Connecting DNA Replication Fork Instability with Novel Chemotherapeutic Strategies for Breast Cancer
Abstract
DNA-damaging chemotherapy and ionizing radiation are widely used breast cancer treatments, but are highly toxic to healthy cells. This problem has been partially overcome by the discovery that patients with deleterious germline mutations in the BRCA1 or BRCA2 genes are particularly sensitive to therapies that target DNA, such as cisplatin, or that inhibit specific repair pathways, such as PARP inhibitors. The efficacy of these therapies is, however, hampered by the development of chemoresistance. Moreover, it remains unclear how to best identify which patients with BRCA1 or BRCA2 mutations will respond to these therapies. This proposal aims to identify molecular determinants of chemotherapy resistance in BRCA2-mutated patients with important implications for prognosis and development of more efficient treatments. Several cancer treatment compounds inhibit DNA replication. They create roadblocks on DNA in the expectation that replication forks collide with these roadblocks and break, killing cancer cells. This proposal is based on the recent discovery that the BRCA2 protein is required to stabilize stalled replication intermediates, in addition to its well-established roles in DNA repair. Specifically, BRCA2 prevents extensive nucleolytic degradation in replication forks that have been stalled or damaged by chemotherapy. Uncontrolled nuclease-dependent degradation of stalled replication intermediates detected in the absence of BRCA2 is rapidly emerging as the leading cause of the chemotherapeutic sensitivity of BRCA2-deficient tumors. These findings led to the revolutionary idea that chemoresistance is associated with the "recovered" ability of BRCA2-deficient cells to protect stalled forks from nucleolytic degradation. We will define mechanisms and factors that promote the extensive over-resection of stalled forks in a BRCA2-deficient background. Next, we will determine whether genetic perturbations in these mechanisms are responsible for the "recovered" ability of BRCA2-deficient cells to protect stalled forks from nucleolytic degradation. This will be achieved using an innovative combination of single-molecule DNA replication and electron microscopy, enabling a unique and direct visualization of replication intermediate structures in different genetic backgrounds. Our group is in the unique position to perform these studies because we are one of the few laboratories that combines these state-of-the-art techniques to visualize DNA replication intermediates. Identifying cellular factors that promote over-resection will provide a clear pathway to identify individuals likely to develop resistance to standard DNA-damaging or PARP inhibitor therapies. To this purpose, we are developing novel tissue microarrays from BRCA2-deficient breast cancers that will be used in the future to screen for mutations or changes in the protein levels and systematically interrogate whether these correlate with chemotherapeutic resistance. These studies will lead to a completely new strategy that therapeutically exploits the mutational signatures of breast cancer patients to develop more effective targeted breast cancer therapies.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 31, 2017
- Source ID
- W81XWH1610377
Entities
People
- Alessandro Vindigni
Organizations
- Saint Louis University
- United States Army