Ubiquitin-dependent regulation of the DNA damage-induced apoptosis and relevance for the chemoresistance of refractory CLL

细胞对DNA损伤的反应，包括DNA损伤诱导的细胞凋亡（DDIA）的表达在转录水平上受到调控，并且依赖于几个关键调控蛋白的翻译后修饰。本项目旨在了解泛素修饰如何调节难治性CLL中的DDIA。拟议的工作将特别关注促凋亡BH 3-only蛋白家族成员NOXA及其抗凋亡对应物MCL 1，这些蛋白先前已被证明在CLL发病机制中发挥关键作用。NOXA代表了一种关键的促凋亡因子，其响应于化疗而转录上调。申请人最近发现，NOXA的加速泛素化和随后的降解代表了一种控制DDR的新分子机制，并解释了耐药肿瘤细胞如何逃避标准化疗。为了探索Noxa泛素化的调控机制，我们采用Ub-PS微阵列技术进行了大量高通量研究，以监测肿瘤样本中泛素-蛋白酶体系统（UPS）的变化，包括升高的Noxa泛素化（化疗耐药肿瘤）。此外，E3连接酶高含量微阵列（鉴定Noxa相互作用物）和基于体内RNAi的候选方法被用于预测C.优雅这些分析一起鉴定了许多Ub修饰剂，包括E3 Ub连接酶、去泛素化酶（DUB）和参与DDR特别是DDIA的辅因子。例如，泛素羧基末端水解酶-L1（UCH-L1）在这些研究中被表征为Noxa特异性DUB，调节Noxa泛素化和周转。化学抗性CLL中UCH-L1的表观遗传沉默导致CLL中的Noxa不稳定治疗抗性。此外，这些数据表明，Noxa泛素化不仅控制其稳定性（K48 Ub链），而且还密切影响其亚细胞定位和功能（非典型Ub链）。因此，泛素化的Noxa与分泌机制相关，并参与响应遗传毒性应激的细胞因子分泌。除了DUB之外，高通量分析还鉴定了E3连接酶CHIP和CARP 1。初步分析已经证明了两种E3连接酶在泛素化Noxa中的能力。该项目旨在利用如何以及哪些因素控制NOXA泛素化和影响DDIA的知识，为CLL患者设计新的治疗策略。本研究将探讨以下具体问题：1）识别和表征触发Noxa泛素化的E3连接酶。2)Noxa泛素化对DNA损伤的调节。3)Noxa泛素化的生理相关性。4)CLL中Noxa泛素化的治疗靶向。通过识别新的因素和新的治疗靶点，这些分析将对CLL患者具有治疗价值。

Orchestration of the cellular responses to DNA damage, including DNA damaged-induced apoptosis (DDIA) is regulated on the transcriptional level as well as relying on posttranslational modifications of several key regulatory proteins. This project aims at understanding how the ubiquitin modification regulates DDIA in refractory CLL. The proposed work will especially focus on the pro-apoptotic BH3-only protein family member NOXA and its anti-apoptotic counterpart MCL1 that have been previously shown to play a critical role in CLL pathogenesis. NOXA represents one of the key proapoptotic factors which are transcriptionally upregulated in response to chemotherapy. The applicants recently found that accelerated ubiquitylation and subsequent degradation of NOXA represents a novel molecular mechanism that controls DDR and explains how chemoresistant tumor cells escape standard chemotherapy. To explore the mechanisms governing Noxa ubiquitylation, we performed a number of high-throughput studies by employing Ub-PS Microarray technology to monitor the changes within the ubiquitin-proteasome system (UPS) in tumor samples comprising elevated Noxa ubiquitylation (chemo-resistant tumors). In addition, E3 ligase high-content microarray (identifying Noxa interactors), and in vivo RNAi-based candidate approaches were employed to predict genotoxic stress sensitivity in C. elegans . Together these analyses identified a number of Ub modifiers, including E3 Ub ligases, deubiquitylases (DUBs), and cofactors involved in the DDR and particularly DDIA. For instance, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) was characterized in these studies as a Noxa-specific DUB, regulating Noxa ubiquitylation and turnover. Epigenetic silencing of UCH-L1 in chemo-resistant CLL results in Noxa destabilization therapy resistance in CLL. Furthermore, these data showed that Noxa ubiquitylation is not only controlling its stability (K48 Ub-chain) but also intimately impacts on its subcellular localization and function (atypical Ub-chain). Accordingly, ubiquitylated Noxa was associated with the secretory machinery and was involved in cytokine secretion in response to genotoxic stress. In addition to DUBs, the high-through-put analyses identified the E3 ligases CHIP and CARP1. Preliminary analysis already demonstrated the capability of both E3 ligases in ubiquitylating Noxa. This project aims to exploit the knowledge of how and which factors control NOXA ubiquitylation and impact on DDIA for the design of novel therapeutic strategies in CLL patients. The following specific issues will be addressed: 1) Identification and characterization of E3 ligase(s) triggering Noxa ubiquitylation. 2) Regulation of Noxa ubiquitylation in response to DNA damage. 3) Physiological relevance of Noxa ubiquitylation. 4) Therapeutic targeting of Noxa ubiquitylation in CLL. By identifying new factors and novel therapeutic targets, these analyses will be of therapeutic value for CLL patients.