Mapping the BRCA2 replication gap suppression domain to uncover themolecular mechanism of chemotherapy response

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Abstract Loss of the breast cancer susceptibility (BRCA1 or BRCA2) genes in hereditary breast and ovarian cancer (HBOC) is characterized by defects DNA repair by homologous recombination (HR) and in the protection of replication forks (known as fork protection (FP)). It is thought that HR and FP deficiencies produce points of vulnerability in cancer cells because they cannot fix or prevent DNA double stranded breaks (DSBs) and therefore cells are sensitive to DNA damaging agents such as to cisplatin and Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Our recent findings provide a counter model in which these therapies induce single stranded DNA (ssDNA) gaps that sensitize BRCA deficient cells due to a defect in gap suppression (GS). Several BRCA mutant cell models support gaps in mediating response, however, each model of resistance maintains at least two functions. Thus, it is not certain which function underlies the resistance, leaving a knowledge gap that limits clinical insight. The development of effective therapies requires identifying whether HR, FP, and/or GS is the fundamental mediator of response. This goal of this study is to systematically disrupt and retain each function (HR, FP, GS) within BRCA2 to define what function is critical for therapy resistance, elucidate a unified mechanism of resistance, and provide insight into inhibiting pathways of resistance to inform therapeutic choices. To do this we aim to determine the molecular mechanism of GS through mapping the GS domain(s) in BRCA2 (Specific Aim 1). In BRCA2 deficient cells complemented with wild-type vs a series of BRCA mutants that either delete or selectively target well-characterized domains (i.e., HR or FP), protein interacting regions, or DNA binding sites, we will analyze gap induction in our routine DNA fiber and immunofluorescence assays. If not already well characterized, we will assess mutants for HR proficiency in standard assays and FP via examination of nascent strand degradation in DNA fiber assays. We will use CRISPR/CAS9 to make additional mutants in the identified GS domain(s) to further characterize the critical residues mediating GS. We will also test PARPi sensitivity of these mutant expressing cells in order to assess the link of HR, FP, or GS to response. We also aim to determine if apoptosis underlies loss of cell viability in BRCA2 deficient cells following genotoxins (Specific Aim 2). Apoptosis will be measured using standard assays in BRCA2 mutants following treatment with cisplatin or PARPi. In addition, we will treat cells with apoptosis inhibitors and determine if sensitivity to PARPi or cisplatin is suppressed. We will verify the time and dose in which DSBs are induced compared to apoptosis and assess if inhibition of apoptosis reduces DSB formation. The rationale for the proposed research is that BRCA2 deficiency will be most effectively treated by therapies that form gaps, gap formation will be a biomarker of tumor response, and to maximize therapy response, pathways limiting gap formation should be targeted. The insight gained from the experiments proposed will have implications for cancer and provide new opportunities for therapeutic intervention.