Combining Homologous Recombination and

Author Manuscript Published OnlineFirst on September 26, 2018; DOI: 10.1158/1541-7786.MCR-17-0357 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk. Ambre Petitalot 1, Elodie Dardillac 2,3, Eric Jacquet 4, Naima Nhiri 4, Josée Guirouilh-Barbat 2,3, Patrick Julien 1, Isslam Bouazzaoui 5, Dorine Bonte 2, Jean Feunteun 2, Jeff A. Schnell 4, Philippe 5 Lafitte 1, Jean-Christophe Aude , Catherine Noguès 1, Etienne Rouleau 1, Rosette Lidereau 1, $ 2,3 Bernard S. Lopez , Sophie Zinn-Justin 5 and Sandrine M. Caputo 1$ on behalf the UNICANCER Genetic Group BRCA network# 1

Service de Génétique, Département de Biologie des Tumeurs, Institut Curie, Paris, France. Institut Gustave Roussy, CNRS UMR 8200, Université Paris-Saclay, Villejuif, France. 3 Team labeled "Ligue 2014" 4 Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Saclay, Gif-sur-Yvette, France. 5 Institut de Biologie Intégrative de la Cellule, CEA, CNRS, Université Paris Sud, UMR 9198, Université ParisSaclay, Gif-sur-Yvette, France #UNICANCER Genetic Group BRCA network: Françoise Bonnet, Natalie Jones, Virginie Bubien, Michel Longy, Nicolas Sévenet: Institut Bergonié - Bordeaux; Sophie Krieger, Laurent Castera, Dominique Vaur: Centre François Baclesse - Caen; Nancy Uhrhammer, Yves Jean Bignon: Centre Jean Perrin - Clermont-Ferrand; Sarab Lizard: CHU de Dijon, Hôpital d'Enfants, Service de Génétique Médicale - Dijon; Aurélie Dumont, Françoise Revillion: Centre Oscar Lambret - Lille; Mélanie Léone, Nadia Boutry-Kryza, Olga Sinilnikova (deceased): Hospices Civils de Lyon and Centre Léon Bérard - Lyon; Audrey Remenieras, Violaine Bourdon, Tetsuro Noguchi, Hagay Sobol: Institut Paoli-Calmettes – Marseille, France; Pierre-Olivier Harmand, Paul Vilquin, Pascal Pujol: Laboratoire de Biologie Cellulaire et Hormonale (CHU Arnaud de Villeneuve) Montpellier; Philippe Jonveaux, Myriam Bronner, Joanna Sokolowska : CHU de Nancy-Brabois - Vandoeuvrelés-Nancy; Capucine Delnatte, Virginie Guibert, Céline Garrec, Stéphane Bézieau: CHU - Institut de Biologie Hôtel Dieu - Nantes; Florent Soubrier, Erell Guillerm, Florence Coulet: Groupe hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Université Pierre et Marie Curie, Laboratoire d’Oncogénétique et Angiogénétique moléculaire - Paris; Cédrick Lefol, Virginie Caux-Moncoutier, Lisa Golmard, Claude Houdayer, Dominique Stoppa-Lyonnet: Institut Curie - Paris; Chantal Delvincourt, Olivia Beaudoux: Institut Jean Godinot Reims; Danièle Muller: Centre Paul Strauss—Strasbourg; Christine Toulas: Institut Claudius Régaud - Toulouse; Marine Guillaud-Bataille, Brigitte Bressac-De Paillerets: Institut Gustave Roussy - Villejuif 2

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Running title: Classification of BRCA1 BRCT Missense VUS

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Disclosure of Potential Conflict of Interest: No potential conflicts of interest were disclosed.

To whom correspondence should be addressed. Tel: + 33 1 72 38 93 67; Fax: + 33 1 53 10 26 65; Email: [email protected] or [email protected]

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Downloaded from mcr.aacrjournals.org on October 1, 2018. © 2018 American Association for Cancer Research.


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Abstract

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BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, in order to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA-repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented based on mutated BRCT-domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide-binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide-binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis.

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Implications: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection.

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Introduction

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BRCA1 encodes a large, 1863-residue protein that functions as a hub, coordinating a large range of cellular pathways including DNA repair, transcriptional regulation, cell cycle control, centrosome duplication and apoptosis (1). Mutations in BRCA1 have been identified that predispose to breast and/or ovarian cancer. Further screening of patients revealed mutations throughout the whole BRCA1 gene sequence. The causal nature of mutations causing a premature stop is generally accepted. Other variants correspond to missense variations, deletions/insertions or intronic variations preserving the reading frame. The causal or neutral nature of these variants is more difficult to establish. Therefore, they are called variants of uncertain significance (VUS). In France, they are detected in about 10% of the tested population in the absence of a causal mutation. Initial attempts to evaluate the clinical significance of VUS in BRCA1/2 were mainly based on family data including family history and cosegregation with the disease. From these data, VUS were classified as either neutral (class 1), likely neutral (class 2), associated to an unknown cancer risk (class 3), likely causal (class 4) and causal (class 5) (2). However, the majority of the VUS are rare, and therefore these family-based clinical analyses often lack statistical power. High throughput experimental assays are then the most reliable way to determine the functional impact of variants with non-truncating changes in protein residue composition.

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Our goal is to improve the understanding of cancer susceptibility caused by BRCA1 gene variations by developing a high-throughput and robust experimental approach for missense VUS functional characterization. At the present time and according to most frequently used BRCA1 genetic testing criteria, a causal mutation used for genetic counselling is found in approximately ~10% of tested index cases (individuals for whom a complete study of both genes is performed; (3)). The carriers from such variants of class 5 affected with breast or ovarian cancer may now benefit from therapies based on platinum agents or PARP inhibitors that are specifically efficient against BRCA1/2related tumors (4,5). Moreover, geneticists can provide informative tests for relatives and adapt their management (reinsurance or preventive management) according to the test results (6). If the variant is classified as neutral (class 1), another molecular etiology for the familial risk should be found. However, today, most variants are assigned to class 3, which corresponds to a lack of knowledge on the VUS impact. Study of the impact of these VUS is based on different approaches (co-segregation, co-occurrence with a causal mutation, family history, RNA stability and quality, amino acid conservation, structural impact of the mutation, functional studies, loss of heterozygosity in tumors and tumor phenotype and the use of multi-factor risk models (7–11)). A combination of structural and functional information is particularly well adapted to the characterisation of rare missense variants, with the aim of evaluating the risk of cancer and taking clinical decisions.

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Several groups have undertaken the description of the structure and function of a set of VUS, in order to predict if the corresponding mutations or deletions/insertions are likely to affect biological pathways involving BRCA1 and lead to tumorigenesis. Most of the studies have analysed the impact of mutations in the N-terminal and C-terminal globular domains, whose functions are largely described: the N-terminal RING domain associated to the protein BARD1 regulates BRCA1 localisation and, through its E3 ubiquitin ligase activity, contributes to the G2/M cell cycle checkpoint, whereas the C-terminal BRCT domains are responsible for transcription activation and binding to phosphorylated proteins involved in DNA double-strand break signalling and repair (12–14). The Cterminal region of BRCA1 encompasses two BRCT repeats (aa 1646-1736 and aa 1760–1855) that adopt similar structures and are packed together in a head-to-tail arrangement (15). These BRCT domains interact specifically with phosphorylated protein targets containing the sequence pSer-x-xPhe (16–20). The structural impact of the BRCT missense variations compiled in the BRCA1 Circos 3



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resource from the ENIGMA consortium was analysed using limited proteolysis and phosphorylatedBACH1 binding assays, and for a subset of these mutations, by expression in bacteria and characterisation of the thermodynamic stability of the purified mutants (21–25). Impact of these mutations in cells was revealed using transcriptional activation assays (23), and for a small subset of these mutations, homology-directed DNA repair (or Homologous Recombination: HR) assays (26). However, a high-throughput approach that provides both structural and functional information on the same VUS and is recognized as sufficient to conclude in most cases about the impact of the VUS on tumorigenesis is still lacking.

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In order to further understand the link between protein stability, phosphopeptide recognition, localization in cells, HR and tumorigenesis, we decided to systematically analyse the 3D structure and function of the 78 BRCT missense variants deposited in the BRCAShare (ex-UMD-BRCA1 database, (27,28)), including all VUS detected in France, except missense mutations with a splice impact (29,30). Therefore, we developed high-throughput assays in order to (1) measure the capacity of the VUS to repair double-strand breaks by HR in cells, (2) test their capacity to relocalize to the nucleus after addition of MMC, (3) measure the thermostability of the corresponding expressed/purified mutated BRCT domains and (4) investigate their binding to a large panel of phosphorylated peptides from different BRCA1 partners (ACC1, BACH1, CtiP and Abraxas). Half of the selected VUS are also listed in other databases such as the BIC, KConfab and ClinVar databases. For these VUS, we were able to compare our experimental data with previously published results obtained on subsets of VUS through either in vitro or in cell approaches, thus validating our results (23,25,26). Our largescale analysis from both in vitro and in cell data provides a solid basis for discussing the impact of the different structural and functional defects on increased cancer risk.

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Materials and Methods

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Lentiviral inducible BRCA1 shRNA system

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An anti-BRCA1 shRNA construct was made targeting the 3’-untranslated region (3’-UTR) of the HsBRCA1 gene (Genbank accession number NC_000017.10). The target sense sequence is 5’TATAAGACCTCTGGCATGAAT-3’ with a loop TTCAAGAGA. The sequence was cloned into AgeI and EcoRI of a pLKO-Tet-On vector purchased from Addgene (31). The pLKO-Tet-On expresses constitutively a tetracycline-controlled transcriptional suppressor Tet which in turn controls expression of the anti-BRCA1 shRNA sequence inserted in the shRNA cloning site of the vector to be under the human H1 promoter. In the absence of doxycycline (a tetracycline derivative), expression of the anti-BRCA1 shRNA is blocked. When doxycycline is added to the culture medium, transcription of the anti-BRCA1 shRNA occurs, which results in the knockdown of the HsBRCA1 gene in a highly dose-dependent manner.

