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Background The majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive.Memory T (TM) cells and TILs rely on fatty acid catabolism to preserve their effector functions due to nutrient competition for glucose with tumor cells. Therefore, enhancing fatty acid catabolism of TILs represents an attractive strategy to increase the efficacy of immunotherapies.Sirt2 is an NAD+ dependent histone deacetylase. We previously showed that upregulation of Sirt2 in human TILs negatively correlates with response to TIL therapy in advanced non-small cell lung cancer (NSCLC) and Sirt2 deficiency leads to hyper-reactive T cells with superior antitumor activity. Methods Sirt2 expression was analyzed by flow cytometry and Western blot. The role of Sirt2 in tumor immunity was studied using in vivo B16F10 tumor challenge models as well as ex vivo analysis including RNA-sequencing, CFSE proliferation assay, DAPI/AnnexinV staining, IFN-γ ELISpot assay, intracellular staining of effector molecules and LDH cytotoxicity assay on WT versus Sirt2KO T cells. Molecular partners of Sirt2 were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. The role of Sirt2 in T cell metabolism was investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. AGK2, a Sirt2 selective inhibitor, was used for Sirt2 blockade in human T cells. Results Sirt2 expression is upregulated during T cell activation, TM stage, and within the TME. Our molecular studies revealed that Sirt2 negatively impacts the acetylation status and the activity of the trifunctional protein, the key enzyme of fatty acid oxidation (FAO). Accordingly, Sirt2 deficiency enhanced FAO and metabolic fitness of activated T cells and mouse TILs isolated from B16F10 tumor nodules. As a consequence of enhanced FAO, Sirt2 deficient mice displayed increased accumulation of TM cells, which was associated with decreased apoptosis and increased survival after tumor challenge leading to superior tumor rejection. Most importantly, pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhanced their metabolic fitness and cytotoxic activity against their autologous tumor cells. Conclusions Our findings indicate Sirt2 as a suppressor of T cell metabolism amenable to therapeutic targeting, and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the hypoglycemic TME, thus, leading to an effective anti-tumor immune response. Acknowledgements This work was supported in part by K08 CA194273, ACS IRG-17-173-22, NCI Cancer Center Support Grant (P30-CA076292) and the Moffitt Foundation.

Background: The majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive. Sirt2 is an NAD+ dependent histone deacetylase and conflicting evidences suggest its tumor-suppressor and oncogenic roles. In inflammatory response, Sirt2 suppresses inflammation via negative regulation of NF-κB subunit; however, the role of Sirt2 in tumor immunity has not been described. Method: Human TILs from non-small cell lung cancer (NSCLC) tumor samples and matched peripheral blood T cells were analyzed for Sirt2 expression by flow cytometry. The role of Sirt2 in anti-tumor immunity was studied by in vivo B16F10 tumor challenge in Wild-type (Wt) and Sirt2 knockout (Sirt2KO) mice. The role of Sirt2 in T cell effector functions was investigated ex vivo by CFSE proliferation assay, IFN-γ ELISpot assay, intracellular staining of effector markers and LDH cytotoxicity assay on Wt versus Sirt2KO T cells. Sirt2 targets were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. T cells metabolic changes were investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. Sirt2 blockade in human T cells was performed using AGK2, a Sirt2 selective inhibitor. Result: We show that Sirt2 expression is upregulated within the TME, and its upregulation in human TILs is associated with a poor clinical response to immunotherapy in a phase I clinical trial in advanced NSCLC. We also show that, Sirt2 deficiency in mice boosts T cell effector functions and tumor rejection in vivo. Our molecular and metabolomic studies revealed multiple metabolic pathways as Sirt2 targets including glycolysis, TCA-cycle, FAO and glutaminolysis. We found that Sirt2 deacetylase deficiency increased acetylation and enzymatic activity of key metabolic enzymes leading to a hyper metabolic status of T cells. Finally pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhances their metabolic fitness and effector functions. Conclusion Our findings indicate Sirt2 as a master suppressor of T cell metabolism amenable to therapeutic targeting and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the metabolically challenging TME, thus, leading to an effective anti-tumor immune response. Citation Format: Imene Hamaidi, Lin Zhang, Nayoung Kim, Min Hsuan Wang, Cristina Iclozan, Bin Fang, Min Liu, John M. Koomen, Anders E. Berglund, Sean J. Yoder, Jiqiang Yao, Robert W. Engelman, Ben C. Creelan, Jose R. Conejo-Garcia, James J. Mulé, Scott J. Antonia, Sungjune Kim. Sirt2 blockade promotes T cell metabolism and restores the anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1635.

Summary Dysregulated metabolism is a key driver of maladaptive tumor-reactive T lymphocytes within the tumor microenvironment. Actionable targets that rescue the effector activity of antitumor T cells remain elusive. Here, we report that the Sirtuin-2 (Sirt2) NAD+-dependent deacetylase inhibits T cell metabolism and impairs T cell effector functions. Remarkably, upregulation of Sirt2 in human tumor-infiltrating lymphocytes (TILs) negatively correlates with response to TIL therapy in advanced non-small-cell lung cancer. Mechanistically, Sirt2 suppresses T cell metabolism by targeting key enzymes involved in glycolysis, tricarboxylic acid-cycle, fatty acid oxidation, and glutaminolysis. Accordingly, Sirt2-deficient murine T cells exhibit increased glycolysis and oxidative phosphorylation, resulting in enhanced proliferation and effector functions and subsequently exhibiting superior antitumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and effector functions. Our findings unveil Sirt2 as an unexpected actionable target for reprogramming T cell metabolism to augment a broad spectrum of cancer immunotherapies.