E3 ligase mediated control of Foxp3 expression and immune suppression-mechanisms and potential as immunotherapeutic target

项目摘要 肿瘤细胞利用免疫调节机制逃避宿主防御的检测和根除。 Foxp3 + CD4 + CD25+调节性T细胞（Treg）介导的免疫抑制对于免疫逃避至关重要， 肿瘤细胞和成功的肿瘤免疫治疗的障碍。因此，破坏Treg功能的能力是 具有重要的治疗意义。我们和其他人最近的工作揭示了关键的Treg转录因子， Foxp3受多聚泛素化依赖的翻译后调节。特别是，我们发现E3 泛素连接酶Stub1，响应于一系列应激信号而被诱导，促进 Foxp3为Treg抑制的动态调节提供了潜在的靶标。在目前的提案中，我们 试图：1）剖析Stub1表达及其翻译后的分子信号通路， 2）了解Treg的生理Stub1诱导和基因缺失的后果 细胞稳态、分化和功能;和3）测试Foxp3泛素化的药理学激活剂， 新的免疫治疗策略，以破坏癌症背景下的免疫抑制。这些研究将 扩大我们对翻译后Foxp3调控机制的理解。具体来说，我们将 进一步探索决定Stub1活性和表达的途径，包括以前未被认识到的 连接酶通过激酶GS3K β磷酸化。为此，我们将利用生物化学方法， 体外和体内Treg功能的良好表征的模型，以建立消融这些功能的结果。 途径。此外，Foxp3的Stub1/泛素依赖性途径的药理学修饰剂 将测试降解（在药物筛选和先前研究中鉴定）作为免疫破坏剂的功效。 抑制-抗癌免疫治疗的主要障碍。这项审查将以积极的方式进行， 鼠黑素瘤模型（体内）以及从健康人中获得的人白细胞的离体研究 捐赠者和晚期癌症患者。在此过程中，我们将确定潜在的治疗应用， 调节Stub1活性以增强抗肿瘤免疫。我们的实验可能揭示新的调节模式， Stub1活性和Foxp3蛋白下调。详细评估Stub 1的生理诱导及其 对Foxp3和Treg功能的影响被预测为证明有效的治疗应用。Stub1的使用- 激活剂与经证实的检查点靶向剂组合可产生甚至更好的抗肿瘤功效。

Project Summary Tumor cells exploit mechanisms of immune regulation to evade detection and eradication by host defenses. Foxp3+CD4+CD25+ regulatory T cell (Treg)-mediated immune suppression is crucial for immune evasion by tumor cells and an obstacle for successful tumor immunotherapy. Hence, the ability to disrupt Treg function is of major therapeutic significance. Recent work by us and others revealed that the key Treg transcription factor, Foxp3 is subject to polyubiquitination-dependent posttranslational regulation. Particularly, we found the E3 ubiquitin ligase Stub1, which is induced in response to a range of stress signals, facilitates the degradation of Foxp3 providing a potential target for dynamic modulation of Treg suppression. In the current proposal, we are seeking to: 1) Dissect molecular signaling pathways involved in Stub1 expression and its post-translational modification; 2) Understand the consequences of physiological Stub1 induction and genetic deletion for Treg cell homeostasis, differentiation and function; and 3) Test pharmacological activators of Foxp3 ubquitination as novel immunotherapic strategies to undermine immune suppression in the cancer setting. These studies will expand our understanding of the mechanisms behind posttranslational Foxp3 regulation. Specifically, we will further explore pathways determining Stub1 activity and expression, including the previously unappreciated phosphorylation of the ligase by the kinase GS3Kβ. To this end, we will utilize biochemical approaches and well-characterized models of in vitro and in vivo Treg function to establish the consequences of ablating these pathways. Furthermore, pharmacological modifiers of the Stub1/Ubiquitin-dependent pathway for Foxp3 degradation (identified in a drug screen and previous studies) will be tested for efficacy as breakers of immune suppression - a major obstacle for anti-cancer immunotherapy. This vetting will be carried out in an aggressive murine melanoma model (in vivo) as well as in ex vivo studies of human leukocytes obtained from healthy donors and advanced cancer patients. In so doing we will determine the potential therapeutic application of modulating Stub1 activity to boost anti-tumor immunity. Our experiments may reveal novel modes of regulating Stub1 activity and Foxp3 protein downregulation. Detailed assessment of physiological Stub1 induction and its impact on Foxp3 and Treg function is predicted to demonstrate a potent therapeutic application. Use of Stub1- activators in combination with proven checkpoint targeting agents may yield even better anti-tumor efficacy.