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Lentivirus production

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The constructed plasmid pLKO-Tet-On/Anti-BRCA1-shRNA, and the plasmids pMD2.G and pCMVdR8.74 (Kindly provided by Anne Galy, Genethon, France) were transfected into the HEK293T cells using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. The plasmid pMD2.G encodes the envelope protein from the vesicular stomatitis virus (VSV-G) and the plasmid pCMVdR8.74 encodes the proteins from the genes Gag and Pol from the human immunodeficiency virus type 1 (HIV-1). The media was changed 24 h post-transfection. Then, the supernatants were collected at 48, 72 and 96 h post-transfection. For each time point, after elimination of the cell debris, the collected supernatant was ultracentrifuged through a sucrose cushion (20%) at 20,000 rpm for 2 h at 12°C. The supernatant was removed and the virus pellet was resuspended in 500 µl of complete media overnight at 4°C. The following days, the virus was pooled in the first tube. Finally, the volume was increased to 1 ml, the solution was filtered, aliquoted, and stored at -80°C.

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Design of the RG37 cell line containing the doxycyclin-inductible shBRCA1 4



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RG37 cells are SV40-transformed human fibroblasts containing a chromosomally integrated DR-GFP substrate that specifically monitors gene conversion (32). RG37 cells were infected with lentiviral particles containing a shRNA directed against the 3’UTR part of the BRCA1 messenger and the puromycin resistance gene. Puromycin was added 3 days after infection and cells were re-seeded at low density to isolate individual clones. Clones were then screened for deficiency in the formation of BRCA1 foci after ionizing radiation, deficiency in HR (using the DR-GFP substrate, figure 2) and for the extinction of BRCA1 expression monitored by western blot. The new cell line was called RG37shBRCA1.

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Plasmid constructions for expression in mammalian cells

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Cloning of the mutated BRCT1-BRCT2 gene into the pcDNA3 (modified)-full length Brca1 was performed by Nested PCR (33). First insert was generated by PCR from the gene coding for GSTBRCT1-BRCT2 using the primers: 5’TCAACAGAAAGGGTCAACAAAAGAAT3’ and 5’GCCGATATCATC GATTCAGTAGTGGCTGTG3’ (1). Second insert was generated by PCR from pcDNA3 (modified)-full length BRCA1 gene using the primers: 5’ACACCCAGGATCCTTTCTTGATTG3’ (2) and 5’ATTCTTTTGTTGACCCTTTCTGTTGA3’. These inserts were associated by PCR using the primers 1 and 3. The new insert was cloned into the BamHI and EcoRV sites of the pcDNA3 (modified)-full length BRCA1. All constructs were verified by DNA sequencing.

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Homologous recombination assays in human RG37-shBRCA1 cells

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Cells were pre-treated (or not) with 10 g/ml doxycyclin 3 days before plating. They were then seeded at 2x105 per well in 6 wells plates. 24 h after plating, expression vectors encoding for BRCA1 (or its variant forms) and the HA-tagged meganuclease I-SceI were co-transfected using JetPEI reagent (PolyPlus, Ozyme). 48 h after transfection, cells were trypsinized and GFP+ cells were directly measured by flow cytometry.

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The expression of BRCA1 and I-SceI was monitored by western blot (Suppl. Figure 1A). Therefore, protein extracts (25–50 μg) were resolved on 9% SDS-PAGE, then transferred to a nitrocellulose membrane (0.22 µm) and probed with the following specific antibodies: anti-BRCA1 (mouse, ab16780, Abcam), anti-HA (mouse, sc-7392, Santa Cruz Biotechnology Inc.), anti-Tubulin (mouse, T5168, Sigma-Aldrich). Immunoreactivity was visualized using an enhanced chemiluminescence detection kit (WesternBright ECL, Advansta Inc.).

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Each batch of assays was run with positive and negative controls and each variant was tested at least in triplicate in at least 4 independent experiments. The statistical analysis was performed using the R language release 3.5.1 (34)) running on a Linux server. We have compared the variant BRCA1 HR to the WT BRCA1 HR using a one-side paired student t-test. To account for the multiple testing, the pvalues were adjusted using the Benjamini-Horchberg method (35); we controlled the false discovery rates at a level =0.05.

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RG37-shB1 cells were treated two days prior with doxycycline before transfection. Cells were seeded at a density of 1x105 per well in six well plates each containing a glass cover slide and tranfected various BRCA1 variant using JetPEI reagent. 24 h later, cells are treated with 2.5 µM mitomycin for 2 h then the medium is changed. After 8 h, cells were fixed (PBS, 2% paraformaldehyde, 10 min) and permeabilized (PBS, 0,1% Triton X-100, 5 min). Cells were then incubated with PBS containing 1% BSA, 0.05% TWEEN and anti-BRCA1 antibody (ab16780 abcam) for 45 min at 37°C. After 3 washing, cells were then incubated with PBS containing 1% BSA, 0.05% TWEEN Alexa 568– conjugated secondary antibodies (Jackson ImmunoResearch). Cells were then stained with DAPI and

Western Blot Analysis of BRCA1 Protein

Statistical analysis

Localization assays

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examined with fluorescence microscope. Statistical significance was calculated using a two-tailed Student t-test with GraphPad.

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Plasmid constructions for bacterial expression

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The gene coding for the BRCA1 region BRCT1-BRCT2 (amino acids 1646 to 1863) was cloned using the LIC technology (Ligase Independent Cloning) into the pETM-10 and pETM-30 vectors (EMBL) thanks to the following primers: forward 5’GGCAGGAGCAGCCTCGGAGAATCTTTATTTT CAGGGCGTCAACAAAAGAATGTCCATGGTGGT 3’ and reverse 5’GCAAAGCACCGG CCTCGttaTCAGTAGTGG CTGTGGGGGATCT3’. All mutations were generated by site-directed mutagenesis (Stratagene) and verified by sequencing.

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Peptides for binding studies

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The following peptides were synthetized by GeneCust Europe (Luxembourg): one control peptide CTRL-P (PTRV-pS-SPVFGAT), 4 mono-phosphorylated peptides CTiP-P (PTRVS-pS-PVFGAT), BACH1-P (ISRST-pS-PTFNKQTK), ACC1-P (DSPPQ-pS-PTFPEAGH), Abraxas-1P (or AB-1P) (GFGEYSR-pS-PTF) and 1 bi-phosphorylated peptide Abraxas-2P (or AB-2P) (GFGEY-pS-R-pSPTF).

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The BRCT variants cloned into the pETM-30 expression vector were introduced into the E. coli BL21(DE3) strain and expressed in 96-well microplates using a self-inducible medium. After lysis, soluble protein fractions were separated from insoluble fractions. Soluble fractions were transferred to new 96-well microplates for gel analysis and the rest of the soluble fraction was incubated with 20 μL Hi-Trap chelating beads, washed and diluted into SB1x. Bacteria pellets were suspended in 50 µL 2% SDS. Protein expression and solubility were further analyzed by SDS-PAGE.

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The BRCTs variants cloned into the pETM-30 expression vector were introduced into the E. coli BL21(DE3) strain and were grown at 37°C in LB medium containing 75µg/ml kanamycin to an OD600nm of 1.0. Protein overproduction was induced at 20°C with 1 mM isopropyl -D-thiogalactoside (IPTG) for 12 h. The bacteria were then harvested by low speed centrifugation at 6000 rpm for 15 min. The bacterial pellet was suspended in 30 ml lysis buffer (100 mM Tris pH 7.5, 150 mM NaCl, 1% (w/v) Triton X-100, 10% glycerol, 1 mM EDTA, 10mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM ATP, 10mM MgSO4) and incubated with 1% lysozyme overnight at 4°C. Then, 1 μl benzonase (Sigma), 2 mM ATP, 10 mM MgSO4 and 10 mM MgCl2 and 10 mM DTT were added. After 30 min, the extract was centrifuged at 20,000 rpm for 30 min, and the soluble protein fraction was loaded on a 30 mL Glutathione sepharose 4FF column previously equilibrated in 50 mM Tris, 150 mM NaCl pH 7.5. The column was washed with a buffer containing 1 M NaCl, then 150 mM NaCl, and then with 50 mM Tris, 150 mM NaCl pH 7.5. The TEV protease was added and incubated overnight at 4°C. After TEV cleavage, the elution from the GST-trap column was loaded onto a HisTrap column equilibrated in 50 mM Tris pH 7.5, 150 mM NaCl. The protein was eluted with 50 mM Tris pH 7.5, 150 mM NaCl and 10 mM imidazole. Fractions highly enriched in BRCT variants were diluted fivefold in 50 mM Tris pH 7.5 and loaded on a 5 mL Q-Sepharose High Performance column equilibrated in 50 mM Tris pH 7.5. Bound proteins were eluted with between 200 and 350 mM NaCl. In both cases, the collected fractions were analysed by 0.1% SDS-15% PAGE, using as a marker the broad range prestained protein marker (Biorad). The purified protein was characterized by electrospray ionization mass spectroscopy.

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ITC was performed using a high-precision VP-ITC calorimetry instrument. To characterize interactions between the BRCT1-BRCT2 domains and peptides, all proteins were dialyzed against 50

Protein expression and solubility assay

Purification of BRCA1 BRCT domain variants

Isothermal Titration Calorimetry.

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mM Tris-HCl pH7.5, 150 mM NaCl, 10 mM β-mercaptoethanol and protease inhibitors (Roche). BRCT1-BRCT2 domains (10-20 μM) in the calorimetric cell at 30°C were titrated with the peptide (at a concentration of 100-200 μM in the injection syringe). Analyses of the data were performed using the Origin software provided with the instrument.

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Size-exclusion chromatography experiments aiming at identifying interactions between BRCT1BRCT2 domains and peptides after ITC were performed using a Superdex-75 10/300 GL column (GE Healthcare) pre-equilibrated in the ITC buffer (50mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM βmercaptoethanol and protease inhibitors (Roche)). Proteins were concentrated after ITC (calorimetric cell) to obtain a volume of 500 μl and were loaded on the column at a flow rate of 0.5 ml/min at 4 °C.

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Thermostability measurements by Fluorescence-based Thermal Shift Assay (FBTSA)

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The FBTSA method is used to monitor the BRCT domain thermal denaturation and changes induced by a mutation and/or a binding event. To measure the thermostability of WT and mutated BRCT domains, we mixed 1 µg of purified protein in 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM ßME and the SYPRO Orange dye (diluted 400-fold from a 5000-fold stock solution, Invitrogen). The same experimental conditions were used to investigate the interaction of the BRCT domains and the phosphorylated peptides CTRL-P, ACC1-P, CTIP-P, BACH1-P, AB-1P (one phosphorylation) and AB-2P (two phosphorylations). The peptides were first diluted in the same buffer as the BRCT domains and then added to the reaction mixture with increasing concentrations up to 256 µM. Reaction mixtures were made in duplicate in a MicroAmp Optical 384-well reaction plate at a final volume of 10 µl (4 µM) and each experiment was repeated at least twice independently. Experiments were carried out in QuantStudio 12KFlex qPCR machine (Applied Biosystems) with a temperature gradient in the range of 15–95 °C at 3 °C/min.

