Abstract 3596: Metabolic reprogramming of the tumor microenvironment to enhance the functionality of TCR-edited T cell in colorectal cancer and liver metastases
Authors: Potenza Alessia , Balestrieri Chiara , Spiga Martina , Albarello Luca , Pedica Federica , Ferrari Arianna , Botrugno Oronza A. , Camisa Barbara , Tiziano Elena , Sirini Camilla , Casucci Monica , Iozzi Chiara , Abbati Danilo , Elmore Ugo , Lullo Giulia Di , Casorati Giulia , Doglioni Claudio , Tonon Giovanni , Dellabona Paolo , Rosati Riccardo , Aldrighetti Luca , Monti Paolo , Ruggiero Eliana , Bonini Chiara 
Published: March 2024

Abstract
In solid malignancies, T cells functionality is 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 revealed extensive transcriptional remodeling across tumors, being metabolic pathways among the major drivers of this variance. Accordingly, extracellular adenosine, accumulating in the TME for the hydrolysis of ATP by CD39 and CD73, has been recognized as a novel inhibitory mediator, which binds preferentially to the A2A receptor (A2Ar) to exert its immunosuppressive activity. By high dimensional flow cytometry, we revealed that CD39 is a major driver of T-cell exhaustion in primary and metastatic CRCs. Therefore, we sought to disrupt CD39 in TCR-edited T cells (TCRED). By CRISPR/Cas9, we disrupted both the α and β chains of the endogenous TCR together with ENTPD1, the gene encoding for CD39, and redirected T cell specificity against the HER2 antigen. TCREDCD39KO T cells outperformed their competent counterpart in eliminating patient-derived organoids (PDOs) from primary and metastatic tumors both in vitro and in vivo. Given the role of CD39 in starting the biochemical cascade leading to adenosine production, we aimed at investigating the direct role of adenosine on the functional advantage observed with TCREDCD39KO T cells. We challenged our cellular products with target cells in the presence or absence of adenosine and observed that TCREDCD39KO T cells are superior to their competent counterpart in the ability to release cytokines, while the effect is completely abolished when adenosine is present. Also, when TCREDCD39KO T cells were challenged with PDOs, the exogenously added adenosine nullifies the functional killing advantage given by the CD39 KO. To gain more insights into the pathway, we also generated TCREDA2ArKO T cells. In line with the different roles of CD39 and A2AR on the ATP/adenosine pathway, the disruption of the adenosine receptor resulted in a significant advantage in PDO killing in the presence of adenosine only. We then evaluated the influence of CD39KO in the metabolic capacity of T cells and revealed that TCREDCD39KO T cells have a greater potential to rely on the mitochondrial other than glycolytic functions, which explains higher functionality and persistence. Further evaluation on the metabolic effect of CD39 KO are currently ongoing. Overall, we showed that the ATP/adenosine cascade is relevant for T cells functionality and metabolic status and can be harnessed in adoptive cell therapy strategies to counteract the immunosuppressive TME.
Citation Format: Alessia Potenza, Chiara Balestrieri, Martina Spiga, Luca Albarello, Federica Pedica, Arianna Ferrari, Oronza A. Botrugno, Barbara Camisa, Elena Tiziano, Camilla Sirini, Monica Casucci, Chiara Iozzi, Danilo Abbati, Ugo Elmore, Giulia Di Lullo, Giulia Casorati, Claudio Doglioni, Giovanni Tonon, Paolo Dellabona, Riccardo Rosati, Luca Aldrighetti, Paolo Monti, Eliana Ruggiero, Chiara Bonini. Metabolic reprogramming of the tumor microenvironment to enhance the functionality of TCR-edited T cell in colorectal cancer and liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3596.