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, which in turn interferes with therapy response 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. Benefiting from the evolving link between basic biology including microarray analyses, mass spectrometry, cell culture, in vitro (de)ubiquitylation studies, C. elegans and mouse animal model systems, and translational clinical research, this project will be able to develop novel therapeutic strategies for the treatment of refractory CLL patients