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Selection of 78 BRCT VUS from the UMD-BRCA1 database

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The VUS were selected from the French UMD-BRCA1 database, which gathered in November 2015, 2020 distinct variants collected from 8446 families, and included 1028 distinct VUS of class 3 (http://www.umd.be/BRCA1/) (27). A large majority of these VUS of class 3 were found in a unique family of patients. The exon containing the largest number of variants (exon 18) corresponded to the first BRCT domain (BRCT1) (27). We focused our study on variants located in the C-terminal BRCT1 and BRCT2 domains of BRCA1 (amino acids 1646 to 1859). We selected the 65 VUS of class 3 that corresponded to a missense variation in the BRCT domains of BRCA1 (Figure 1A; Suppl. Table I). We also selected 6 variants of class 5 (V1665E, R1699W, G1706R, A1708E, S1715N in BRCT1 and M1775R in BRCT2) (=causal variants) and 1 variant of class 4 (G1706E in BRCT1) (=likely causal variant) as well as 3 variants of class 2 (M1652T in BRCT1, R1751Q in linker, M1783T in BRCT2) (=likely neutral variants) and 3 variants of class 1 (M1652I, T1720A in BRCT1, V1804D in BRCT2) (=neutral variants), to serve as controls for our experiments (Figure 1A). The 78 selected mutations are distributed all along the BRCT domain sequence. They are also located on the whole 3D structure: in the BRCT domain hydrophobic cores, at the interface between the 2 BRCT domains, as well as in solvent-exposed exposed regions and in particular the phosphopeptide-binding region (Figure 1B). All 78 VUS were evaluated through a dedicated high-throughput workflow combining tests performed in cells (HR assay, localization after DNA damage) and in vitro (protein stability upon expression in bacteria and binding to a set of 5 phosphopeptides) in order to provide a comprehensive description of their function.

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Impact of the missense mutations on Homologous Recombination

Size-exclusion chromatography.

Results

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To address this question, we designed a novel cell line: RG37-shB1. This cell line was derived from RG37 cells, which are SV40-transformed human fibroblasts containing an integrated DR-GFP substrate to specifically monitor gene conversion upon expression of the meganuclease I-SceI (Figure 2A) (32). RG37 were infected with a retroviral construct containing a doxycycline-induced shRNA raised against the 3’-UTR of the endogenous BRCA1 gene (RG37-shB1cells). Therefore, supplying doxycycline specifically silenced the endogenous BRCA1 without affecting the expression of the exogenous transfected BRCA1 cDNA. Figure 2B shows that supplying doxycycline strongly decreased the expression of BRCA1 and 2-fold reduced the efficiency of I-SceI-induced HR. Cotransfection of a wild-type BRCA1 cDNA with the I-SceI coding plasmid stimulated the frequency of HR in cells unexposed to doxycycline, as already described with RG37 cells, and, importantly, rescued HR frequency in doxycycline treated cells (Figure 2B). Interestingly, all tested BRCA1 variants reported as causal (V1665E, R1699W, G1706R, A1708E, S1715N, M1775R) or likely causal (G1706E) were unable to complemented BRCA1 deficiency for HR (Figure 2C).

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Using the same assay, we measured the capacity of the 78 missense variants (including the 7 causal mutations shown above) to complement BRCA1 silencing for I-SceI-induced HR. Either WT BRCA1 or the VUS were expressed in this system, as checked by Western blot using anti-BRCA1 antibodies (Suppl. Figure 1A). Variation of HR-efficiency as a function of the position of the variation in the sequence did not reveal any functionally critical hot spot (Suppl. Figure 1B). Expression of variants reported as neutral (M1652Ib due to G>A, T1720A, V1804D) or likely neutral (M1652T, R1751Q, M1783T) systematically complemented HR-efficiency at least as much as WTBRCA1 (Figure 3A and Table I). VUS M1652Ia (due to G>T; class 3) was tested, which also affects position 1652: it consistently showed a WT HR-efficiency. Based on this unique assay, we could already confirm the link between causality and HR defect with a sensitivity of 100% (7/7 causal variants show an HR defect) and a specificity of 100% (6/6 neutral variants have no HR defect). This first analysis confirmed the essential role of HR-defect in tumorigenesis.

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Global analysis of the HR assay results performed on the 78 variants revealed that 31 VUS (40%) failed to complement BRCA1 deficiency for HR (Figure 3A). These VUS include the 7 mutations of class 4 or 5, 24 VUS of class 3 and no VUS of class 1 or 2. Positions that are mutated in defective VUS are distributed all along the BRCA1 BRCT domains, from amino acid 1655 to amino acid 1837. However, 19 amongst the 31 defective VUS are mutated within BRCT1. Moreover, most mutated residues corresponding to defective VUS are buried in the 3D structure of the BRCT domains (25 within 31 are less than 20% solvent accessible), whereas only half of the positions mutated in variants characterized by a normal HR are buried in the hydrophobic cores of the BRCT domains or at the BRCT1/BRCT2 interface. This analysis shows that if a position is either in BRCT1 or buried, its mutation is more likely to negatively affect HR.

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At given positions, several mutations were identified, corresponding to different VUS. For example, position 1665 is mutated in 3 VUS that showed either no defect (V1665M, V1665L) or a significant defect (V1665E, causal) in HR-efficiency, suggesting that any hydrophobic residue is tolerated at this buried position but the introduction of a charged amino acid causes a HR defect. At position 1699, which is solvent-exposed and is responsible for contacts with the phosphopeptide main chain (Fig. 5B), variation to Q had the same functional impact as the causal mutation to W. Impact of these mutations on BRCA1 structure and phosphorylated BACH1 binding are consistent with the analysis of Coquelle et al. (21), who showed that the R1699W mutation destabilized the BRCA1 protein structure and likely interfered with the docking of the peptide phenylalanine into the BRCT peptide-binding groove, whereas the R1699Q mutation caused a steric clash between the glutamine side-chain carbonyl and the main-chain carbonyl of the peptide phenylalanine residue. At position 8



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1706, which is buried, variation to A did not affect HR, whereas the causal mutations to charged residues (G1706E and G1706R) did. Similarly, at position 1708, which is also buried, variation to V did not affect HR, whereas the causal mutation to R or E did. Such analysis underlines the consistency of our HR assay results and highlights the variability of the functional consequences of variations at a same position as a function of the physico-chemical properties of the WT versus variant amino acid. Clearly, at buried positions, mutation to a hydrophobic residue is tolerated but mutation to a charged residue impairs HR-efficiency. At solvent-exposed positions, if the mutated residue is involved in the recognition of a functionally essential partner, then any type of replacement might impair recognition and increase cancer risk.

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Impact of the missense mutations on BRCA1 nuclear localization after DNA damage in mammalian cells

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A first explanation for the deleterious effect of a mutation on HR is the overall low concentration and/or the decreased nuclear localization after DNA damage of the corresponding variant. As no significant difference in protein amounts was observed throughout the VUS by Western Blot, we further revealed by immunofluorescence BRCA1 localization in RG37-shB1 cells after addition of mitomycin C. This molecule is a chemotherapeutic drug that upon reduction is converted into a highly reactive intermediate alkylating DNA. We observed that WT BRCA1 as well as a large set of VUS correctly localized at the nucleus after addition of this drug. However, 34 variants were not correctly localized in response to mitomycin C (Table I, Figure 3B and 3C). These variants include 4 causal variants (all except R1699W and A1708E), the likely causal G1706E, and 29 VUS of class 3. Within these 29 variants, 18 are defective for HR. The remaining 11 VUS are capable of compensating their localization defect in order to carry out normal DNA repair by HR. They might still show other functional defects. The 6 neutral or likely neutral VUS showed no localization defect. Finally, 6 VUS of class 3 showed HR defects but no localization defects, demonstrating that localization defect is not the only mechanism leading to impaired HR. In summary, this second assay enabled classification of 5/7 causal variants (sensitivity=71.4%) and 6/6 neutral variants (specificity=100%), but only 18 VUS of class 3 are identified as both HR-defective (within 24 VUS) and localization defective (within 29 VUS), suggesting that the two assays provide only partially overlapping conclusions.

29 30

Impact of the missense mutations on solubility in E. coli and thermostability of recombinant BRCT domains

31 32 33 34 35

Another explanation for the loss of HR-activity of 40% of the VUS is the impact of BRCA1 variations on the BRCT domain thermostability leading to a global loss of function of these domains. We measured the structural impact of the 78 missense variations by expressing the mutated domains, purifying the soluble domains and measuring the thermostability of the resulting mutated BRCT domains by fluorescence-based thermal shift assay (FBTSA).

36 37 38 39 40 41 42 43 44 45

First, 23 mutated proteins were insoluble in E. coli (Table I, Figure 4A and Suppl. Figure 2). We classified these proteins as highly unstable, and no further work was carried out on them. They corresponded to 4 variants of class 5, 1 variant of class 4, 17 VUS of class 3, and 1 variant of class 2. Interestingly, 21 out of 23 (91%) of these VUS also showed significant HR defects. Only two of these VUS shows a normal HR function: M1652T that is classified as likely neutral and A1752E that belongs to class 3. Thus, a very large majority of highly unstable BRCT domains correspond to VUS with a defective HR activity. Reciprocally, only 4 VUS with impaired HR activities (13% of the HRdefective VUS) correspond to domains that could be purified in vitro: R1699W, R1699Q, M1775R and V1833M. These results revealed a nice correlation between the HR-activity of the whole BRCA1 VUS and the solubility of the mutated BRCT fragments, in our conditions. 9



1 2 3 4 5

When going further into this in vitro analysis, 13 poorly soluble mutated BRCT domains could not be purified because of aggregation, 26 could be expressed and purified but with low yields, and only 16 were obtained with a yield close to that of the WT-BRCT domains (Table I and Figure 4A). This classification seems to correlate with HR results, because 46% of the aggregating mutants, but only 12% of the poorly soluble mutants and 6% of the WT-like mutants are HR-defective.

