Project Summary Mobilization of both adaptive and innate immune cells has considerable utility in the treatment of breast cancer and other cancers. Here we provide a novel approach. Our central goal is to demonstrate how ablation of the protein acyl transferase, DHHC3, can enhance both adaptive and innate anti-cancer immunity. Towards achieving this goal, we have already shown that ZDHHC3 ablation i) markedly diminishes levels of checkpoint blockade molecule PD-L1 on tumor cells, and ii) disrupts CMTM6, a molecule needed to maintain PD-L1 expression, such that CMTM6 palmitoylation is abolished and subcellular localization markedly altered. Furthermore, iii) restoration with wild type DHHC3, but not enzymatically inactive DHHC3, restores PD-L1 levels. In addition, iv) ZDHHC3 ablation markedly reduces tumor xenograft growth in nude mice (but not in vitro) by a mechanism involving elevated oxidative stress, senescence, and tumor clearance by innate immune cells. Because the DHHC3 enzyme may regulate both adaptive and innate anti-cancer immunity, but is not needed for normal mouse physiology, it may be a useful therapeutic target. Our central guiding hypotheses are i) that DHHC3 ablation prevents CMTM6 palmitoylation, leading to degradation of unprotected PD- L1 on tumor cells, which enhances adaptive immunity and ii) that DHHC3 ablation also enhances innate immunity, with key regulators of oxidative stress (e.g. ERGIC3, TXNIP) and innate immune cells (e.g. NK cells) playing major roles. These hypotheses will be tested as follows: Aim 1, We will use a variety of in vitro and in vivo experiments to establish the extent to which DHHC3 ablation enhances adaptive immunity by a mechanism involving loss of CMTM6 palmitoylation leading to diminished PD-L1 expression. Aim 2, We will assess relative in vivo effects of mammary tumor cell DHHC3 ablation on innate and adaptive anti-breast cancer immunity, and we will evaluate key contributions of oxidative stress, senescence, and NK cells to innate immunity. Expected Impact: Results should support the utility of DHHC3 as a novel tumor target in breast cancer and other cancers, as DHHC3 ablation from tumor cells simultaneously enhances both adaptive and innate immunity, with CMTM6, PD-L1, oxidative stress, and NK cells playing key roles.