Abstract: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is an important regulator of cellular metabolism. Though classically associated with neurons and macrophages, our data show that CaMKK2 is also expressed in tumor-infiltrating natural killer (NK) cells and may regulate the anti-tumor activity of these cells. Previous publications have shown that CaMKK2 expression enhances breast tumor growth in mice by polarizing tumor-associated macrophages toward a pro-tumor M2 phenotype. However, our preliminary data suggest that CaMKK2 plays the opposite role in NK cells: CaMKK2 signaling seems to enhance NK cell survival, proliferation, migration, and anti-metastatic immune surveillance. We postulate that CaMKK2 enhances NK cell functions by activating the AKT1, AMPK, and mTORC1 pathways. Using a genetic knock-out mouse model of CaMKK2 and common in vitro assays, we intend to firmly delineate the effects of CaMKK2 on a variety of NK cellular functions. Using small-molecule inhibitors or activators, we will determine the role of the AKT1, AMPK, and mTORC1 pathways in CaMKK2-mediated phenotypes. Because CaMKK2 is selectively expressed in NK cells under tumor conditions, we plan to use ATAC sequencing to identify pathways and tumor factors responsible for CaMKK2 upregulation. Finally, we will use the Lox-Cre system to selectively ablate CaMKK2 in murine NK cells and determine the overall effect of this ablation on tumor metastasis. If the loss of CaMKK2 proves detrimental, we will engineer the NK cell line NK-92 to overexpress CaMKK2 and assay its anti-tumor efficacy when transfused into mice with metastatic lesions. A variety of CaMKK2 inhibitors and degraders are being developed for cancer treatment, and some have already proven efficacious in murine breast cancer models. If we demonstrate that loss or inhibition of CaMKK2 suppresses NK cell function as hypothesized, then our research will be instrumental in determining the target patient population for these new drugs. Patients with NK-cell sensitive tumors (e.g. MHC Class I-negative) or patients taking NK-cell mediated therapies (e.g. monoclonal antibody therapies) should avoid CaMKK2 inhibitors. Our research may also prompt strategies for decoupling the effects of CaMKK2 inhibitors on NK cells and other cell types. We believe that much of the therapeutic efficacy of CaMKK2 inhibitors comes from their effects on macrophages, so macrophage-specific delivery strategies for CaMKK2 inhibitors should be prioritized. Furthermore, our research suggests a novel strategy for enhancing the activity of NK cell transfusions. Immortalized human NK cell lines such as NK-92 are under active investigation as cancer therapies. If CaMKK2 expression enhances NK cell anti-tumor activity, then an NK cell line could be genetically modified to artificially overexpress CaMKK2, enhancing the efficacy of the NK cell transfusion.