6 7 8 9 10 11 12 13 14 15

Finally, we measured the thermostability of the 42 purified mutated domains using a highthroughput fluorescence assay by FBTSA (Suppl. Figure 3). The melting temperature measured for the WT is 52°C+/- 0.3°C. No clear distinction could be observed between the results obtained for the 2 causal variants and the 2 likely neutral variants: causal variants M1775R and R1699W showed melting temperatures of 41.9 and 43.8 +/- 0.1°C, respectively, whereas likely neutral mutants R1751Q and M1783T showed melting temperatures of 42.7 and 44.9 +/- 0.1°C, respectively. However, more generally, the 4 HR-defective variants for which a melting temperature could be measured are distributed within the 45% variants with the lowest melting temperatures: their melting temperatures range between 40.4 and 48.8°C. Also, the 3 neutral variants have significantly higher melting temperatures comprised between 50.5 and 51.6°C.

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

In summary, the causality and HR assay results are strongly related to the observation of insolubility versus solubility of the BRCT domains in bacteria. We observed that 5 upon 7 causal variants (that are all HR-defective) exhibit BRCT domains insoluble in bacteria (sensitivity=71.4%), whereas 5 upon 6 neutral variants (that all show WT HR activity) have BRCT domains that are soluble in bacteria (specificity=83%). Moreover, amongst the 24 HR-defective VUS of class 3, 16 correspond to insoluble domains, 6 to domains that aggregate during purification, 1 to a poorly soluble domain and 1 to a WT-like domain and 1 to a WT-like domain. We have represented on the 3D structure of the BRCA1 BRCT domains the localization of the mutated positions corresponding to both HR deficient VUS and insoluble (brown) or aggregating (yellow) BRCT domains (Figure 4B). Clearly, these positions are mainly found in BRCT1 and in particular close to the phosphorylated serine of the BRCA1 partner, underlying the necessity for BRCA1 to interact with phosphorylated partners to promote HR. Fragment from residue 1685 to residue 1708, comprising a -strand, a loop and a -helix interacting with the consensus motif Ser-X-X-Phe of BRCA1 phosphorylated partners (further named the phosphopeptide binding loop), concentrates 13 within the 27 identified positions colored in brown on Figure 4B.

31

Impact of the missense variants on the binding of the BRCT domains to phosphorylated peptides

32 33 34 35 36 37 38 39 40 41 42 43 44 45

The BRCA1 BRCT domains bind to phosphoproteins such as Abraxas, ACC1, BACH1, CtiP, which share a common Ser-X-X-Phe motif phosphorylated on the serine residue (17–19). Because several mutations leading to an increased cancer risk disrupt the binding surface of the BRCT domains to phosphorylated peptides, it was suggested that BRCA1 phosphopeptide-binding is essential for BRCA1's tumor suppressor function (26,36). Moreover, a previous study of mice carrying a BRCT mutant of BRCA1 that is defective in recognition of phosphorylated proteins also suggested that BRCT phosphoprotein recognition is required for BRCA1 tumor suppression (14). Using the same high-throughput fluorescence assay as for the thermostability measurements, revealing here thermostability shifts due to peptide binding, we tested binding of the 42 purified mutated BRCT domains to 5 different phosphopeptides. These are fragments of the DNA repair protein Abraxas (belonging to the so-called BRCA1-A complex), the acetyl-CoA carboxylase 1 (ACC1), the DNA helicase BACH1 (belonging to the so-called BRCA1-B complex) and the transcriptional corepressor CtiP (belonging to the so-called BRCA1-C complex). ACC1 is an enzyme essential for cancer cell survival that catalyses fatty acid biosynthesis; phosphopeptide (ACC1-P) recognition is important for 10



1 2 3 4 5 6 7

the regulation of fatty acid biosynthesis (37). BACH1 phosphopeptide (BACH1-P) recognition is involved in G2/M checkpoint control and DSB repair and CtiP phosphopeptide (CTIP-P) recognition is essential for DNA end resection of DSBs during HR. Abraxas and the BRCA1-A complex recruit BRCA1 to DNA double-strand-break sites (DSBs) through an ATM-dependent ubiquitin-mediated signalling pathway. The interaction of phosphorylated Abraxas (mono-phosphorylated peptide AB-1P and di-phosphorylated peptide AB-2P) with BRCA1 is critical for the function of Abraxas in DNA repair of DSBs and maintenance of genomic stability (23).

8 9 10 11 12 13 14 15 16 17 18 19

First, in order to confirm the binding capacity of the purified WT BRCT domains, we measured their affinity for a set of phosphorylated peptides using isothermal titration calorimetry (ITC). We measured the affinities of the interactions between the BRCT domains and either ACC1-P or BACH1-P. We obtained affinities of 2.1 +/- 0.2 M and 0.19 +/- 0.01 M respectively, consistently with the literature (Suppl. Figure 4A,B; (37,38)). We also measured the affinities of the interactions between the BRCT domains and either AB-1P or AB-2P. We obtained affinities of 25 +/- 2 nM and 6.2 +/- 1.2 nM, respectively (Suppl. Figure 4C,D). These last interactions are characterized by a large affinity increase compared to the interactions with ACC1-P and BACH1-P. Finally, we ran sizeexclusion chromatography experiments on these complexes. The BRCA1 and BRCA1+BACH1-P elution volumes correspond to the molecular mass of a monomer whereas that of BRCA1+AB-1P is indicative of a monomer-dimer equilibrium and that of BRCA1+AB-2P reveals a dimer, which is also consistent with the literature (Suppl. Figure 5A,B; (23)).

20 21 22 23 24 25 26 27

Second, we verified using our FBTSA assay that binding of the WT BRCA1 BRCT domains to ACC1-P, BACH1-P, CtiP-P, AB-1P and AB-2P induced a measurable increase in the BRCT thermostability that rose with peptide concentration (Suppl. Figure 6A). Indeed, addition of ACC1-P and CtiP-P caused a maximal stability increase of 5.9 and 6.2 +/- 0.1 °C at 256 M, respectively, whereas addition of the same amount of BACH1-P, AB-1P and AB-2P increased the WT BRCT thermostability by 8.2, 14.1 and 15.1 +/- 0.1 °C, respectively. Control experiments showed that addition of a peptide with the same sequence as CtiP but phosphorylated on the other serine residue (CTRL-P; see Figure 5A) increased the thermostability by only 0.1°C.

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

From this description of the WT BRCT domain binding properties, it was possible to design a large high throughput experiment aiming at identifying the VUS defective in phosphopeptide-binding. Figure 5A shows the thermostability shifts caused by binding of the peptides CTRL-P, ACC1-P, BACH1-P, CtiP-P, AB-1P and AB-2P to the BRCT domains of WT BRCA1 and the 42 mutants that could be expressed and purified. Clearly addition of either ACC1-P, CtiP-P, BACH1-P, AB-1P or AB2P generally have the same impact on the thermostability of the mutated BRCT domains. This confirms that the 5 peptides bind to the same BRCA1 site. Five variants significantly lost affinity for the peptides: R1699W and M1775R (Suppl. Figure 6B) are the two only causal variants that could be purified; mutants R1699Q, F1717Y and H1746N correspond to VUS of class 3. In the case of mutants F1717Y and H1746N, no binding of the peptides to the BRCT domains could be detected (Figure 5A). Representation of the 4 positions mutated in these 5 variants on the 3D structure of the BRCT domains revealed that Arg1699 and Met1775 are directly involved in peptide binding whereas Phe1717 and His1746 are further from the binding site and probably indirectly contribute to peptide recognition (Figure 5B) (20). These residues might be critical for correctly positioning the phosphopeptide binding loop (amino acids 1685 to 1708), as they interact with Lys1690 and Tyr1703, respectively. Altogether, the variants of classes 4 and 5 are all HR-deficient and when tested are phosphopeptide-binding deficient, whereas the variants of classes 1 and 2 have a WT HR activity and when tested show a WT phosphopeptide-binding capacity. By taking into account the results of the 11



1 2

cellular HR assay and the in vitro binding assay, it is possible to classify 7/7 causal variants (sensibility=100%) and 6/6 neutral variants (specificity=100%).

3 4

Discussion

5 6 7 8 9 10 11 12 13 14

In this study, we tested 78 BRCA1 variants using 4 assays that focus on different properties previously shown as related to BRCA1 oncogenic function. Our challenge was to set up a high throughput protocol in order to (1) measure BRCA1 HR capacity in 6 well plates through a fluorescence reading, (2) test bacterial expression of the BRCT domains in 96 well plates and read the stability and phosphopeptide-binding of the domains using fluorescence-based thermal shift assays. Thus, it was possible to provide results for the 4 assays on a very large set of VUS from the UMDBRCA1 database, corresponding to all VUS that are mutated in the BRCT domains (Table I). We will now compare these results to the published data available on other VUS databases (Table II). We will discuss the relationship existing between the measured properties of our VUS and predisposition to cancer and suggest related mechanisms of tumorigenesis.

15

Comparison with the results obtained on the VUS from BIC, KConfab and ClinVar databases

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Over the past few years, several functional assays for classification of BRCA1 VUS have been developed. In particular, a fast cDNA-based functional assay was reported, based on the VUS ability to functionally complement BRCA1-deficient mouse embryonic stem cells, i.e. restore the proliferation defect due to the absence of BRCA1 expression and survive to addition of cisplatin (39). Seventy-four VUS distributed all along the BRCA1 gene were tested using this assay. This analysis strongly suggested that causal missense variants are confined to BRCA1 RING and BRCT domains. Several other groups restricted their analyses to BRCA1 proteins mutated in the evolutionarily conserved RING or BRCT domains. They often aimed at understanding the impact of mutations on protein stability, phosphopeptide-binding and transcriptional activation (21,23–25,40–43). They suggested that phosphopeptide-binding and transcription assays gave results that were correlated to cancer risk, with specificity and sensitivity higher than 80%. Additional studies reported the activity of a subset of VUS in HR, which was also closely associated with cancer risk (39,44,45). Finally, characterization of 12 BRCT domain variants performed both in vitro and in cells showed that all of the variants, regardless of how profound their destabilizing effects are in vitro, retained some DNA repair activity and thereby partially rescued the chicken BRCA1 knockout (26). By contrast, the variant R1699L, which disrupts the binding to phosphorylated proteins (but which is not destabilizing), was completely inactive. These studies raised the question of the link between protein stability, phosphopeptide-binding, HR activity and cancer risk.

