Regulation of CD8+T cells by Zbtb42

摘要： 促进抗肿瘤反应的效应CD 8 +T细胞功能失调并衰竭。但关键 定义CD 8 +T细胞的效应子与耗尽状态之间的微妙平衡的机制仍然难以捉摸。 我们的初步研究表明，在早期肿瘤中，T-box转录因子的类小泛素化， bet，促进其与Zbtb 42的关联，Zbtb 42是ThPOK转录因子家族的成员。Zbtb42/T- bet复合物促进CD 8+肿瘤浸润淋巴细胞（TIL）的效应子功能和抗肿瘤活性。 相反，在晚期肿瘤中，泛素连接酶Trim 37在耗尽的CD 8 + TIL中上调， 靶向Zbtb 42降解并破坏Zbtb 42/T-bet复合物。重要的是，CRISPR-Cas9介导 Trim 37的抑制拯救了耗尽的CD 8 + TIL并恢复了它们的效应子功能。这些关键发现使我们 假设Zbtb 42/T-bet复合物的泛素化和类小泛素化是关键的分子事件， 决定了效应物与CD 8 + TIL的耗尽，其可以是治疗靶向的。 在Aim 1中，我们将确定Zbtb 42/T-bet复合物促进效应CD 8 +T细胞增殖的机制。 功能和抗肿瘤反应。我们将使用新产生的Zbtb 42-/-和T-bet-K208 R-KI小鼠来研究 T-bet在Lys（K）-208处的SUMO化如何促进Zbtb 42/T-bet复合物的形成 Zbtb 42内的相互作用基序（SIM）。此外，我们将描述Zbtb 42/T-bet 复合物协同结合并反式激活IFN-γ启动子。在目标2中，我们将研究Trim 37， 在晚期肿瘤中耗尽的（PD 1 + Tim 3+）CD 8 + TIL中上调，通过其meprin与Zbtb 42结合 和TRAF同源（MATH）结构域，并靶向Zbtb 42在K164处的泛素化。使用新生成的 在Trim 37-/-小鼠中，我们将研究Zbtb 42/T-bet复合物的破坏如何导致抑制性转录。 在晚期肿瘤中耗尽的CD 8 +T细胞的概况。在目标3中，我们将靶向Zbtb 42-Trim 37通路， CAR-T细胞促进肿瘤消退。我们将测试阻断Zbtb 42泛素化的治疗潜力。 在MC 38和患者来源的异种移植物（PDX）中，CAR-T细胞对抗癌胚抗原（CEA） 结肠癌模型。 这些研究的完成将导致1）发现新的Zbtb 42/T-bet复合物，其对于效应子的产生至关重要。 CD 8 +T细胞功能，2）确定Trim 37介导的泛素化如何破坏这种复合物，导致 在耗尽的CD 8 + TIL中的替代转录谱，和3）评估靶向Zbtb 42/T-bet的手段。 Trim 37通路克服目前CAR-T细胞治疗实体瘤的局限性。

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.