Investigating the role of neddylation in the repair of topoisomerase I inhibitor-induced replication damage in colorectal cancer

PROJECT SUMMARY Colorectal cancer (CRC) is one of the most lethal cancers. The topoisomerase I (TOP1) inhibitor irinotecan is a major component of the current chemotherapeutic regimens for treatment of CRC. TOP1 is an enzyme that resolves DNA topological issues by transient DNA breakage. TOP1-DNA cleavage complexes (TOP1ccs) are covalent protein-DNA intermediates formed during TOP1 activity. Clinically active TOP1 inhibitors convert the transient TOP1ccs into long-lived TOP1 DNA-protein crosslinks (TOP1-DPCs). TOP1-DPCs interfere with DNA metabolic processes and induce cell death. Cells can repair TOP1-DPCs using mechanisms such as the ubiquitin-proteasome system (UPS), and enhanced repair capability can result in reduced clinical efficacy of TOP1 inhibitors. Targeting the UPS-dependent repair may improve the therapeutic benefits of TOP1 inhibitors. Following high-throughput screens in CRC cells, I identified pevonedistat, a NEDD8-activating enzyme inhibitor, as a cytotoxic agent inducing synergistic cytotoxicity with irinotecan and other TOP1 inhibitors. I also found that inhibition of NEDD8 modification (neddylation) blocked the proteasomal degradation of TOP1-DPCs. I hypothesize that the DDB1-CUL4-RBX1 complex (CRL4), a ubiquitin E3 ligase that is activated upon neddylation and is important for DNA replication and repair, ubiquitylates TOP1-DPCs leading to their degradation. I found that DCAF13, a DDB1-CRL4 associated factor, binds to TOP1 and is required for TOP1- DPC ubiquitylation, suggesting that DCAF13 recruits TOP1-DPC to CRL4. I will systematically assess this hypothesis and its implications during this award via three specific aims. During the K99 phase, I will work closely with my mentor, Dr. Yves Pommier, a world-renowned expert in topoisomerases and cancer chemotherapy. During my K99 training, I will assess the combination of irinotecan plus pevonedistat in CRC patient-derived preclinical models (Aim 1). In specific aim 2, I will determine if the CRL4 complex acts as a ubiquitin ligase for TOP1-DPCs and determine if DCAF13 is sufficient to recruit TOP1-DPCs for CRL4- mediated ubiquitylation. In addition, I will study the mechanism by which neddylation activates the CRL4 complex for TOP1-DPC repair. Work in aim 2 will continue the independent R00 phase of this grant. A major goal for this latter phase will be to determine CRL4-targeted ubiquitylation sites on TOP1-DPCs using mass spectrometry. These results will be important for understanding mechanisms the allow cells to distinguish between TOP1ccs and long lasting TOP1-DPCs Further experiments in the R00 phase (Aim 3) will interrogate the role of neddylation in the repair of TOP1-DPC-induced DNA double-strand breaks (DSBs) during replication in CRC cells. I will determine if neddylation modifies histones to recruit factors involved in homologous recombination (HR) and if neddylation modifies p53 to enhance p53-regulated HR gene expression to facilitate the DSB repair. Thus, support through the K99/R00 award will be instrumental in completing this important research and will help me successfully transition as an independent researcher.