Znf740 in the regulation of CD8+T cell exhaustion

摘要： CAR-T细胞疗法是一种新兴的癌症治疗选择，但其疗效有限，特别是在固体 肿瘤，因为效应器CD8+T细胞在肿瘤微环境中变得功能失调并耗尽 (TME)。然而，定义效应器和疲惫状态之间微妙平衡的关键途径 CD8+T细胞仍不清楚。我们的初步数据表明，锌指的一个新成员--Znf740 转录因子家族，对效应性CD8+T细胞至关重要。Znf740与SuMOylated T-bet结合 促进CD8+肿瘤浸润性淋巴细胞(TILs)的效应功能和抗肿瘤活性。 相反，在耗尽的PD1+TIM3+CD8+T细胞中，Znf740的表达下调，这破坏了 锌锌740：T-BET络合物。重要的是，重新构建Znf740表达可以挽救耗尽的CD8+TIL和 恢复其效应器功能。此外，在CD8+T细胞中转基因表达Znf740导致肿瘤减少 生长与TIL产生的干扰素-增加有关。这些重要的发现让我们提出了假设 锌740：T-bet复合体对于CD8+T细胞的效应功能是至关重要的，而CD8+T细胞的破坏 PD1+TIM3+细胞中的这种复合体促进CD8+TIL的耗竭，这可以作为治疗的靶点。 在AIM1中，我们将研究Znf740如何促进效应器CD8+T细胞功能和抗肿瘤反应。vbl.使用 新产生的Znf740-/-和T-bet-K208R敲入小鼠，我们将描述 通过其相扑相互作用基序(SIM)与T-bet结合，形成Znf740：T-bet复合体，并反式激活 干扰素-启动子在效应T淋巴细胞中的表达。在目标2中，我们将以Znf740为靶点，以克服T细胞耗竭并促进 肿瘤消退。我们将研究在晚期肿瘤中破坏Znf740：T-bet复合体是如何促进 Pd1+TIM3+耗竭TIL的交替转录图谱。使用新产生的T细胞特异性Znf740 转基因小鼠，我们将检测在CAR-T细胞中过表达的Znf740对癌胚胎的影响 MC38结肠癌模型中的抗原(CEA)。最后，过表达Znf740基因对人卵巢癌的治疗潜力 CAR-T细胞将在结肠癌患者来源的异种移植(PDX)模型中进行测试。 这些研究的完成将导致1)一种新的Znf740：T-bet络合物的建立，该络合物对 效应器CD8+T细胞的功能，2)决定了降低的Znf740表达如何破坏这一复合体，从而导致 晚期肿瘤患者衰竭的CD8+TIL中的交替转录谱，以及3)评估靶向的方法 目前实体肿瘤的CAR-T细胞治疗的局限性。这可能会导致临床 使用“抗衰竭CAR-T细胞”改善晚期肿瘤患者预后的试验。

ABSTRACT: CAR-T cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors, because the effector CD8+T cells become dysfunctional and exhausted in the tumor microenvironment (TME). However, the key pathways that define the delicate balance between the effector vs. exhausted state of CD8+T cells remain unclear. Our preliminary data demonstrate that Znf740, a novel member of the zinc finger family of transcription factors, is critically essential for effector CD8+T cells. Znf740 binds to SUMOylated T-bet and promotes the effector function and anti-tumor activity of CD8+ tumor-infiltrating lymphocytes (TILs). Conversely, in exhausted PD1+Tim3+CD8+T cells, Znf740 expression is downregulated, which disrupts the Znf740:T-bet complex. Importantly, reconstitution of Znf740 expression rescues exhausted CD8+TILs and restores their effector function. Further, transgenic expression of Znf740 in CD8+T cells resulted in reduced tumor growth which was associated with elevated IFN- production by TILs. These key findings led us to hypothesize that the Znf740:T-bet complex is critically essential for the effector function of CD8+T cells, and the disruption of this complex in PD1+Tim3+ cells promotes exhaustion of CD8+TILs which can be therapeutically targeted. In Aim1, we will investigate how Znf740 promotes effector CD8+T cell function and anti-tumor response. Using newly generated Znf740-/- and T-bet-K208R knock-in mice, we will delineate the mechanism by which Znf740 binds to T-bet through its SUMO-interacting motif (SIM) to form the Znf740:T-bet complex and transactivates the IFN- promoter in effector CD8+TILs. In Aim 2, we will target Znf740 to overcome T cell exhaustion and promote tumor regression. We will investigate how disruption of the Znf740:T-bet complex in advanced tumors promotes an alternate transcription profile of PD1+Tim3+ exhausted TILs. Using newly generated T cell-specific Znf740 transgenic mice, we will test the effect of overexpressing Znf740 in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 colon cancer model. Finally, the therapeutic potential of overexpressing Znf740 in CAR-T cells will be tested in colon cancer patient-derived xenograft (PDX) models. Completion of these studies will result in the establishment of 1) a novel Znf740:T-bet complex that is critical for the effector CD8+ T cell function, 2) determine how reduced Znf740 expression disrupts this complex leading to alternate transcription profile in exhausted CD8+ TILs in advanced tumors, and 3) evaluate the means to target the Znf740 to overcome the current limitations of CAR-T cell therapy for solid tumors. This could lead to clinical trials using "Exhaustion Resistant CAR-T cells" for improved outcomes in patients with advanced tumors.