34 35 36 37 38 39 40 41 42 43 44

We have compared our results to the data available from these studies (Supplementary Table I). Our VUS list contains 42 VUS also present in the BIC, KConFab or ClinVar databases, which were characterized by the teams of L.S. Itzhaki, J.N.M. Glover, J. Jonkers and A.N. Monteiro (Table II). In particular, the thermodynamic stability of 17 mutated BRCT domains and the effects of 4 of the corresponding variants on BRCA1-mediated DNA repair by HR were measured by Itzhaki’s team and our team (25,26). The resulting data are independent measurements obtained on a subset of VUS that can be used to evaluate the robustness of the approaches. The 7 mutants described as insoluble by Itzhaki’s team are mostly insoluble or aggregating during purification in our experimental set up. The 10 remaining mutants are always soluble and could be purified in both teams. Within the 4 VUS studied in cells by both teams, one is defective for HR in both assays (A1708E, causal), 2 VUS have a WT DNA repair activity in both assays (M1783T, likely neutral and S1841N) and 1 VUS shows a 12



1 2 3

moderate DNA repair activity in Itzhaki’s assay and a WT in our assay (M1652Ib). This last variant, M1652Ib, is already classified as neutral. We can conclude that the results obtained by the two teams are in general consistent.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

A large scale analysis of the sensitivity to proteases, phosphorylated peptide binding and in cell transcriptional activity of 117 VUS corresponding to mutations in the BRCT domains of BRCA1 was performed by Glover’s team (21,23). Most of the in vitro analysis was performed on BRCT domains produced by in vitro transcription/translation, which is an approach complementary to our study. Comparison of all these results provides essential arguments to guide further interpretation of our results (Table II). Thirty-nine VUS were studied by both Glover’s team and our team. Within these 39 mutants, 20 could be purified by us. Eighteen were resistant to degradation by proteases in Glover’s team. The VUS H1746N and V1833M were sensitive to proteases in Lee et al. (23), and are significantly less stable (by more than 5°C) than the WT BRCT domains in our study. In the case of the 19 mutants that we could not be obtained using a bacterial expression system, 17 were sensitive to proteases, which confirm that these mutants were unstable. Only VUS V1713A and M1652T were not sensitive to proteases in Lee et al. (23). In summary, our results on solubility in bacteria and results of Lee et al. on protease sensitivity are mostly consistent, however, because of the different experimental set up used to produce the proteins, some differences are observed for a few VUS (23). Furthermore, Glover’s team could measure the binding of 19 variants insoluble in bacteria to a phosphorylated BACH1 peptide. They revealed that 17 of these variants have a defective phosphorylated peptide binding capacity (only mutants M1652T and V1833M bind to phospho-BACH1). This analysis shows that mutated BRCT domains insoluble in bacteria, when produced in other systems, are in general also defective in phosphopeptide recognition.

23

Relationship between phosphopeptide-binding defects and high cancer risk

24 25 26 27 28 29 30 31 32 33

From our data, it is particularly clear that the results from the phosphopeptide-binding assays are highly correlated with cancer risk. These assays generally gave similar results using any of the 5 tested peptides CTiP-P, BACH1-P, ACC1-P, AB-1P and AB-2P and provided information on 42 mutants including 5 neutral variants that showed WT BRCT binding properties and 2 causal variants that exhibited defective BRCT phosphopeptide-binding capacities. One VUS had a different behavior when tested for binding against the 5 phosphopeptides: E1836K showed improved binding to CtiP-P, ACC1-P, AB-1P and AB-2P but reduced binding to BACH1-P (very close to that of the causal R1699W; Suppl. Figure 6C) (46). It had a normal HR activity and was correctly localized after addition of MMC. From our data, it is still unclear if reduced binding to only BACH1-P is sufficient to significantly increase the cancer risk.

34 35 36 37 38 39 40 41 42 43 44

We could not test the binding properties of 36 mutated BRCT domains because there were insoluble in bacteria. Comparison of Glover’s team results with our results suggests that most (89%) mutated BRCT domains insoluble in bacteria in our study are also defective in phosphopeptidebinding. In particular, by producing proteins using in vitro transcription/translation, Glover’s team was able to characterize the phosphopeptide-binding capacities of 5 of our 7 causal mutants. R1699W and M1775R (consistently with our study) as well as G1706E show large binding defects. A1708E and S1715N show smaller but still significant binding defects. Glover’s team also tested the binding properties of our insoluble BRCT mutant corresponding to a likely neutral VUS (M1652T) and showed that this mutant binds phosphorylated BACH1 as the WT BRCT domains. This analysis strongly suggests that all causal mutations lead to binding defects and all neutral mutations do not impact BRCT phosphopeptide-binding properties. It was recently consistently published from the

13



1 2

study of mice carrying a BRCT mutant of BRCA1 that is defective in recognition of phosphorylated proteins that BRCT phosphoprotein recognition is required for BRCA1 tumor suppression (14).

3 4 5 6 7 8 9 10 11 12

It was also proposed that a significant defect in phosphopeptide-binding could completely abolish HR (26). From Glover’s team results and our data, we do not observe a complete correlation between the results of the binding and HR assays. R1699W (causal), R1699Q and M1775R (causal) both show clear binding and HR defects (see consistently for M1775R reports of teams of S. Pellegrini and JD Parvin, and inconsistently report of the team of M.A. Caligo: (23,44,47)). However, E1836K is only defective for BACH1-P binding and exhibits a WT HR efficiency. More strikingly, F1717Y and H1746N show strong binding defects to all 5 phosphopeptides together with a WT HR activity. The functional consequences of these binding defects and their putative contribution to tumorigenesis are unclear. Based on the systematic binding defects measured for causal variants, impaired phosphopeptide-binding might be sufficient per se to be associated with increased cancer risk.

13

Classification of variants

14 15 16 17 18 19 20 21 22 23 24 25 26 27

Most of the mutations responsible for familial breast or ovarian cancers affect genes that control HR and/or the replication/HR interface directly or indirectly (48,49). The two most often mutated genes, BRCA1 and BRCA2, are major players in HR (50,51). This overrepresentation of genes involved in the response to DNA damage and the communication between replication and recombination highlights the importance of these specific pathways in the etiology of breast cancer. We here present one of the first high-throughput structural and functional analyses of a large set of BRCA1 BRCTs VUS, which provides information on the cellular localization and DNA repair capacity of all variants, as well as the thermal stability and binding properties of a subset of variants. Analysis of these results together with the data published in the literature on overlapping subsets of variants enabled to correlate for the first time on a large-scale protein stability, phosphopeptidebinding, HR activity and cancer risk. From this analysis, 3 measurements seem essential to predict cancer risk: (1) BRCT phosphopeptide-binding affinities, (2) BRCT capacity to be produced and purified from bacteria, (3) VUS HR activity. Combining these 3 measurements as well as results from the literature, we sorted the 65 BRCA1 VUS of class 3 into 3 groups (Table II).

28 29 30 31 32 33 34 35

Thirty-three VUS show no HR and phosphopeptide-binding defect in our assays, they are proposed to have no impact on cancer risk (1P). They include all variants of classes 1-2 but one (M1652T, previously classified as likely neutral) and 28 VUS of class 3 (43% of this class). We cannot further classify the likely neutral VUS M1652T because its BRCT domains were insoluble in E. coli in our assays. Glover and co-workers reported that this variant binds to BACH1-P (23), however its binding to other phosphopeptides has not been tested. Supporting our classification of 28 VUS of class 3 as 1P, it was recently shown from genetic data that G1706A and L1844P can be assigned to neutral (personal communication).

36 37 38 39 40 41 42 43 44

Thirty VUS exhibit several defects including systematic HR defect and either BRCT insolubility in bacteria or phosphopeptide binding defect, they are proposed to have a severe impact on cancer risk (3P). They include all variants of classes 4-5 as well as 23 VUS of class 3 (35% of this class). In one case (S1655A), the VUS also showed a decreased binding to a library of phosphopeptides containing the Ser-X-X-Phe motif (46). Moreover the corresponding mouse mutation caused a hypersensitivity to genotoxic stress and a defective HR DNA repair (14). Further supporting our classification, L1764P, which is HR-deficient and shows insoluble BRCT domains in bacteria, was classified as causal based on genetic data during our study (11). C1697R is also HR-deficient and exhibits insoluble BRCT domains in bacteria. We could recently calculate a causality score for C1697R from the French and

14



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literature data as described by D. Goldgar, and we classified this variant as causal (Suppl. Table II) (11,53).

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Only 15 VUS are identified as having an intermediate impact (2P). One of them (M1652T) was already classified as likely neutral and cannot be yet further classified, as explained above. For 3 other VUS of class 3, no HR defect was observed, and either we did not find the same binding defect as a function of the tested phosphopeptide (E1836K), or we measured a WT binding for the 5 phosphopeptides but a BACH1-P binding defect was reported by others (A1708V, A1789S; (23)). Here again, complementary experiments are needed in order to confirm that these VUS are neutral. In the case of the 11 other VUS, strong defects were observed by us, which suggests that these VUS could be causal. V1833M shows an HR defect, even if no binding defect could be measured by us and others (23). The mechanism leading to V1833M HR defect is yet unknown. G1770V, S1722Y, E1735K and A1752E have insoluble BRCT domains but show no HR defect. G1770V was recently classified as causal based on genetic data (personal communication). D1739G, D1739V, W1837G and S1841N have insoluble BRCT domains that were reported by others as defective in binding BACH1-P (23) but also show no HR defect. Finally, H1746N and F1717Y have soluble BRCT domains that poorly bind to the 5 tested phosphopeptides but show no HR defect. We propose that such VUS with a strong binding defect are likely causal and should systematically be monitored because of their association with high cancer risk.

19

Conclusion

20 21 22 23 24 25 26 27 28 29 30

Our analysis strongly suggests that VUS exhibiting WT HR efficiency as well as WT phosphopeptide binding properties are neutral. Also, HR-defective VUS are generally causal. Association of HR-deficiency with a binding defect is strongly associated to causality. Moreover, our study reveals that VUS with BRCT domains that are insoluble in bacteria are often causal. Their phosphopeptide-binding properties should systematically be characterized, and if a defect is identified, they should similarly be considered as increasing cancer risk. In particular, we identified 2 new variants that show a strong binding defect without being HR deficient and our analysis stresses that these variants are likely causal. From these new guidelines, we believe that it is possible to improve the clinical interpretation of BRCA1 variants, and select a larger group of patients for treatment with therapies based on platinum agents or PARP inhibitors that are specifically efficient against BRCA1/2related tumors.

