Introduction: Adoptive cell therapy (ACT) has emerged as a promising approach to cancer treatment. Our group reported a novel class of anti-tumor cells, termed Induced-T-to-NK cells (ITNKs), by inactivating BCL11B in mature human T cells. ITNKs exhibit characteristics of both T-cell and NK-cell, enabling them to efficiently recognize and lyse solid tumors in vitro and in vivo. The phase I clinical trial (NCT:03882840) demonstrated that patients with late-stage solid tumors benefitted from autologous ITNK infusion without any severe side effects. Notably, one patient with metastatic colorectal cancer (CRC) achieved partial remission (PR). However, ITNKs also exhibit limitations in their in vivo persistence, which remains to be addressed for ultimate treatment benefits. Cellular metabolism plays a vital role in the reprogramming of induced T cells to ITNKs. An increase of oxidative phosphorylation (OXPHOS) in ITNKs promotes NK-related gene expression and augments their antitumor efficacy by elevating acetyl-CoA levels. T cells with high mammalian target of rapamycin-1 (mTOR) activity elevate glycolysis and render short-term effector capacity. We hypothesize that the PI3K/AKT/mTOR pathway impairs the persistence of ITNKs through the increased glycolytic metabolism.
Objective: In this study, we aim to elucidate the mechanism by which the increase in lactate production via glycolysis promotes the expression of exhaustion-related genes in ITNKs. The plausible mode of action through epigenetic modifications was also explored.
Methods: ITNKs were analyzed by RNA-seq, ATAC-seq, CUT&Tag-seq, and RT-qPCR. Cell surface markers and proteins were detected by Flow cytometry and Western blot. Cell-derived xenograft mouse models were used to evaluate the in vivo antitumor activity of ITNKs. Glycolysis metabolism was measured by the Extracellular Acidification Rate (ECAR). Lactate levels were quantified using a lactate assay kit.
Results: PI3K/AKT/mTOR signaling pathway and glycolytic activity were upregulated in ITNKs compared to T cells. Inhibition of mTORC1 with Rapamycin reduced the expression of exhaustion markers, including PD-1, TIM3, and LAG3, and promoted the stem cell memory phenotype (CD45RO+CCR7-). Rapamycin-pretreated ITNKs demonstrated long-term antitumor activity and increased tumor infiltration in mouse models. Our findings indicate that PI3K/AKT/mTOR pathway activation enhances glycolytic activity in ITNKs, which elevates the lactate levels and modulates the histone lactylation modifications.
Conclusion: The PI3K/AKT/mTOR signaling pathway is activated in ITNKs. This is accompanied by upregulation of glycolytic metabolism. The high glycolytic activity results in elevated cellular lactate levels, leading to an increase in histone lactyaltion modifications and subsequent exhaustion features in ITNKs.

Rui LIAO

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