DNA topoisomerase I (Top1) is an essential enzyme in humans and is the molecular target of the anticancer agent camptothecin (CPT), and its analogues (topotecan and irinotecan) that are used to treat different cancers. Camptothecins reversibly stabilize Top1-cc by converting them into replication mediated double strand breaks (DSB). Although Top1 inhibitors show broad spectrum antitumor activity, the therapeutic efficacies of these agents are highly variable due to mostly undefined reasons. The cytotoxicity of DNA damage inducing drugs can be enhanced by inhibiting the DNA damage response and repair pathways that respond to them. The long-term objective of our studies is to define the molecular mechanisms involved in repair of Top1-inhibitors induced lesions with the goals of identifying novel drug targets to effectively kill tumor cells by combination therapies. In this direction, our studies have identified a novel role for E3 ubiquitin ligase Rad18 in activation of FA pathway in repair and cell tolerance to CPT-induced damage. However, the mechanisms by which Rad18 activates FA pathway and its putative substrates in the specific context of CPT- induced DNA damage are unknown. Moreover, the FA pathway is known to be activated by replication stress, but its precise roles in DNA damage response to CPT are unknown.
The specific aim 1 of this proposal will focus on determining the precise roles of Rad18 and FA pathway in repair of CPT-induced DSB. Techniques such as DR-GFP reporter system and sister chromatid exchange assays will be used to address these questions. Using various biochemical and proteomic approaches, the specific aim 2 will focus on determining the mechanisms by which Rad18 activates FA pathway and identifying the putative Rad18 substrates that are responsible for the activation of FA pathway in response to CPT.
The specific aim 3 of this proposal will focus on determining roles of Rad18 and FA pathway in cell recovery from CPT induced S-phase checkpoint, by using state of the art biochemical and DNA combing assays. Since Rad18 is a critical regulator of these pathways, the outcomes from these proposed studies will establish a rationale for exploiting Rad18 as a therapeutic target in combination therapies with Top1 inhibitors. Moreover, the putative Rad18 substrates that we propose to identify may represent novel tumor suppressive mechanisms and can potentially lead to discovery of novel therapeutic targets.
The long term goal of this proposal is to understand the molecular mechanisms that repair DNA damage induced by anticancer drug camptothecin. The outcomes from these studies may identify novel therapeutic targets to effectively kill tumor cells by combination therapies with Top1 inhibitors and may identify a novel tumor suppressive mechanism.
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|Tripathi, Kaushlendra; Mani, Chinnadurai; Barnett, Reagan et al. (2014) Gli1 protein regulates the S-phase checkpoint in tumor cells via Bid protein, and its inhibition sensitizes to DNA topoisomerase 1 inhibitors. J Biol Chem 289:31513-25|