31 32 33 34 35 36 37 38 39 40 41 42 43

Acknowledgments The authors thank the French oncogeneticists, the UNICANCER Genetic Group BRCA network led by Dr Catherine Noguès and probands for their cooperation. This work was supported by a joint translational research grant of the French National Cancer Institute (2011-1-PL BIO-09-IC-1 to A.P.) and the ‘Direction Générale de l’Offre des Soins’ (INCa-DGOS: PRTK2011-046 to A.P., E.D., P.J., I.B., P.L.and PRT-K 14 134 to P.L.) and by the Association d’Aide à la Recherche Cancérologique de Saint-Cloud (ARCS). Lopez’s team is team labeled “Ligue 2014”. We gratefully acknowledge Sylvie Mazoyer for the BRCA1 full-length plasmid; Carine Tellier and Sylvaine Gasparini for their help during the recombinant plasmid construction, Damarys Loew and Vanessa Masson for their help with mass spectroscopy and Sophie Demontety for her help with causality scores.

15



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Millot G, Carvalho MA, Caputo SM, Vreeswijk MPG, Brown MA, Webb M, et al. A Guide for Functional Analysis of BRCA1 Variants of Uncertain Significance (VUS). Hum Mutat. 2012;33:1526–37.

26 27

25.

Rowling PJE, Cook R, Itzhaki LS. Toward classification of BRCA1 missense variants using a biophysical approach. J Biol Chem. 2010;285:20080–7.

28 29

26.

Gaboriau DCA, Rowling PJE, Morrison CG, Itzhaki LS. Protein stability versus function: effects of destabilizing missense mutations on BRCA1 DNA repair activity. Biochem J. 2015;466:613–24.

30 31 32

27.

Caputo S, Benboudjema L, Sinilnikova O, Rouleau E, Béroud C, Lidereau R. Description and analysis of genetic variants in French hereditary breast and ovarian cancer families recorded in the UMD-BRCA1/BRCA2 databases. Nucleic Acids Res. 2012;40:D992–1002.

33 34

28.

Béroud C, Letovsky SI, Braastad CD, Caputo SM, Beaudoux O, Bignon YJ, et al. BRCA Share: A Collection of Clinical BRCA Gene Variants. Hum Mutat. 2016;

35 36

29.

Gaildrat P, Krieger S, Giacomo DD, Abdat J, Révillion F, Caputo S, et al. Multiple sequence variants of BRCA2 exon 7 alter splicing regulation. J Med Genet. 2012;49:609–17.

37 38

30.

Rouleau E, Lefol C, Moncoutier V, Castera L, Houdayer C, Caputo S, et al. A missense variant within BRCA1 exon 23 causing exon skipping. Cancer Genet Cytogenet. 2010;202:144–6. 17



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31.

Wiederschain D, Wee S, Chen L, Loo A, Yang G, Huang A, et al. Single-vector inducible lentiviral RNAi system for oncology target validation. Cell Cycle Georget Tex. 2009;8:498–504.

3 4 5

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Dumay A, Laulier C, Bertrand P, Saintigny Y, Lebrun F, Vayssière J-L, et al. Bax and Bid, two proapoptotic Bcl-2 family members, inhibit homologous recombination, independently of apoptosis regulation. Oncogene. 2006;25:3196–205.

6 7

33.

Scully R, Chen J, Plug A, Xiao Y, Weaver D, Feunteun J, et al. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell. 1997;88:265–75.

8

34.

Team RC. R: A language and environment for statistical computing. 2013;

9 10

35.

Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc Ser B Methodol. 1995;57:289–300.

11 12

36.

Glover JNM. Insights into the molecular basis of human hereditary breast cancer from studies of the BRCA1 BRCT domain. Fam Cancer. 2006;5:89–93.

13 14

37.

Shen Y, Tong L. Structural evidence for direct interactions between the BRCT domains of human BRCA1 and a phospho-peptide from human ACC1. Biochemistry (Mosc). 2008;47:5767–73.

15 16 17

38.

Botuyan MVE, Nominé Y, Yu X, Juranic N, Macura S, Chen J, et al. Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains. Struct Lond Engl 1993. 2004;12:1137–46.

18 19 20

39.

Bouwman P, van der Gulden H, van der Heijden I, Drost R, Klijn CN, Prasetyanti P, et al. A highthroughput functional complementation assay for classification of BRCA1 missense variants. Cancer Discov. 2013;3:1142–55.

21 22 23

40.

Williams RS, Chasman DI, Hau DD, Hui B, Lau AY, Glover JNM. Detection of protein folding defects caused by BRCA1-BRCT truncation and missense mutations. J Biol Chem. 2003;278:53007–16.

24 25 26

41.

Carvalho MA, Marsillac SM, Karchin R, Manoukian S, Grist S, Swaby RF, et al. Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis. Cancer Res. 2007;67:1494–501.

27 28

42.

Drikos I, Nounesis G, Vorgias CE. Characterization of cancer-linked BRCA1-BRCT missense variants and their interaction with phosphoprotein targets. Proteins. 2009;77:464–76.

29 30 31

43.

Woods NT, Baskin R, Golubeva V, Jhuraney A, De-Gregoriis G, Vaclova T, et al. Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance. Npj Genomic Med. 2016;1:16001.

32 33 34

44.

Towler WI, Zhang J, Ransburgh DJR, Toland AE, Ishioka C, Chiba N, et al. Analysis of BRCA1 variants in double-strand break repair by homologous recombination and single-strand annealing. Hum Mutat. 2013;34:439–45.

35 36

45.

Chang S, Biswas K, Martin BK, Stauffer S, Sharan SK. Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations. J Clin Invest. 2009;119:3160–71.

37 38

46.

Campbell SJ, Edwards RA, Glover JNM. Comparison of the structures and peptide binding specificities of the BRCT domains of MDC1 and BRCA1. Struct Lond Engl 1993. 2010;18:167–76. 18



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Guidugli L, Rugani C, Lombardi G, Aretini P, Galli A, Caligo MA. A recombination-based method to characterize human BRCA1 missense variants. Breast Cancer Res Treat. 2011;125:265–72.

3 4 5

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Walsh T, Casadei S, Lee MK, Pennil CC, Nord AS, Thornton AM, et al. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci U S A. 2011;108:18032–7.

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Castéra L, Krieger S, Rousselin A, Legros A, Baumann J-J, Bruet O, et al. Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes. Eur J Hum Genet EJHG. 2014;22:1305–13.

9 10

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Moynahan ME, Chiu JW, Koller BH, Jasin M. Brca1 controls homology-directed DNA repair. Mol Cell. 1999;4:511–8.

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Moynahan ME, Cui TY, Jasin M. Homology-directed dna repair, mitomycin-c resistance, and chromosome stability is restored with correction of a Brca1 mutation. Cancer Res. 2001;61:4842–50.

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Williams RS, Lee MS, Hau DD, Glover JNM. Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1. Nat Struct Mol Biol. 2004;11:519–25.

16 17 18

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19 20

54.

Pierce AJ, Johnson RD, Thompson LH, Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 1999;13:2633–8.

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24

19


Table IA UMD-BRCA1 Class

Expression in E. Localisation after coli & Stability if MMC Soluble

HGVS DNA nomenclature c.

Amino Acid Change

c.4949T>C

M1650T

c.4955T>C

M1652T

2

c.4956G>A

M1652I b

1

c.4956G>T

M1652I a

c.4963T>G

S1655A

c.4991T>C

L1664P

c.4993G>A

V1665M

c.4993G>C

V1665L

c.4994T>A

V1665E

c.4999A>G

K1667E

like wt

c.5005G>T

A1669S

c.5008A>G

R1670G

c.5053A>G

T1685A

0,660550459

C

c.5057A>G

H1686R

0,622568093

c.5060T>A

V1687D

0,776176929

c.5062G>T

V1688F

0,598949212

c.5068A>G

K1690Q

0,638795987

c.5071A>G

T1691A

1,05

c.5072C>A

T1691K

0,711409396

C

c.5072C>G

T1691R

0,658291457

c.5072C>T

T1691I

0,59

c.5085T>A

F1695L

HR

like wt

0,88 ns

like wt

HGVS DNA nomenclature c.

