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 studies demonstrate that sumoylation of the T-box transcription factor, Eomesodermin (Eomes), facilitates its association with Zbtb44, a member of the ThPOK family of transcription factors. The Zbtb44-Eomes complex promotes the effector function and anti-tumor activity of CD8+ tumor infiltrated lymphocytes (TILs). In exhausted CD8+ TILs, the ubiquitin ligase Trim47 targets Zbtb44 for degradation and disrupts the Zbtb44-Eomes complex. Furthermore, CRISPR-Cas9-mediated inhibition of Trim47 rescues exhausted CD8+ TILs and restores their effector function. These preliminary findings led us to hypothesize that ubiquitination and sumoylation of the Zbtb44/Eomes complex are critical molecular events that dictate the effector vs exhaustion of CD8+ TILs which can be therapeutically targeted. In Aim1, we will determine the mechanism by which the Zbtb44-Eomes complex promotes effector CD8+T cell function and anti-tumor immunity. We will use newly generated Zbtb44-/- mice to investigate how sumoylation of Eomes at Lys(K)-446 facilitates the formation of the Zbtb44-Eomes complex via the SUMO interacting motif (SIM) within Zbtb44. Further, we will delineate the mechanism by which the Zbtb44-Eomes complex cooperatively binds to and transactivates the IFN- promoter. In Aim 2, we will determine the mechanism by which Trim47-mediated ubiquitination of Zbtb44 leads to dysfunction of CD8+T cells. We will investigate how Trim47, which is upregulated in exhausted (PD1+Tim3+) CD8+ TILs, targets Zbtb44 for ubiquitination at K139 and promotes its degradation. Using newly generated Trim47-/- mice, we will determine how disruption of the Zbtb44- Eomes complex leads to the inhibitory transcriptional profile of exhausted CD8+T cells. In Aim 3, we will target the Zbtb44-Trim47 pathway to promote anti-tumor immunity. We will test the therapeutic potential of blocking Zbtb44 ubiquitination in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 and in a patient- derived xenograft (PDX) colon cancer model. Completion of these studies will lead to: 1) dissection of the novel Zbtb44-Eomes complex that is critical for effector CD8+ T cell function, 2) determination of how Trim47-mediated ubiquitination disrupts this complex leading to alternate transcription profile in exhausted CD8+ TILs, and 3) evaluate the means to target the Zbtb44- Trim47 pathway to overcome the current limitations of CAR-T cell therapy for solid tumors.