Clinical evidence supports the notion that T cell exhaustion and terminal differentiation pose challenges to the persistence and effectiveness of chimeric antigen receptor-T (CAR-T) cells. MEK1/2 inhibitors (MEKIs), widely used in cancer treatment due to their ability to inhibit aberrant MAPK signaling, have shown potential synergistic effects when combined with immunotherapy. However, the impact and mechanisms of MEKIs on CAR-T cells remain uncertain and controversial. To address this, we conducted a comprehensive investigation to determine whether MEKIs enhance or impair the efficacy of CAR-T cells. Our findings revealed that MEKIs attenuated CAR-T cell exhaustion and terminal differentiation induced by tonic signaling and antigen stimulation, thereby improving CAR-T cell efficacy against hematological and solid tumors. Remarkably, these effects were independent of the specific scFvs and costimulatory domains utilized in CARs. Mechanistically, analysis of bulk and single-cell transcriptional profiles demonstrates that the effect of MEK inhibition was related to diminish anabolic metabolism and downregulation of c-Fos and JunB. Additionally, the overexpression of c-Fos or JunB in CAR-T cells counteracted the effects of MEK inhibition. Furthermore, our Cut-and-Tag assay revealed that MEK inhibition downregulated the JunB-driven gene profiles associated with exhaustion, differentiation, anergy, glycolysis, and apoptosis. In summary, our research unveil the critical role of the MAPK-c-Fos-JunB axis in driving CAR-T cell exhaustion and terminal differentiation. These mechanistic insights significantly broaden the potential application of MEKIs to enhance the effectiveness of CAR-T therapy.

临床证据支持 T 细胞耗竭和终末分化对嵌合抗原受体 T （CAR-T） 细胞的持久性和有效性构成挑战的观点。MEK1/2 抑制剂 （MEKIs） 因其抑制异常 MAPK 信号传导的能力而广泛用于癌症治疗，当与免疫疗法联合使用时，已显示出潜在的协同效应。然而，MEKI 对 CAR-T 细胞的影响和机制仍不确定且存在争议。为了解决这个问题，我们进行了一项全面的调查，以确定 MEKI 是增强还是损害 CAR-T 细胞的功效。我们的研究结果表明，MEKI 减弱了强直信号传导和抗原刺激诱导的 CAR-T 细胞耗竭和终末分化，从而提高了 CAR-T 细胞对血液和实体瘤的疗效。值得注意的是，这些效应与 CAR 中使用的特定 scFv 和共刺激域无关。从机制上讲，对大量和单细胞转录谱的分析表明，MEK 抑制的作用与减少合成代谢和下调 c-Fos 和 JunB 有关。此外，CAR-T 细胞中 c-Fos 或 JunB 的过表达抵消了 MEK 抑制的作用。此外，我们的 Cut-and-Tag 检测显示，MEK 抑制下调了与耗竭、分化、无反应、糖酵解和细胞凋亡相关的 JunB 驱动的基因谱。总之，我们的研究揭示了 MAPK-c-Fos-JunB 轴在驱动 CAR-T 细胞耗竭和终末分化中的关键作用。这些机制见解显着拓宽了 MEKI 在提高 CAR-T 疗法有效性方面的潜在应用。