Abstract 2405: Rad18 regulates epistatic relationship between FA-BRCA and homologous recombination pathways to repair camptothecin induced DSB
Authors: Tripathi Kaushlendra , Mani Chinnadurai , Clark David , Barnett Reagan , Palle Komaraiah 
Published: May 2015

DNA topoisomerase 1 (Top1) inhibiting anticancer agents such as camptothecin (CPT) show broad spectrum antitumor activity. The therapeutic activities of CPT and its analogues are attributed to prolonged stabilization of Top1-DNA covalent complexes and converting them into replication mediated double strand breaks (DSB). In spite of their therapeutic success, acquired tumor resistance poses major challenges for their therapeutic efficacy. Combination therapies using DNA repair inhibitors can increase the cytotoxic effects of these drugs and aid in overcoming such problems. Homologous recombination (HR) is the predominant DNA repair pathway involved in the repair of CPT induced DSB. Therefore, inhibition of either HR or tumors that are defective in HR could be effectively killed by Top1 inhibitors. In the same way, cells deficient in HR (ex. Rad51) or its associated proteins (such as Rad18, Fanconi anemia (FA) and breast cancer associated (BRCA1, BRCA2) proteins) are known to exhibit hypersensitivity to CPT. Specifically, FA proteins are known to associate with replication forks and implicated in repair of fork stalling lesions by HR in association with BRCA proteins. Thus, an epistatic relationship between FA and BRCA pathway has been suggested in repair of fork stalling lesions, but molecular basis for this cross-talk is not known. We previously shown that Rad18 regulates FANCD2 monoubiquitination (a key step in FA pathway) to promote HR mediated repair of CPT-induced DSB and cell survival. To further characterize the molecular interactions between Rad18 and FA-BRCA pathway in regulating HR, we have assessed their role in HR by DR-GFP reporter assays by downregulating Rad18 and FANCD2 proteins by siRNAs either alone or in combination. Consistent with survival data, cells deficient in Rad18 or FANCD2 exhibited reduced level of HR. Likewise, cells deficient in both the genes also exhibited similar results of single gene deficient cells, suggesting these two proteins work in a common pathway that promotes HR. Further biochemical and biophysical studies in Rad18 and FANCD2 deficient cells revealed dramatic decrease in nuclear localization of Rad51 and its CPT induced foci formation. Since, FANCD2 is required for BRCA2 recruitment to the sites of DSB, and BRCA2 known to associate with Rad51 and stabilize it on single stranded DNA, we further analyzed BRCA2 foci formation and its colocalization with Rad51 in response to CPT. Interestingly, our studies revealed reduced BRCA2 and Rad51 foci and their colocalization in response to CPT in Rad18 and FANCD2 deficient cells. Consistent with their role in HR, cells deficient in either of these genes exhibited chromosomal aberrations, implicating their role in accurate repair of replication coupled DSB. Together these data support that Rad18 regulates epistatic relationship between FA-BRCA and HR pathways in response to replication coupled DSB induced by CPT.

Citation Format: Kaushlendra Tripathi, Chinnadurai Mani, David Clark, Reagan Barnett, Komaraiah Palle. Rad18 regulates epistatic relationship between FA-BRCA and homologous recombination pathways to repair camptothecin induced DSB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2405. doi:10.1158/1538-7445.AM2014-2405