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.