver and release IL-12 with limited success. Therefore, there is a significant need to better understand the biology of IL-12 regulation in dendritic cells to identify novel, safe and effective strategies of targeting this pathway as an anti-cancer strategy. Our group previously identified PIKfyve lipid kinase as a novel target of the highly efficacious, well- tolerated and orally administered multi-kinase inhibitor, ESK981. We demonstrated the ability of this drug to reduce tumor growth in multiple syngeneic mouse models of cancer and enhance anti-PD1 therapy. Interestingly, drug efficacy was dependent on the presence of CD8+ T cells, and the IL-12 and IFN𝛾𝛾 and signaling pathways in vivo. Furthermore, ESK981 could directly enhance IL-12 signaling in primary dendritic cells in vitro and in vivo through transcriptional regulation of IL12B. Therefore, the overall goal of this project is to define the mechanism of IL-12 regulation by PIKfyve and demonstrate the nature of PIKfyve inhibition, as an anti-cancer therapy, in anti-tumor immune responses. The overarching hypothesis is that PIKfyve inhibition induces IL-12 signaling in dendritic cells to enhance antigen- specific, CD8+ T cell responses in cancer. Experiments in Aim 1 will investigate the effect of Pikfyve knockdown and conditional knock-out in mouse dendritic cells on the genes and protein subunits in the IL-12 signaling pathway in vitro and in vivo. To examine dendritic cell subsets, such as CD8α+ and CD103+ cDCs, mice with genetic knock-out of Batf3 will be included for in vitro and in vivo studies. Experiments in Aim 2 will study the potential anti-tumor effect of PIKfyve inhibitors, including ESK981and Apilimod, on dendritic cell Pikfyve conditional knock-out, Batf3 knock-out, and wild type mice. We will monitor tumor progression and examine T cell responses in the tumor draining lymph nodes and tumor microenvironment, including OVA model antigen and neoantigen specific CD8+ T cell activation using tetramers available for the MC38 tumor model. The proposed study will provide greater insight into the biology of IL-12 signaling in dendritic cells an...