Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.

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PARP inhibitor (PARPi) therapy targets BRCA1/2 mutant tumor cells, but acquired resistance limits its effectiveness. In this issue of Molecular Cell, Marzio et al. ( 2019) identify a novel mechanism of resistance to PARPi through regulation of RAD51 protein stability via an SCF ubiquitin ligase dependent on EMI1.

Breast and ovarian cancers are prevalent among women, and hereditary breast and ovarian cancers (HBOCs) have been associated with germline mutations in genes such as BRCA1 and BARD1. BRCA1 and BARD1 form an obligate heterodimer, and the BRCA1/BARD1 complex is required for tumor suppression functions. Our lab has tested hundreds of BARD1 and BRCA1 variants and identified many that are deficient in homologous recombination, which we have shown to accurately predict cancer predisposition in the clinic. Several of these functionally defective BRCA1 mutants map to a pocket of amino acids on the surface of the protein that does not have any known binding partners. Repair-deficient mutations are also present on the surface of the BARD1 protein in domains that are not known to be associated with DNA repair. We hypothesize that the DNA repair deficiencies mediated by these BRCA1 and BARD1 mutants are due to differences in protein binding when compared to wild-type protein. We have found that BRCA1 mutants in this protein pocket do not phosphorylate and do not localize to the nucleus following DNA damage, both of which are characteristic of the DNA damage response. However, BARD1 repair-deficient mutants still bind phosphorylated BRCA1, indicating that their deficiencies are not due to loss of BRCA1 function. To investigate protein interaction differences we are creating fusion proteins of wild-type and mutant BRCA1 with BioID2, which will biotinylate proteins that bind to BRCA1. We will then be able to identify, via avidin purification and mass spectrometry, protein interactions that are present in the wild-type but absent in the mutant BRCA1. Novel proteins that we identify will be tested for DNA repair and tumor suppressor function. This information will allow us to better understand both BARD1 and BRCA1 function and the mechanism of homologous recombination. Citation Format: Aleksandra Adamovich, Margaret Wingo, Tapahsama Banerjee, Miranda Gardner, Michael Freitas, Jeffrey Parvin. BRCA1 and BARD1 protein interactions that are required for DNA repair function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1367.