Regulation of Zbtb44-Eomes complex in CD8+T cells and anti-tumor immunity

摘要 CAR-T细胞疗法是癌症治疗的新兴选择，但其疗效有限，尤其是在实体瘤中 因为效应CD 8 +T细胞在肿瘤微环境（TME）中变得功能失调并耗尽。 然而，定义效应细胞与CD 8 +T细胞耗竭状态之间微妙平衡的关键途径， 细胞仍然不清楚。 我们的初步研究表明，T-box转录因子Eomesodermin（Eomes）的sumoylation， 促进其与Zbtb 44（转录因子的ThPOK家族的成员）的缔合。Zbtb44-Eomes 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。在 当CD 8 + TIL耗尽时，泛素连接酶Trim 47靶向Zbtb 44降解并破坏Zbtb 44-Eomes 复杂.此外，CRISPR-Cas9介导的Trim 47的抑制拯救了耗尽的CD 8 + TIL并恢复了CD 8 + TIL。 其效应器功能。这些初步的发现使我们假设，泛素化和类小泛素化的蛋白质， Zbtb 44/Eomes复合物是决定CD 8 + TIL的效应子与耗竭的关键分子事件 其可以是治疗靶向的。 在Aim 1中，我们将确定Zbtb 44-Eomes复合物促进效应CD 8 +T细胞增殖的机制。 功能和抗肿瘤免疫。我们将使用新产生的Zbtb 44-/-小鼠来研究类小泛素化是如何影响小鼠的免疫应答的。 赖氨酸（K）-446位的Eomes通过SUMO相互作用基序促进Zbtb 44-Eomes复合物的形成 (SIM)在ZBTB 44中。此外，我们将描述Zbtb 44-Eomes复合物 协同结合并反式激活IFN-γ启动子。在目标2中，我们将通过以下方式确定机制： Trim 47介导的Zbtb 44泛素化导致CD 8 +T细胞功能障碍。我们将调查如何 Trim 47在耗尽的（PD 1 + Tim 3+）CD 8 + TIL中上调，靶向Zbtb 44在K139处的泛素化， 促进其退化。使用新生成的Trim 47-/-小鼠，我们将确定如何破坏Zbtb 44- Eomes复合物导致耗尽的CD 8 +T细胞的抑制性转录谱。在目标3中，我们将针对 Zbtb 44-Trim 47通路促进抗肿瘤免疫。我们将测试阻断 在MC 38和患者中CAR-T细胞中针对癌胚抗原（CEA）的Zbtb 44泛素化 衍生的异种移植物（PDX）结肠癌模型。 这些研究的完成将导致：1）对新的Zbtb 44-Eomes复合物的解剖，该复合物对于 效应CD 8 + T细胞功能，2）确定Trim 47介导的泛素化如何破坏该复合物 导致耗尽的CD 8 + TIL中的替代转录谱，和3）评估靶向Zbtb 44-TIL的方法， Trim 47通路克服目前CAR-T细胞治疗实体瘤的局限性。

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.