Amino Acid Change

1P

c.5192A>G

E1731G

1,12 ns

1,12 ns

B

-2,7

1P

NA

2P

c.5192A>T

E1731V

1,2 ns

1,2 ns

B

-0,9

1P

like wt

B

-0,3

1P

c.5203G>A

E1735K

0,97

0,97

B-

NA

2P

B

-0,4

1P

c.5213G>A

G1738E

0,828877005

B-

NA

3P

C

NA

3P

c.5213G>T

G1738V

0,828877005

0,97

B-

NA

3P

B

-1,3

1P

c.5216A>G

D1739G

1,27 ns

like wt

B-

NA

2P

1,25 ns

like wt

B-

NA

2P

like wt

A

8

2P

like wt

B

-1,8

1P

like wt

0,828877005

like wt

B

-0,9

1P

c.5216A>T

D1739V

1,07

like wt

B

-0,1

1P

c.5236C>A

H1746N

C

NA

3P

c.5252G>A

R1751Q

like wt

A

-0,01

1P

c.5254G>A

A1752T

0,828877005

0,828877005

B-

NA

3P

1,04

like wt

B

-0,01

1P

c.5254G>C

A1752P

0,597

0,828877005

B-

NA

3P

0,98

like wt

B

like wt

1P

c.5255C>A

A1752E

0,95

0,828877005

C

NA

2P

NA

3P

c.5282T>C

F1761S

0,619369369

0,828877005

C

NA

3P

C

NA

3P

c.5291T>C

L1764P

0,828877005

0,828877005

C

NA

3P

C

NA

3P

c.5309G>T

G1770V

0,95

0,828877005

B-

NA

2P

C

NA

3P

c.5321A>G

N1774S

A

-1,2

1P

like wt

C

NA

3P

c.5324T>G

M1775R

like wt

B

1P

c.5344T>C

W1782R

NA

3P

c.5348T>C

M1783T

C

NA

3P

c.5355G>T

Q1785H

C

NA

3P

c.5359T>G

C1787G

0,97

1P

c.5365G>T

A1789S

1,02

0,632716049

probl em of re localisation

like wt

like wt

like wt

like wt

NA

3P

c.5371G>C

V1791L

B

4,5

3P

c.5411T>A

V1804D

R1699Q

0,713567839

like wt

A

5,9

3P

c.5419A>G

c.5099C>T

T1700I

0,649425287

like wt

C

NA

3P

c.5116G>A

G1706R

5

0,659090909

C

NA

c.5117G>A

G1706E

4

0,56

C

c.5117G>C

G1706A

c.5123C>A

A1708E

c.5123C>T

c.5096G>A

Classification

like wt

like wt

0,821672355

5

Loss of phosphopeptide binding

-0,8

C

R1699W

Expression in E. Localisation after coli & Stability if MMC Soluble

C

like wt

C1697R

c.5095C>T

HR

B

0,663316583

c.5089T>C

UMD-BRCA1 Class

like wt

like wt

5

Classification

like wt

like wt

0,62974268

Loss of phosphopeptide binding

2

1,08403589883057 ns

0,89ns

5 2

like wt

1,02

0,828877005

B

6,8

3P

1,11442786069652 ns

problem of re localisation

B

-0,8

1P

like wt

B

-1,2

1P

like wt

A

-0,8

1P

like wt

A

-0,4

1P

B

-1,6

2P*

1,11442786069652 ns

0,94

0,828877005

0,98

like wt

A

-0,4

1P

1,11324376199616 ns

like wt

B

-0,4

1P

I1807V

1,02

like wt

A

-0,01

1P

c.5426T>G

V1809G

0,596928983

C

NA

3P

3P

c.5429T>G

V1810G

0,86 ns

A

-0,01

1P

NA

3P

c.5432A>G

Q1811R

0,614203455


C

NA

3P


A

-0,2

1P

1

0,828877005

like wt

0,890909090909091ns

like wt

B

-0,8

1P

c.5458G>A

G1820S

1,05

0,585014409

like wt

C

NA

3P

c.5461T>C

F1821L

1,1

like wt

A

-0,01

1P

A1708V

1,10384 ns

like wt

B

-1,7

2P*

c.5474G>A

G1825E

1,12ns

like wt

A

-1,5

1P

c.5129G>A

G1710E

0,97

like wt

B

-2,5

1P

c.5497G>A

V1833M

0,828877005

like wt

B

-3,3

2P

c.5138T>C

V1713A

0,678341272

0,678341272

C

NA

3P

c.5498T>G

V1833G

0,739626556

NA

3P

c.5144G>A

S1715N

0,703305352

0,678341272

C

NA

3P

c.5506G>A

E1836K

0,91

3,7

2P

c.5150T>A

F1717Y

1,22 ns

B

8,2

2P

c.5509T>C

W1837R

0,828877005


B-

NA

3P

c.5155G>T

V1719L

1,1

B

-0,01

1P

c.5509T>G

W1837G

0,836785162287481 ns


B-

NA

2P

c.5158A>G

T1720A

B

-0,3

1P

c.5522G>A

S1841N

0,893877551020408ns


B-

NA

2P

c.5165C>A

S1722Y

NA

2P

c.5531T>C

L1844P

0,931313131

like wt

A

-0,01

1P

c.5177G>T

R1726I

-1,7

1P

c.5566C>T

P1856S

1,03

like wt

A

-0,4

1P

5

5

1

1,03

like wt


like wt

0,94

1,22 ns

B-


B

20



like wt

B-

A


Table IB

Phosphopeptide binding in vitro

Function in cells

Solubility in bacteria and during purification

Defect scoring

BACH1-P

ACC1-P/CTIP-P

AB-1P/AB-2P

HR

1

1

1

3

2

 1P

2P

0

Binds to the 5 phosphopeptides in this study

Binds to phosphopeptides as reported in the literature

No impact

3P

0-1

2

2-3

3

5-6

Contradictory binding data

Binds to phosphopeptides as reported in the literature

Do not bind to phosphopeptides as reported in the literature

Do not bind to the 5 phosphopeptides in this study

All cases

Undetermined

Undetermined

Undetermined

Likely Causal

Causal

21


1

Table II DNA nomenclature

Class

Protease sensitivity

Expression in E. Coli & Stability if Soluble

Expression in E. Coli & Stability if Soluble

BACH1-P peptide bdg

N,M,F,FF = increasing sensitivity

S, S-,S--,I = decreasing solubility

S, S-,S--,I = decreasing solubility

N, M,F,FF = decreasing affinity

Lee et al. 2010

Rowling et al. 2010

our study

Lee et al. 2010

Loss of Loss of affinity Loss of affinity Transcription phosphopepti for BACH1-P for Opt-P assay de binding

N = native-like

N,F = decreasing affinity

N,M,F = decreasing activity

Coquelle et al. Rowling et al. 2011 2010

our study

Lee et al. 2010

NO BDG

300

Cisplatin resistance & HR

Formation of foci after IR & HR

Transcription assay

Localisation after MMC

HR

Resist.: N,F HR: +,-

Foci: N,F HR: +,-,--

N,M,F = decreasing activity

N,F = native, non native localization

N+,N,F = enhanced, normal, defective HR

our study

our study

F

N

F

F

F

F

F

F

F

F

F

F

F

F

F

Bouwman et Gaboriau et al. Woods et al., al., 2013 2015 2016

c.5095C>T

R1699W

3P

N

S--

S-

FF

F

F

c.5324T>G

M1775R

3P

N

S-

S-

FF

F

F

c.5117G>A

G1706E

3P

FF

I

FF

-

M

F / HR-

c.5123C>A

A1708E

3P

FF

I

I

F

-

F

F

c.5144G>A

S1715N

3P

F

I

I

M

-

F

F / HR-

F / HR--

c.4994T>A

V1665E

3P

I

-

F

F

c.5116G>A

G1706R

3P

I

-

F

F

c.5096G>A

R1699Q

3P

N

F

N

F

c.5053A>G

T1685A

3P

c.5072C>A

T1691K

c.5089T>C

S+

S

FF

F

I

3P

FF

C1697R

3P

FF

28,7µM/F

100

F

F

F / HR+

FF

-

F

F

F

F

I

FF

-

F

F

F

F

I

M

-

F

F

F

F

c.5138T>C

V1713A

3P

N

I

F

-

F

F

F

F

c.5254G>C

A1752P

3P

FF

S--

FF

-

F

F

F

F

c.5282T>C

F1761S

3P

F

I

FF

-

F

F

F

F

c.5291T>C

L1764P

3P

F

I

F

-

F

F

F

F

c.5432A>G

Q1811R

3P

F

I

M

-

F

F

F

F

c.5509T>C

W1837R

3P

FF

S--

FF

-

F

F

F

F

c.5213G>A

G1738E

3P

FF

S--

Loss of specificity

-

F

F

F

F

c.4963T>G

S1655A

3P

I

N

F

c.5057A>G

H1686R

3P

I

-

F

F

c.5060T>A

V1687D

3P

I

-

F

F

c.5062G>T

V1688F

3P

I

-

F

F

c.5068A>G

K1690Q

3P

I

-

N

F

c.5072C>G

T1691R

3P

I

-

F

F

c.5072C>T

T1691I

3P

I

-

F

F

c.5099C>T

T1700I

3P

I

-

N

F

c.5213G>T

G1738V

3P

S--

-

N

F

c.5254G>A

A1752T

3P

S--

-

F

F

c.5426T>G

V1809G

3P

I

-

F

F

c.5498T>G

V1833G

3P

S--

-

F

F

c.5236C>A

H1746N

2P

N

N

c.5150T>A

F1717Y

2P

N

N

c.5216A>G

2

Amino Acid Change

I I

F

S

Loss of binding (Botuyan et al, 2004)

FF

D1739G

2P

FF

D1739V

2P

FF

c.5509T>G

W1837G

2P

FF

I

c.5522G>A

S1841N

2P

FF

I

c.5309G>T

G1770V

2P

S--

-

c.5165C>A

S1722Y

2P

S--

-

c.5203G>A

E1735K

2P

S--

-

c.5255C>A

A1752E

2P

I

-

c.5497G>A

V1833M

2P

I

F

F

F

M

M

F

c.5216A>T

F

F

-

F

S-

F / HR-

S--

FF

-

F

F

F

N

N

S--

FF

-

F

F

F

N

N

S--

F

-

F

F

F

N

S--

F

-

F

F

F

N

F / HR-

F

N

Not Clear

N

N

F

N

S-

N

c.5123C>T

A1708V

2P

F

S-

F

c.5506G>A

E1836K

2P

N

S

FF

c.5365G>T

A1789S

2P

N

S-

M

c.5158A>G

T1720A

1P

N

S-

N

c.5252G>A

R1751Q

1P

M

S-

N

c.5411T>A

V1804D

1P

N

S-

c.4956G>A

M1652Ib

1P

N

S-

c.5348T>C

M1783T

1P

N

c.4991T>C

L1664P

1P

N

N

N / HR+

N

F

F

N

F

N

F

M

N

N

F/N

M

F

N

N

N

N

N

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

S-

N

N

N

N

N

N

S--

S-

M

N

N

M

M

N

N

S-

S-

N

N

N

N

N

F

N

N

N

N

N

S-

2µM/M

N

22

N N N / HR+

Downloaded from mcr.aacrjournals.org on October 1, for Cancer Research. c.4993G>A V1665M 1P N SSN 2018. © 2018 NAmerican N Association N M c.5005G>T

