Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor.

While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.

Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation.

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.

The Role of PALB2 in the DNA Damage Response and Cancer Predisposition.

The deoxyribonucleic acid (DNA) damage response (DDR) is a major feature in the maintenance of genome integrity and in the suppression of tumorigenesis. PALB2 (Partner and Localizer of Breast Cancer 2 (BRCA2)) plays an important role in maintaining genome integrity through its role in the Fanconi anemia (FA) and homologous recombination (HR) DNA repair pathways. Since its identification as a BRCA2 interacting partner, PALB2 has emerged as a pivotal tumor suppressor protein associated to hereditary cancer susceptibility to breast and pancreatic cancers. In this review, we discuss how other DDR proteins (such as the kinases Ataxia Telangiectasia Mutated (ATM) and ATM- and Rad3-Related (ATR), mediators BRCA1 (Breast Cancer 1)/BRCA2 and effectors RAD51/DNA Polymerase η (Polη) interact with PALB2 to orchestrate DNA repair. We also examine the involvement of PALB2 mutations in the predisposition to cancer and the role of PALB2 in stimulating error-free DNA repair through the FA/HR pathway.

DNA repair genes PAXIP1 and TP53BP1 expression is associated with breast cancer prognosis.

Despite remarkable advances in diagnosis, prognosis and treatment, advanced or recurrent breast tumors have limited therapeutic approaches. Many treatment strategies try to explore the limitations of DNA damage response (DDR) in tumor cells to selectively eliminate them. BRCT (BRCA1 C-terminal) domains are present in a superfamily of proteins involved in cell cycle checkpoints and the DDR. Tandem BRCT domains (tBRCT) represent a distinct class of these domains. We investigated the expression profile of 7 tBRCT genes (BARD1, BRCA1, LIG4, ECT2, MDC1, PAXIP1/PTIP and TP53BP1) in breast cancer specimens and observed a high correlation between PAXIP1 and TP53BP1 gene expression in tumor samples. Tumors with worse prognosis (tumor grade 3 and triple negative) showed reduced expression of tBRCT genes, notably, PAXIP1 and TP53BP1. Survival analyses data indicated that tumor status of both genes may impact prognosis. PAXIP1 and 53BP1 protein levels followed gene expression results, i.e., are intrinsically correlated, and also reduced in more advanced tumors. Evaluation of both genes in triple negative breast tumor samples which were characterized for their BRCA1 status showed that PAXIP1 is overexpressed in BRCA1 mutant tumors. Taken together our findings indicate that PAXIP1 status correlates with breast cancer staging, in a manner similar to what has been characterized for TP53BP1.

Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance.

Variants of Uncertain Significance (VUS) are genetic variants whose association with a disease phenotype has not been established. They are a common finding in sequencing-based genetic tests and pose a significant clinical challenge. The objective of this study was to assess the use of functional data to classify variants according to pathogenicity. We conduct functional analysis of a large set of BRCA1 VUS combining a validated functional assay with VarCall, a Bayesian hierarchical model to estimate the likelihood of pathogenicity given the functional data. The results from the functional assays were incorporated into a joint analysis of 214 BRCA1 VUS to predict their likelihood of pathogenicity (breast cancer). We show that applying the VarCall model (1.0 sensitivity; lower bound of 95% confidence interval (CI) = 0.75 and 1.0 specificity; lower bound of 95% CI = 0.83) to the current set of BRCA1 variants, use of the functional data would significantly reduce the number of VUS associated with the C-terminal region of the BRCA1 protein by ~ 87%. We extend this work developing yeast-based functional assays for two other genes coding for BRCT domain containing proteins, MCPH1 and MDC1. Analysis of missense variants in MCPH1 and MDC1 shows that structural inference based on the BRCA1 data set can aid in prioritising variants for further analysis. Taken together our results indicate that systematic functional assays can provide a robust tool to aid in clinical annotation of VUS. We propose that well-validated functional assays could be used for clinical annotation even in the absence of additional sources of evidence.