53BP1 is a nuclear scaffolding protein and is involved in DNA repair and cell cycle checkpoint control. Upon induction of DNA double strand breaks, 53BP1 is rapidly phosphorylated by ATM/ATR, and recruited to sites of DNA breaks where it co-localizes with phosphorylated histone H2AX and other DNA repair proteins such as BRCA1. Women with functional loss of BRCA1 are at significantly higher risk for developing breast or ovarian malignancies. Cancer cells that are deficient in BRCA1 have significant homologous recombination mediated DNA repair (HDR) defect, genomic instability, and sensitivity to PARP inhibitors (PARPi) and cisplatin. The current model is that cells with BRCA1 deficiency have unconstrained 53BP1 activity. 53BP1, by yet uncharacterized mechanism, inhibits end resection at DNA double strand breaks and thus, promoting non- homologous end joining (NHEJ) and suppressing HDR. Interestingly, cells with both BRCA1 and 53BP1 deficiencies have partial restoration of HDR and resistance to PARPi and cisplatin. These observations have given rise to notion that 53BP1 and BRCA1 are critical regulators of DNA repair choice. The mechanism, however, by which 53BP1 executes its function in inhibiting end-resection and HDR, and promoting NHEJ is completely unknown. 53BP1 has no known enzymatic function, and is thought to function as a scaffold protein that acts as a platform for protein-protein interactions. We propose that 53BP1 interacts with a set of proteins that are necessary for its role in inhibiting end resection. These interactions are critical for the suppression of HDR seen in BRCA1-/- cells. Loss of 53BP1 interacting proteins will restore HDR in BRCA1 -/- cells and lead to chemoresistance. Recent studies have identified RAD50, a component of MRN complex, and TopBP1, a regulator of CtIP, as 53BP1 interacting proteins. As the MRN complex and CtIP plays critical role in end resection and HDR, it is plausible that 53BP1 mediates the DNA repair phenotype in Brca1 - /- cells via RAD50 and TopBP1. siRNA knockdown will be performed on 53BP1 interacting proteins in Brca1 -/- cell line to measure the restoration of HDR and resistance to PARPi and cisplatin. Furthermore, we will use unbiased high-throughput proteomics approach to identify novel 53BP1-interacting proteins in both Brca1 +/+ and Brca1-/- cell lines. Role of these candidate genes will be analyzed in DNA repair using specific GFP-based assays for HR and NHEJ. Expression of known and novel 53BP1 interacting proteins in human and murine BRAC1-related cancers will be determined using immunohistochemistry, RT-PCR, and genomic sequencing. The ultimate aim of this proposal is to gain new insight into how 53BP1 functions to regulate DNA repair choice in both BRCA1-mutant and BRCA1-wt cells. This will lead to new understanding of how DNA repair pathways are regulated as well as innovative approaches to target chemo resistance in human cancers.
The aim of this proposal is to gain a new insight into how 53BP1 and 53BP1 interacting proteins regulate the DNA repair choice in BRCA1 -/- and BRCA1 +/+ cells. This will lead to a better understanding of how DNA repair pathways can be altered and how chemoresistance is acquired in BRCA1-related cancers.
