Project 2: Targeting DNA replication in BRCA-associated breast cancer

PROJECT SUMMARY Germline mutations in BRCA1 and BRCA2 and PALB2 predispose to breast cancer and also increase the risks of ovarian, pancreatic, prostate, and other cancers. The products of the 3 genes constitute a BRCA1-PALB2- BRCA2 axis that plays essential roles in the DNA damage response, particularly in DNA double strand break repair by homologous recombination (HR). In addition, BRCA1 and BRCA2 have also been implicated in the regulation of DNA replication after DNA damage, as their mutant cells are defective in the intra-S phase checkpoint, which slows down DNA synthesis after the damage presumably to allow time for DNA repair and minimize the generation of mutations. However, the mechanisms by which the two proteins regulate DNA synthesis have remained elusive, and the impact of such “unrestrained” DNA synthesis on replication fidelity and genome stability after DNA damage remains unknown. In our preliminary studies, we found that BRCA2 interacts with an essential DNA replication factor, MCM10, and that either loss of BRCA2 or disruption of the BRCA2- MCM10 interaction results in unrestrained replication fork speed, reduced fork stalling, and reduced origin firing after DNA damage and replication stress. Moreover, we also found that loss of BRCA1 leads to reduced replication fork speed both before and after DNA damage. In this proposal, we will determine how BRCA2 and BRCA1 regulate DNA replication kinetics, the impact of unrestrained DNA synthesis on replication fidelity and therapy response, and the potential of replication stress inducers as a targeted therapy for BRCA and PALB2 mutant cancers. In Aim 1, we will define the mechanisms of BRCA2 and BRCA1 in the regulation of replication fork speed, fork stalling and origin firing after replication stress induced by topoisomerase inhibitors. In Aim 2, we will engineer triple negative breast cancer (TNBC) cell lines to assess the impact of replication kinetics dysregulation on replication fidelity, cancer cell response to topoisomerase inhibitors and potential relapse after treatment. In Aim 3, we will use conditional knockout and syngeneic mouse models to determine the efficacy of replication stress inducers on Brca1, Brca2 and Palb2 null mammary tumors.