Abstract 3693: Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells

Abstract

Abstract Adoptive Cell Therapy (ACT) holds great promise to transform cancer treatments. However, applying ACT to a broad patient population still faces major challenges. The significant barriers to the success of ACT have been attributed to multiple factors, including functional exhaustion, poor persistence, and the inability of T cells to efficiently compete against tumor cells for metabolic resources in the tumor microenvironment (TME). These functional deficiencies highlight the importance of targeting T cell metabolism to improve ACT treatment outcomes. In the TME, tumor cells preferentially utilize glycolysis to rapidly generate ATP, converting pyruvate into lactate and creating a hypoxic environment that is inconducive for effector T cells due to a lack of access to the metabolites needed for sustenance, leading to T cell exhaustion, and declined proliferation. Consequently, T cells lose their function and are unable to persist against tumor cells. The expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells has been shown to improve their persistence, polyfunctionality, and anti-tumor effects. Our hypothesis is that the improved polyfunctionality and persistence of CASTAT5-CD4+ T cells are partly due to the reprogramming of their metabolic landscape by CASTAT5, resulting in improved metabolic fitness. Single-cell metabolic pathway enrichment analysis of the identified polyfunctional CASTAT5-CD4+ T cells revealed increased capacity for glycolysis, electron transport chain, oxidative phosphorylation, and amino acid metabolism. Single-cell Flux Estimation Analysis (scFEA) further revealed increased metabolic fluxes in amino acid uptake and synthesis, glycolysis, tricarboxylic acid (TCA) cycle, and polyamine synthesis. Interestingly, CASTAT5-CD4+ T cells showed increased polyamine (spermine and spermidine) synthesis, with the upregulation of the metabolic fluxes from putrescine to spermine, catalyzed by spermidine synthase (Srm) and spermine synthase (Srs), both of which are also upregulated in the polyfunctional CD4+ T cell populations. Spermidine and spermine have been shown to enhance mitochondrial metabolism in T cells, increasing their proliferation and survival. Here, we show that there is increased synthesis of spermine in CASTAT5-CD4+ T cells, which may contribute to their increased capacity for oxidative phosphorylation and TCA cycle, leading to the observed improvements in their persistence and anti-tumor function. These findings suggest a role for the polyamine synthesis pathway in the improvement of T cell function. Further studies modulating targets within the polyamine synthesis pathway will help provide an improved understanding of the underlying mechanisms affecting T cell function, thus, unveiling potential metabolic targets to improve the efficacy of tumor-specific T cells. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Zhi-Chun Ding, Gang Zhou, Huidong Shi. Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3693.