DMA topoisomerase I (Topi) plays important roles in DMAreplication, transcription and recombination and is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CRT poisons Topi by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DMAlesions that signal cell cycle arrest and cell death. Although it is generally accepted that Topi targeted drugs induce DNA damage in S-phase, it is clear that signaling pathways activated in response to damage ultimately dictate cellular fate. Using yeast as a model system, conserved components of the replication machinery, CDC45 and DPB11(TopBP1), protect cells from Topi damage. Rapamycin-sensitive TOR signaling also protects yeast cells from cytotoxic DNA lesions during S-phase. Our data support a model whereby TOR acts as a survival pathway in response to genotoxic stress by maintaining replication fork stability and the dNTP pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA damaging agents coincides with increased mutation rates, which may contribute to the acquisition of drug resistance.
Three specific aims are proposed to investigate conserved aspects of the replication machinery and TOR signaling that maintain cell survival in response to cytoxic agents, suca at CRT.
In Aim 1, a combination of yeast genetics and chromatin immunoprecipitates to query high-density tiling arrays (ChlP-chip experiments) will investigate the mechanism by which rapamycin-sensitive TOR signaling maintains replication fork stability and regulates DNA damage-induced mutagenesis.
Aim 2 proposes to determine if rapamycin- sensitive mTOR signaling regulates human cell sensitivity to cyotoxic chemotherapeutics and the acquisition of drug resistance. A DNA fiber labeling strategy will determine if rapamycin treatment affects replication fork progression and stability in the presence of DNA damage, while the extent of DNA damage induced will be defined by yH2AX staining. siRNA-based approaches will determine if S-phase checkpoint function is required for the protective function of mTOR.
In Aim 3, an analysis of synthetic lethal interactions will define pathway interactions of the conserved human DNA replication proteins, CDC45 and TopBPI, in regulating cell sensitivity to CRT and rapamycin. These studies will provide critical insights into the function of the TOR pathway in modulating cellular responses to DNA damage, while will impact the clinical development of rapamycin in combination with topoisomerase l-targeted therapeutics. The potential to block drug-induced mutations that confer resistance represents a unique application of rapamycins with clinical importance for the treatment of pediatric malignancies.
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