Abstract LB361: CD39 disruption boosts TCR-edited T cells by metabolic reprogramming
Authors: Alessia Potenza, Laura Conte, Chiara Balestrieri, Martina Spiga, Lorenzo Da Dalt, Fabrizia Bonacina, Paolo Monti, Danilo Norata, Eliana Ruggiero, Chiara Bonini
Journal: Cancer Research (2025)

Abstract In solid malignancies, T cells functionality is closely intertwined with metabolic reprogramming within the tumor microenvironment (TME), which supports the increased energy request of cancer cells and suppresses the effector function and cytokines production of the immune infiltrate. In primary colorectal cancer (CRCs) and related liver metastases, we previously revealed extensive transcriptional remodeling across tumors, being metabolic pathways one of the major drivers of this variance. Among these, the adenosine (Ado) pathway has been shown to impact on the functionality of T cells infiltrating primary and metastatic colorectal cancer. Accordingly, upon T cell receptor (TCR) engagement, T cells release a substantial amount of adenosine triphosphate (ATP) in the TME, which is converted into Ado by CD39 and CD73. While ATP is necessary for T cells’ survival and activation, Ado dampens T cells anti-tumor activity. We previously demonstrated that the genetic disruption of CD39, which has the dual role of maintaining high levels of ATP while inhibiting Ado production, improves the activity of TCR-edited (TCRED) T cells in the adoptive T cell therapy (ACT) context. However, the mechanisms by which CD39 affects T cell behavior is not fully elucidated. Since CD39 activity depends on ATP availability, determined by the balance between catabolic and anabolic pathways, we investigated whether changes in T cells’ energetic metabolism could occur in TCRED T cells after CD39 disruption. By exploiting the Seahorse assay, we compared TCREDCD39COMPHER2 T cells and TCREDCD39KOHER2 T cells’ energetic profiles upon antigen-specific stimulation. We observed a reduced oxygen consumption rate and extracellular acidification rate in TCREDCD39KOHER2 T cells as well as a limited production of ATP, comparable to unstimulated cells. This suggests a metabolic adaptation of TCREDCD39KOHER2 cells towards glycolysis, a less efficient but oxygen-independent ATP production pathway. To investigate the metabolic adaptation of TCREDCD39KOHER2 T cells in a hostile TME, we challenged them in the absence of glucose and oxygen; we noticed a strongly diminished production of reactive oxygen species by TCREDCD39KOHER2 T cells, with the absence of oxygen being the most influencing factor, suggesting less reliance of these cells on mitochondrial activity. To gain a deeper knowledge of how the mitochondrial activity changes upon CD39 disruption, we performed morphological analyses by transmission electron microscopy and observed a rearrangement of TCREDCD39KOHER2 T cells’ mitochondrial cristae upon antigen-specific stimulation. Overall, we linked metabolic reprogramming in TCREDCD39KOHER2 T cells to improved anti-tumor performances. Single-cell RNA sequencing analysis is ongoing to understand the specific pathways involved in this metabolic shift and how can be harnessed to potentiate ACT strategies. Citation Format: Alessia Potenza, Laura Conte, Chiara Balestrieri, Martina Spiga, Lorenzo Da Dalt, Fabrizia Bonacina, Paolo Monti, Danilo Norata, Eliana Ruggiero, Chiara Bonini. CD39 disruption boosts TCR-edited T cells by metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB361.