Gabrielle Zuern

Abstract A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo 1 . The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy 2–6 , suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7 ) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

<h3>Background</h3> Poor CAR T cell persistence is a major factor limiting durable responses to CAR T cell therapy. TCF1 is a transcription factor that regulates the expression of memory-related genes and its expression correlates with clinical CAR T persistence, making it a promising target for enhancing CAR T efficacy. Despite TCF1’s established role in memory and stemness, overexpression of TCF1 alone does not enforce a memory-like phenotype or improve CAR T potency,¹ indicating that the function and relevance of TCF1 in human CAR T cells is unclear. These results highlight the need to more carefully interrogate the role of TCF1 in engineered human T cells. Prior work has shown that TCF1 is regulated in a context-specific manner by the presence or absence of its co-activator, β-catenin. Our central hypothesis is that TCF1 activity is governed by direct binding of TCF1 to β-catenin in CAR T cells, which can be leveraged to enforce memory programs and enhance CAR T cell potency. <h3>Methods</h3> To test whether TCF1/β-catenin binding is sufficient to enforce CAR T memory, we engineered and tested several variants of a chimeric TCF1 factor (TCF1fusion) in which β-catenin is covalently linked to TCF1. TCF1fusion expression and activity was assessed by Western blot and a TCF1 reporter assay, respectively. Human T cells were co-transduced to express a CAR and TCF1-related factors, phenotyped using flow cytometry and qRT-PCR. CAR T cell antitumor function was assessed <i>in vitro</i> by tumor cell co-culture experiments and <i>in vivo</i> using a leukemia xenograft tumor model in immunocompromised NSG mice. <h3>Results</h3> TCF1fusion expression results in as much as &gt;40-fold increased TCF1 reporter activity and increased expression of endogenous TCF1 target genes, including memory-associated markers CCR7 and IL-7Rα, compared to full-length TCF1 and negative controls. Interestingly, TCF1fusion-CAR T cells exhibit impaired expansion and antitumor activity both <i>in vitro</i> and in leukemia xenograft models, suggesting that constitutive TCF1 activity is deleterious to CAR T cell. <h3>Conclusions</h3> Our results suggest that TCF1/β-catenin binding is sufficient to promote high expression of putative TCF1 target genes and a memory-like phenotype, but might lock CAR T cells into a memory state, thereby blunting CAR T antitumor function. Our findings generate new insight into human TCF1 biology and inform ongoing approaches to leverage TCF1 to enhance the efficacy of CAR T cell therapeutics and other cancer immunotherapies. <h3>Reference</h3> Doan AE, <i>et al</i>. FOXO1 is a master regulator of memory programming in CAR T cells. <i>Nature</i> 2024;<b>629</b>:211–218. <h3>Ethics Approval</h3> Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee approval has been obtained for all mouse experiments (IAC 24-000232).