Tuesday, June 6, 2023
6/6/2023
Summary: The objective of this proposal is to understand the mechanisms that govern DNA replication fork stability upon treatment with multiple-drug doses in BRCA-mutant tumors. Mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 are associated with several forms of cancer, including breast and ovarian cancers. BRCA proteins are required for the maintenance of replication fork stability following treatment with chemotherapeutics such as cisplatin, a DNA cross-liking agent widely used for cancer treatment. Replication forks can reverse to aid the repair of DNA damage induced by chemotherapeutics and BRCA proteins are key to protecting the reversed structures from nucleolytic degradation. In absence of BRCA, reversed replication forks are extensively degraded by nucleases, leading to chemosensitivity. However, the molecular basis of the DNA-damaging drug sensitivity in BRCA-mutant tumors remain unclear. Defining these mechanisms is crucial for basic research to inform and improve current clinical oncology regimens based on DNA replication inhibitors. Most studies focus on the analysis of replication perturbations following a single-dose treatment. For the first time, we investigated replication fork perturbations in BRCA1-deficient cells treated with cisplatin 24 hours after pre-exposure to this drug. Our preliminary data challenge the dogma that DNA-damaging drug sensitivity originates from the extended replication fork degradation phenotype observed after a single-dose treatment in BRCA1-deficient cells. We found that fork degradation is no longer detectable when using multiple cisplatin doses. This effect depends on the overexpression and DNA primase activity of the PrimPol polymerase. Based on this premise, we hypothesize that a PrimPol-dependent pathway rescues replication fork degradation following multiple rounds of cisplatin treatment and modulates cisplatin sensitivity in BRCA1-deficient cells. We also posit that cancer cell reliance on fork repriming is enhanced under any condition that leads to reversed fork degradation¾¿e.g., BRCA1 or BRCA2 protein deficiency. Aim 1 will define the function of the dual enzymatic activity of PrimPol in replication fork stability in BRCA1- deficient cells following treatment with multiple cisplatin doses. Aim 2 will determine whether PrimPol-mediated repriming rescues fork degradation by suppressing fork reversal, which would otherwise lead to extensive nascent strand degradation in BRCA-mutants. Aim 3 will determine the impact of the cisplatin-induced PrimPol overexpression on genomic instability and BRCA1-deficent cancer cell viability. This will be achieved by using a unique combination of single-molecule DNA replication and electron microscopy approaches available in our laboratory. These studies will establish a new paradigm for the PrimPol polymerase in replication fork protection and genomic stability. They will also introduce the novel concept that multiple-drug doses need to be used to fully understand how cells respond to chemotherapeutics, and they will offer new insights for the treatment of BRCA-mutant cancer patients by targeting the PrimPol-dependent pathway.
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