Regulation of CD8+T cells by Zbtb42

ABSTRACT: Effector CD8+T cells that promote anti-tumor response become dysfunctional and exhausted. However, the key mechanisms that define the delicate balance between effector vs. exhausted state of CD8+T cells remain elusive. Our preliminary studies demonstrate that in early-stage tumors, sumoylation of the T-box transcription factor, T- bet, facilitates its association with Zbtb42, a member of the ThPOK family of transcription factors. The Zbtb42/T- bet complex promotes the effector function and anti-tumor activity of CD8+ tumor-infiltrating lymphocytes (TILs). Conversely, in advanced tumors, the ubiquitin ligase Trim37 is upregulated in exhausted CD8+TILs, which targets Zbtb42 for degradation and disrupts the Zbtb42/T-bet complex. Importantly, CRISPR-Cas9-mediated inhibition of Trim37 rescues exhausted CD8+TILs and restores their effector function. These key findings led us to hypothesize that ubiquitination and sumoylation of the Zbtb42/T-bet 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 Zbtb42/T-bet complex promotes effector CD8+T cell function and anti-tumor response. We will use newly generated Zbtb42-/- and T-bet-K208R-KI mice to investigate how sumoylation of T-bet at Lys(K)-208 facilitates the formation of the Zbtb42/T-bet complex via the SUMO interacting motif (SIM) within Zbtb42. Further, we will delineate the mechanism by which the Zbtb42/T-bet complex co-operatively binds to and transactivates the IFN-γ promoter. In Aim 2, we will investigate how Trim37, which is upregulated in exhausted (PD1+Tim3+) CD8+TILs in advanced tumors, binds to Zbtb42 via its meprin and TRAF homology (MATH) domain and targets Zbtb42 for ubiquitination at K164. Using newly generated Trim37-/- mice, we will examine how disruption of the Zbtb42/T-bet complex leads to the inhibitory transcriptional profile of exhausted CD8+T cells in advanced tumors. In Aim 3, we will target the Zbtb42-Trim37 pathway in CAR-T cells to promote tumor regression. We will test the therapeutic potential of blocking Zbtb42 ubiquitination in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 and a patient-derived xenograft (PDX) colon cancer model. Completion of these studies will lead to 1) discovery of the novel Zbtb42/T-bet complex that is critical for effector CD8+T cell function, 2) determination of how Trim37-mediated ubiquitination disrupts this complex leading to alternate transcription profile in exhausted CD8+TILs, and 3) evaluate the means to target the Zbtb42/T-bet- Trim37 pathway to overcome the current limitations of CAR-T cell therapy for solid tumors.