Multiplexed assay of variant effect reveals residues of functional importance in the BRCA1 coiled-coil and serine cluster domains.

Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.

DNA repair function scores for 2172 variants in the BRCA1 amino-terminus.

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1.

DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus.

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1 . AUTHOR SUMMARY: Most missense substitutions in BRCA1 are variants of unknown significance (VUS), and individuals with a VUS in BRCA1 cannot know from genetic information alone whether this variant predisposes to breast or ovarian cancer. We apply a multiplexed functional assay for homology directed repair of DNA double strand breaks to assess variant impact on this important BRCA1 protein function. We analyzed 2172 variants in the amino-terminus of BRCA1 and demonstrate that variants that are known as pathogenic have a loss of function in the DNA repair assay. Conversely, variants that are known to be benign are functionally normal in the multiplexed assay. We suggest that these functional determinations of BRCA1 variants can be used to augment the information that clinical cancer geneticists provide to patients who have a VUS in BRCA1 .

The functional impact of BRCA1 BRCT domain variants using multiplexed DNA double-strand break repair assays.

Pathogenic variants in BRCA1 are associated with a greatly increased risk of hereditary breast and ovarian cancer (HBOC). With the increased availability and affordability of genetic testing, many individuals have been identified with BRCA1 variants of uncertain significance (VUSs), which are individually detected in the population too infrequently to ascertain a clinical risk. Functional assays can be used to experimentally assess the effects of these variants. In this study, we used multiplexed DNA repair assays of variants in the BRCA1 carboxyl terminus to functionally characterize 2,271 variants for homology-directed repair function (HDR) and 1,427 variants for cisplatin resistance (CR). We found a high level of consistent results (Pearson's r = 0.74) in the two multiplexed functional assays with non-functional variants located within regions of the BRCA1 protein necessary for its tumor suppression activity. In addition, functional categorizations of variants tested in the multiplex HDR and CR assays correlated with known clinical significance and with other functional assays for BRCA1 (Pearson's r = 0.53 to 0.71). The results of the multiplex HDR and CR assays are useful resources for characterizing large numbers of BRCA1 VUSs.

Modulation of Early Mitotic Inhibitor 1 (EMI1) depletion on the sensitivity of PARP inhibitors in BRCA1 mutated triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) represents approximately 10-15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 inhibitor. Notably, increasing the RAD51 protein expression only recapitulated some, but not all, of the effects of EMI1 depletion in conferring to the cell resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking mitotic entry. We found that combining CHK1 inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.

Functional analysis of BARD1 missense variants in homology-directed repair and damage sensitivity.

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.

F-Box Protein-Mediated Resistance to PARP Inhibitor Therapy.

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.

A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function.

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.

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation.

Mesodermal cells signal to neighboring epithelial cells to modulate their proliferation in both normal and disease states. We adapted a Caenorhabditis elegans organogenesis model to enable a genome-wide mesodermal-specific RNAi screen and discovered 39 factors in mesodermal cells that suppress the proliferation of adjacent Ras pathway-sensitized epithelial cells. These candidates encode components of protein complexes and signaling pathways that converge on the control of chromatin dynamics, cytoplasmic polyadenylation, and translation. Stromal fibroblast-specific deletion of mouse orthologs of several candidates resulted in the hyper-proliferation of mammary gland epithelium. Furthermore, a 33-gene signature of human orthologs was selectively enriched in the tumor stroma of breast cancer patients, and depletion of these factors from normal human breast fibroblasts increased proliferation of co-cultured breast cancer cells. This cross-species approach identified unanticipated regulatory networks in mesodermal cells with growth-suppressive function, exposing the conserved and selective nature of mesodermal-epithelial communication in development and cancer.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms.

Cancer cells require telomere maintenance to enable uncontrolled growth. Most often telomerase is activated, although a subset of human cancers are telomerase-negative and depend on recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). ALT depends on proteins that are essential for homologous recombination, including BLM and the MRN complex, to extend telomeres. This study surveyed the requirement for requisite homologous recombination proteins, yet to be studied in human ALT cell lines, by protein depletion using RNA interference. Effects on ALT were evaluated by measuring C-circle abundance, a marker of ALT. Surprisingly, several proteins essential for homologous recombination, BARD1, BRCA2, and WRN, were dispensable for C-circle production, while PALB2 had varying effects on C-circles among ALT cell lines. Depletion of homologous recombination proteins BRCA1 and BLM, which have been previously studied in ALT, decreased C-circles in all ALT cell lines. Depletion of the non-homologous end joining proteins 53BP1 and LIG4 had no effect on C-circles in any ALT cell line. Proteins such as chromatin modifiers that recruit double-strand break proteins, RNF8 and RNF168, and other proteins loosely grouped into excision DNA repair processes, XPA, MSH2, and MPG, reduced C-circles in some ALT cell lines. MSH2 depletion also reduced recombination at telomeres as measured by intertelomeric exchanges. Collectively, the requirement for DNA repair proteins varied between the ALT cell lines compared. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways.

HDAC10 as a potential therapeutic target in ovarian cancer.

