Base Excision Repair Deficiency as a Risk Modifier in BRCA2 Associated Cancer

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Germline mutations in BRCA2 confer exceptionally high risk for breast, ovarian and multiple other cancers. There is strong epidemiological evidence that these risks are modified by genetic factors, however, currently there is no clear understanding of which genetic factors modify the risk of cancer in BRCA2 mutation carriers. An insight into the early driver events will be critical to help design more effective therapeutic, and importantly, preventive strategies for individuals with mutations in BRCA2. In the project outlined here, we will be investigating one such genetic factor that can influence cancer predisposition and/or response to chemotherapeutics in BRCA2 mutation carriers. We have identified members of base excision repair (BER) pathway, which, when lost, alter sensitivity of BRCA2 deficient cells to DNA damaging agents – especially those that cause stalled replication forks. We have identified BER proteins, APE1 (apurinic/apyrimidinic endonuclease) and UNG (Uracil-DNA glycosylase), which, when depleted, rescue sensitivity of BRCA2 deficient cells to stalled fork inducing agents. Depletion of these proteins also reverses chromosomal aberrations, a marker of genomic instability, in BRCA2 depleted cells. Importantly, we find that endogenous base damage is highly elevated in BRCA2 depleted cells. Based on our preliminary data, we hypothesize that altered BER activity and/or APE1 or UNG levels affect cancer risk predisposition in BRCA2 mutation carriers. In keeping with these hypotheses, a 5-year survival analysis of women with BRCA2-mutant ovarian cancer shows that patients with low APE1 levels in their tumor have a far worse prognosis compared to those with high APE1 levels. In order to investigate these hypotheses, we propose to determine whether APE1 and/or UNG affect stalled fork resolution in BRCA2 depleted cells (Aim 1). We will also determine the basis for increased base damage in replicating BRCA2 depleted cells, and the molecular mechanism by which APE1 and UNG affect the resolution of DNA damage in these cells (Aim 2). Finally, we will also determine whether phenotypically normal BRCA2mut/+ mammary epithelial cells (MECs) demonstrate altered stalled fork resolution upon loss of APE1 and/or UNG and how response to therapeutics in BRCA2 mutant cancers is affected by levels of APE1 and/or UNG in those cells (Aim 3). Successful completion of this work will give us an insight into a) a heretofore unknown function of BER proteins in affecting resolution of fork associated DNA damage in BRCA2 depleted cells , b) whether alterations in BER pathway has the ability to influence BRCA2 mutation driven tumorigenesis, and/or the response of BRCA2 tumors to chemotherapeutics, d) source of increased base damage in BRCA2 depleted cells, and c) the mechanism that drives these phenomena. Such work will lay the foundation for targeted, future preventive and therapeutic strategies for BRCA2 mutation associated cancer.