FOXO1 is a master regulator of CAR T memory programming [RNA_FOXO1_OE]

Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states. Transcriptomic study of HA.28z or CD19.28z CAR T cells overexpressing empty vector (NGFR) or FOXO1 or TCF1 transcription factors, 3 independent donors per condition

嵌合抗原受体T细胞（CAR T）的持久性不足，限制了其在B细胞恶性肿瘤与实体瘤治疗中的应用[1,2]。记忆相关基因（如TCF7，其蛋白名称为TCF1）的表达与患者的治疗应答及长期持久性密切相关[3-7]，由此提示记忆程序与治疗疗效存在关联。本研究证实，先驱转录因子（pioneer transcription factor）FOXO1可促进人CAR T细胞的记忆程序，并抑制其耗竭。对人CAR T细胞内源性FOXO1进行药物抑制或基因编辑，会降低记忆相关基因的表达，诱导出耗竭样表型，并削弱其在体内外的抗肿瘤活性。FOXO1过表达可诱导出与T细胞记忆相符的基因表达程序，并增强FOXO1结合基序处的染色质开放程度。在慢性刺激条件下，过表达FOXO1的CAR T细胞可维持自身功能、记忆潜能与代谢适配性，并在体内表现出更强的持久性与抗肿瘤活性。与之相反，过表达TCF1并无法强化典型记忆程序，也不能提升CAR T细胞的治疗效能。值得注意的是，患者体内内源性FOXO1的活性与CAR T细胞及肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocyte, TIL）的应答情况相关，这凸显了其在癌症免疫治疗中的临床价值。本研究结果表明，通过FOXO1实现记忆重编程，可增强人CAR T细胞的持久性与治疗效能；同时也证实，结合致密染色质并诱导局部表观遗传重塑的先驱转录因子，可用于优化治疗性T细胞的功能状态。本研究的转录组学分析对象为：过表达空载体（NGFR）、FOXO1或TCF1转录因子的HA.28z或CD19.28z CAR T细胞，每组设置3名独立供体的生物学重复。