Melanoma, one of the most aggressive and fatal neoplasms, is responsible for over 80% of skin cancer-related deaths. As melanoma mostly expresses tumor-specific neo-antigens, immunotherapy such as adoptive cell transfer-based therapy (ACT) is considered a promising and effective treatment for this malignancy. Nevertheless, due to a variety of factors that weaken antitumor immunity, the clinical outcome of this therapy remains less satisfactory. Accumulating evidence suggests that metabolic status of both immune cells and tumor cells have great impact on effectiveness of ACT, and metabolic reprogramming may represent a new viable target for reinforcing the efficacy of immunotherapy. The objective of this project is to understand the role and importance of eukaryotic elongation factor-2 kinase (eEF-2K), a unique enzyme that regulates protein synthesis and is highly expressed in several types of neoplasm including melanoma, in regulation of anti-tumor immunity, and to determine the impact and implication of this kinase in immunotherapy for melanoma. We have revealed a critical role for eEF-2K in promoting aerobic glycolysis (Warburg effect) in cancer, and the underlying mechanism. Intriguingly, our recent preliminary studies showed that eEF-2K is critical for the survival of certain suppressive immune cells. As metabolic reprogramming such as aerobic glycolysis plays crucial roles in regulating immune response and tumor sensitivity to therapeutic intervention, we propose to investigate the roles and implication of eEF-2K in melanoma ACT. We hypothesize that the activity of eEF-2K promotes survival of suppressive immune cells and melanoma cells; targeting this kinase can reduce resilience of tumor and suppressive immune cells, hasten tumor cell death via enhancing the cytotoxicity mediated by CD8+ T cells. To test this hypothesis, we propose to investigate the precise role of eEF-2K in modulating tumor cell response to ACT, with the intent to devise novel approaches to improving the outcome of immunotherapy for patients with advanced melanoma. In Aim #1, we will investigate whether and how tumoral expression of eEF-2K affect tumor cell response to ACT; in Aim #2, we wish to assess and optimize the strategy of targeting eEF-2K for improving ACT against melanoma. The small molecule inhibitors of eEF-2K that we discovered (NH125) and obtained from Janssen (JNJ-978) may provide an exciting opportunity to test their effectiveness as novel modulating agents in immunotherapy. We anticipate that the proposed studies will not only reveal eEF-2K as a unique molecular determinant of anti-tumor immunity, but also lead to identification of novel and effective immune modulators (i.e., inhibitors of eEF-2K) for treatment of this intractable tumor through improving immunotherapy.