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损伤诱导的凋亡(DDIA)，在转录水平上受到调控，也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的BH3-Only蛋白家族成员NOXA和其抗凋亡的对应物MCL1，它们在CLL的发病机制中发挥着关键作用。NOXA是一种关键的促凋亡因子，在化疗后转录上调。申请人最近发现，加速泛素化和随后的NOXA降解代表了一种控制DDR的新的分子机制，并解释了耐药肿瘤细胞如何逃脱标准化疗。为了探索控制Noxa泛素化的机制，我们通过使用Ub-PS微阵列技术进行了许多高通量研究，以监测包含Noxa泛素化升高的肿瘤样本(化疗耐药肿瘤)中泛素-蛋白酶体系统(UPS)的变化。此外，E3连接酶高含量微阵列(识别NoxA交互作用)和基于体内RNAi的候选方法被用于预测线虫的遗传毒性应激敏感性。这些分析共同确定了一些Ub修饰物，包括E3Ub连接酶、脱泛素酶(DUBS)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)在这些研究中被鉴定为Noxa特异的DUB，调节Noxa的泛素化和周转。在化疗耐药的CLL中，UCH-L1的表观遗传沉默导致CLL中的NOXA不稳定治疗抵抗。此外，这些数据表明，Noxa泛素化不仅控制其稳定性(K48 Ub链)，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与分泌机制有关，并参与了响应遗传毒性应激的细胞因子分泌。除了DUBS，高通量分析还发现了E3连接酶芯片和CARP1。初步分析已经证明了这两种E3连接酶泛素化Noxa的能力。该项目旨在利用知识如何以及哪些因素控制NOXA泛素化及其对DDIA的影响，以设计新的CLL患者的治疗策略。将解决以下具体问题：1)触发Noxa泛素化的E3连接酶(S)的鉴定和性质。2)DNA损伤对Noxa泛素化的调控。3)NOXA泛素化的生理相关性。4)慢性淋巴细胞性白血病中NOXA泛素化的治疗靶向性。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.