OBJECTIVE: We analyzed histone deacetylase 10 (HDAC10) for function in the context of the DNA damage response in BRCA1-null ovarian cancer cells as well as evaluated the potential of general HDAC inhibitors in primary ovarian carcinoma cells. HDAC10 had previously been shown to be highly stimulatory to the process of homology directed repair in HeLa cells, and in this study we investigated whether HDAC10 could impact in vitro the response to anticancer therapies. We hypothesized that the loss of HDAC10 would sensitize cells to platinum therapy. METHODS: We combined informatics analysis of large DNA sequencing datasets from ovarian cancer tumors with tissue culture based assays of primary and established cell lines to test for sensitivity to platinum therapy if HDAC10 activity was inhibited or depleted. RESULTS: Using The Cancer Genome Atlas (TCGA) dataset, we found that deep deletions in HDAC10 occurred in 5-10% of ovarian cancer tumors. From the TCGA data we found that low HDAC10 mRNA levels correlated with platinum sensitivity of the tumors. Cell proliferation and DNA damage assays in a BRCA1-null ovarian carcinoma cell line demonstrated reduced DNA repair capacity and sensitization of platinum therapy. Similarly, primary ovarian carcinoma cells demonstrated a sensitization to platinum therapies when treated with HDAC inhibitors. CONCLUSIONS: From the results of this study, we suggest that the inhibition of HDAC10 may potentiate the effects of platinum therapies in ovarian tumors.

Integrative analysis of somatic mutations and transcriptomic data to functionally stratify breast cancer patients.

BACKGROUND: Somatic mutations can be used as potential biomarkers for subtyping and predicting outcomes for cancer patients. However, cancer patients often carry many somatic mutations, which do not always concentrate on specific genomic loci, suggesting that the mutations may affect common pathways or gene interaction networks instead of common genes. The challenge is thus to identify the functional relationships among the mutations using multi-modal data. We developed a novel approach for integrating patient somatic mutation, transcriptome and clinical data to mine underlying functional gene groups that can be used to stratify cancer patients into groups with different clinical outcomes. Specifically, we use distance correlation metric to mine the correlations between expression profiles of mutated genes from different patients. RESULTS: With this approach, we were able to cluster patients based on the functional relationships between the affected genes using their expression profiles, and to visualize the results using multi-dimensional scaling. Interestingly, we identified a stable subgroup of breast cancer patients that are highly enriched with ER-negative and triple-negative subtypes, and the somatic mutation genes they harbor were capable of acting as potential biomarkers to predict patient survival in several different breast cancer datasets, especially in ER-negative cohorts which has lacked reliable biomarkers. CONCLUSIONS: Our method provides a novel and promising approach for integrating genotyping and gene expression data in patient stratification in complex diseases.

Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development.

The breast cancer susceptibility gene BRCA1 is well known for its function in double-strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. COBRA1 (also known as NELF-B) is a BRCA1-binding protein that regulates RNA polymerase II (RNAPII) pausing and transcription elongation. Here we interrogate functional interaction between BRCA1 and COBRA1 during mouse mammary gland development. Tissue-specific deletion of Cobra1 reduces mammary epithelial compartments and blocks ductal morphogenesis, alveologenesis and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout are largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restores developmental transcription at puberty, alters luminal epithelial homoeostasis, yet remains deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription programme during mammary gland development.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells.

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

Roles for SUMO in pre-mRNA processing.

When the small ubiquitin-like modifier (SUMO)-1 protein is localized on the genome, it is found on proteins bound to the promoters of the most highly active genes and on proteins bound to the DNA-encoding exons. Inhibition of the SUMO-1 modification leads to reductions in initiation of messenger RNA (mRNA) synthesis and splicing. In this review, we discuss what is known about the SUMOylation of factors involved in transcription initiation, pre-mRNA processing, and polyadenylation. We suggest a mechanism by which SUMO modifications of factors at the promoters of high-activity genes trigger the formation of an RNA polymerase II complex that coordinates and integrates the stimulatory signals for each process to catalyze an extremely high level of gene expression. WIREs RNA 2016, 7:105-112. doi: 10.1002/wrna.1318 For further resources related to this article, please visit the WIREs website.

RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins.

During mitosis the chromatin undergoes dramatic architectural changes with the halting of the transcriptional processes and evacuation of nearly all transcription associated machinery from genes and promoters. Molecular bookmarking of genes during mitosis is a mechanism of faithfully transmitting cell-specific transcription patterns through cell division. We previously discovered chromatin ubiquitination at active promoters as a potential mitotic bookmark. In this study, we identify the enzymes involved in the deposition of ubiquitin before mitosis. We find that the polycomb complex proteins BMI1 and RING1A regulate the ubiquitination of chromatin associated proteins bound to promoters, and this modification is necessary for the expression of marked genes once the cells enter G1. Depletion of RING1A, and thus inactivation of mitotic bookmarking by ubiquitination, is deleterious to progression through G1, cell survival and proliferation. Though the polycomb complex proteins are thought to primarily regulate gene expression by transcriptional repression, in this study, we discover that these two polycomb proteins regulate the transcription of active genes during the mitosis to G1 transition.

Patterns and functional implications of rare germline variants across 12 cancer types.

Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine.

Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks.

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.

Massively Parallel Functional Analysis of BRCA1 RING Domain Variants.

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.