The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

Genshen Zhong^1,2,3*Biao Liu^3,4Xue Gong³Qi Wang³Shuyin Zheng²Xinyu Luo²Xingya Zhang²Biliang Hu⁵Minna Wu²Zhiguo Chen²

• ¹Changsha University, Changsha, China
• ²College of Biological and Chemical Engineering, Changsha, China
• ³Xinxiang Medical University, Xinxiang, China
• ⁴Xinxiang Vocational and Technical College, Xinxiang, China
• ⁵Hunan Siweikang Pharmaceutical Co., Ltd, Changsha, China

Abstract Background: Chimeric Antigen Receptor-T (CAR-T) cell therapy has demonstrated remarkable success in hematological malignancies but remains limited in the treatment of solid tumors. This study investigates the role of ATP synthase inhibitory factor 1 (ATPIF1) in modulating the efficacy of Her2-targeted CAR-T cells against solid tumors through metabolic reprogramming and the mitochondrial DNA (mtDNA)-stimulator of interferon genes (STING) signaling pathway. Methods: Her2-targeted CAR-T cells with ATPIF1 overexpression (Her2-IF1 CAR-T) or knockdown (Her2-shIF1 CAR-T) were generated, and their antitumor activity was evaluated in vitro and in vivo. The underlying mechanisms were also elucidated. Results: In vitro, ATPIF1 overexpression enhanced CAR-T cell function, including increased tumor lysis, cytokine secretion (IL-2, IFN-γ), and oxidative phosphorylation (OCR). Conversely, ATPIF1 knockdown impaired these functions. Surprisingly, in vivo results revealed the opposite trend: Her2-shIF1 CAR-T cells exhibited superior tumor inhibition, while Her2-IF1 CAR-T cells showed reduced efficacy despite their prolonged persistence. Mechanistically, ATPIF1 knockdown increased mitochondrial membrane potential (MMP), promoted survival under hypoxic conditions (1% O₂), and enhanced CAR-T infiltration into tumors. This was linked to mitochondrial permeability transition pore (mPTP) opening and mtDNA leakage, which activated the STING pathway, further amplifying T cell migration and antitumor responses. Inhibition of STING with H151 reversed these effects, confirming its critical role in modulating ATPIF1-mediated functions in Her2-targeting CAR-T cells. Conclusion: Our findings highlight the dual role of ATPIF1 in CAR-T cell therapy: while its overexpression boosts metabolic activity in vitro, its knockdown enhances adaptability to the hypoxic tumor microenvironment in vivo, indicating the paradox for modulating the antitumor activities of CAR-T cells via the metabolic remodeling for the treatment of solid tumor These insights suggest that targeting ATPIF1 or the STING pathway could optimize CAR-T cell efficacy in solid tumors, bridging the gap between in vitro performance and in vivo outcomes.