A1669S

1P

N

S

S-

N

N

N

N

M

Impact

Control group of CAUSAL variants

INSOLUBLE or BINDING DEFECT AND HR DEFECTIVE

STRONG BINDING DEFECT

INTERMEDIATE IMPACT AND DEFECTIVE BINDING

INTERMEDIATE IMPACT AND NO BINDING DATA

WT BINDING AND HR DEFECT

UNCONSISTENT BINDING DATA

Control group of NEUTRAL variants

c.5497G>A

V1833M

2P

F

I

S-

N

c.5123C>T

A1708V

2P

F

S-

F

c.5506G>A

E1836K

2P

N

S

FF

c.5365G>T

A1789S

2P

N

S-

M

c.5158A>G

T1720A

1P

N

S-

N

c.5252G>A

R1751Q

1P

M

S-

N

S-

N

2µM/M

N

F

F

N

F

N

F

M

N

N

F/N

M

F

N

N

N

N

N

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

c.5411T>A

V1804D

1P

N

S-

N

N

N

c.4956G>A

M1652Ib

1P

N

S-

S-

N

N

N

c.5348T>C

M1783T

1P

N

S--

S-

M

N

N

M

M

N

N

c.4991T>C

L1664P

1P

N

S-

S-

N

N

N

N

N

F

N

c.4993G>A

V1665M

1P

N

S-

S-

N

N

N

N

M

N

N

c.5005G>T

A1669S

1P

N

S

S-

N

N

N

N

M

N

N

c.5085T>A

F1695L

1P

N

S

N

N

N

M

N

N

c.5355G>T

Q1785H

1P

N

S

N

N

N

M

N

N

c.5566C>T

P1856S

1P

N

S

N

N

N

N

N

N

c.5117G>C

G1706A

1P

N

S-

N

N

N

N

N

N

c.4949T>C

M1650T

1P

S-

N

N

N

c.4956G>T

M1652Ia

1P

S-

N

F

N

c.4993G>C

V1665L

1P

S-

N

N

N

c.4999A>G

K1667E

1P

S

N

N

N

c.5008A>G

R1670G

1P

S-

N

N

N

c.5071A>G

T1691A

1P

S-

N

N

N

c.5129G>A

G1710E

1P

S-

N

N

N

c.5158A>G

V1719L

1P

S-

N

F

N

c.5177G>T

R1726I

1P

S-

N

F

N

c.5192A>G

E1731G

1P

S-

N

N

N

c.5192A>T

E1731V

1P

S-

N

N

N

c.5321A>G

N1774S

1P

S

N

N

N

c.5359T>G

C1787G

1P

S

N

N

N

c.5371G>C

V1791L

1P

S

N

N

N

c.5419A>G

I1807V

1P

S

N

N

N

c.5458G>A

G1820S

1P

S

N

N

N

c.5461T>C

F1821L

1P

S

N

N

N

c.5474G>A

G1825E

1P

S

N

N

N

c.5531T>C

L1844P

1P

S

N

N

N

c.5344T>C

W1782R

1P

S-

N

F

N

c.5429T>G

V1810G

1P

N

S

N

N

F

F

N

N

c.4955T>C

M1652T

2P

N

I

N

-

N

N

N

N

S-

N

N N / HR+

N / HR+ N

N

N

N

1

23


WT BINDING AND HR DEFECT

UNCONSISTENT BINDING DATA

Control group of NEUTRAL variants

NO IMPACT

INTERMEDIATE IMPACT AND WILD-TYPE BINDING


Table and Legends Table I: A. Summary of the experimental results presented in this study. In column “HR”, variants with defective HR are indicated in red and variants with WT HR in blue. In column “localization after MMC”, variants with defective localization are again in red. In column “expression in E. coli and stability during purification if soluble”, VUS with BRCT domains that were mostly insoluble in E. coli appear in red, poorly soluble mutated domains that could not be purified because of aggregation in light red, domains that could be expressed and purified but with low yields in light blue and mutated domains that were obtained with a yield close to that of the WT BRCT domains in blue. In column “loss of phosphopeptide-binding”, VUS with domains binding as the WT BRCT domains are indicated in blue and VUS with domains that do not bind in red (light red: defect in BACH1-P binding; bright red: binding defect observed with the 5 phosphopeptides). Boxed VUS are mutated at positions buried in the 3D structure of the BRCA1 BRCT domains. In column “classification”, we have distributed the 78 variants in 3 distinct classes (1P no impact, 2P intermediate impact, 3P severe impact) as a function of our results, as explained in Table IB. Red stars associated to the class type indicate that inconsistent binding data were previously published. Therefore, the corresponding VUS were classified as 2P. B. Classification of the variants. The scoring scheme is a result of our analysis. First, because of the strong relationship observed between both BRCA1 HR defect and BRCA1 phosphopeptide binding defect and tumorigenesis, our score largely depends on these BRCA1 properties (+3 for an HR defect and +3 if binding to all 5 phosphopeptides is impaired). Also, as impaired solubility in bacteria is generally associated with causality, we scored +2 in case of insolubility or aggregation during the purification. VUS with no observed defect (score=0) were classified as 1P, with an intermediate impact (scores between 1 and 3) as 2P and with defects observed both in vitro and in cells (scores higher than 4) as 3P. Moreover, VUS with a score of 0 for which we measured WT binding properties that are not consistent with the literature were classified as 2P. Table II: Summary of the structural and functional data available on the 78 VUS, based on this study as well as on previously published studies. Experimental results essential for classification are boxed in red and blue. The 4 columns with a yellow background correspond to the data obtained in this study. Nine additional columns present the results of other studies on subset of variants. The 78 studied VUS are presented as a function of their impact. All causal control variants are identified as 3P. A group of 12 VUS of class 3, which exhibit defective HR and either a strong phosphopeptide binding defect in our study or insoluble/aggregating BRCT domains in our study and a binding defect in the literature, are also classified as 3P. They show, when measured, defective transcription activation and cisplatin resistance (17–19). For an additional group of 11 VUS, data are only available from this study; however, these VUS have a defective HR activity and their BRCT domains are poorly soluble in bacteria; therefore, they are also identified as 3P. The 15 VUS identified as 2P (intermediate impact) are presented as a function of their phosphopeptide binding properties. One of these VUS is M1652T of class 2. Within the 14 VUS of class 3, 9 VUS exhibit defective binding, either in this study or in previously published assays. One VUS (V1833M) binds with a WT affinity to the 5 tested phosphopeptide, and for the 5 remaining VUS no binding data are available. Several VUS from this group present transcription activation defects and, when tested, cisplatin resistance defects (17–19). All control variants but one (M1652T, see above) are identified as 1P. A group of 28 VUS showing wild-type binding to the 5 tested phosphopeptides and normal HR efficiency in cells are classified as 1P. Five of them still show a localization defect in our assays. Figure 1: Distribution of the 78 variations corresponding to the selected VUS. (A) Position of the mutations along the BRCA1 sequence. On the upper view, the whole BRCA1 sequence is displayed, with its 2 globular domains in red (N-terminal RING domain) and orange (C-terminal BRCT region), 24



and its nuclear export/import signals in black (NES) and green (NLS), respectively. The lower view is a zoom of the BRCA1 BRCT region. It contains 2 BRCT domains indicated in grey. The 78 variations corresponding to the selected VUS are indicated on this zoom. The 7 causal and 6 neutral selected variants are displayed in red and blue, respectively. (B) Position of the mutated residues in 3D structure of the BRCT domains (in dark gray) in complex with a phosphorylated BACH1 peptide (in light gray) (PDBcode 1T15). Mutated positions are found throughout the whole structure. Those corresponding to VUS of classes 1 and 2 are colored in blue, classes 4 and 5 in red, and class 3 in yellow. VUS of classes 1, 2, 4 and 5 are labeled. Figure 2: Description of the high-throughput cellular assay set up for measurement of VUS Homologous Recombination efficiency. (A) Description of the HR substrate (DR-GFP). Two inactive GFP genes are organized into direct repeats. The 5’ GFP cassette is inactivated because of deletions in both the 5’ and the 3’ sequences. Expression of I-SceI generates a cleavage (DSB) targeted into the substrate. HR between the 2 GFP genes, with I-SceI, can generate a functional GFP gene through gene conversion without crossing over. Recombinant cells are thus GFP-positive (GFP+) and can be monitored by FACS (54). The DR-GFP substrate is stably integrated into a chromosome of SV40-transformed fibroblasts in the RG37 cell line (25). (B) Inducible knockdown of endogeneous BRCA1. One cell line containing a doxycyclin-inducible shRNA against the endogenous BRCA1 was derived from the RG37 cell lines (which bear the HR substrate): the RG37 cells have been transduced with a lentivirus coding for a shRNA against the 3’UTR of the endogenous BRCA1 mRNA. This allows to specifically targeting the endogenous BRCA1 and not the exogenous BRCA1 that will be then used. In addition, expression of this shRNA is inducible by the doxycyclin. Supplying doxycyclin (DOX) induces the expression of the shRNA leading to the silencing of the expression of the endogenous BRCA1 (left panel) and to reduced HR frequencies, monitored using the DR-GFP (right panels). This cell line (named RG37shB1) was then used to test all the BRCA1 variants. Because this shRNA does not affect the expression of exogenous BRCA1, expression of exogenous wild-type BRCA1 (WT-BRCA1) is able to stimulate HR frequency, as already shown for BRCA1 overexpression in RG37 cells (55), and to rescue decreased HR frequency in DOX-exposed cells, i.e. silenced for the expression of the endogenous BRCA1. The values correspond to at least 3 independent experiments. (C) Impact of BRCA1 bearing causal mutations on the rescue of HR in cells silenced for the endogenous BRCA1. The values are shown normalized to WT-BRCA1 (in black). They correspond to 4 to 8 independent experiments. Figure 3: Impact of the BRCA1 missense mutations on Homologous Recombination. (A) Plot of the HR efficiencies measured after expression of either WT BRCA1 or VUS normalized to the HR efficiency after expression of WT BRCA1. Statistical significance was calculated using a oneside paired student t-test. To account for the multiple testing, the p-values were adjusted using the Benjamini-Horchberg method at a level =0.05. HR efficiencies significantly different from the WT value are marked by asterisks. They are indicated by * if p < 0.05 and ** if p < 0.01. All 7 (likely) causal (noted LC or C) variants (in red) cause a significantly decreased HR efficiency whereas all 6 (likely) neutral (noted LN or N) variants (in blue) cause no significant HR difference. Twenty-one variants could not provide WT HR efficiency. (B) Identification of the VUS defective for nuclear localization after addition of MMC, plotted as a function of increasing HR efficiencies as in (A). For observing VUS nuclear localization defects, plates were examined after addition of mitomycin to RG37 cells transfected with VUS plasmids. Cells were immunostained with an anti-BRCA1 antibody followed by an Alexa Fluor 488-conjugated secondary antibody and the nucleus was stained with DAPI. BRCA1 localization was visualized by fluorescence microscopy. Statistical significance was calculated using a two-tailed Student t-test with GraphPad. This analysis revealed that 34 VUS were not correctly addressed to the nucleus. The bars corresponding to these VUS are colored in green. A more quantitative report of the percentage of nuclear fluorescence measured for each VUS can be found in Suppl. Table 3. As an illustration, in the upper part of panel B, images from control experiments showing that BRCA1 WT is correctly localized in the nucleus after mitomycin treatment 25



(lane 1) as well as images revealing the localization of a neutral (M1652T; lane 2) and a causal (V1665E; lane 3) VUS are displayed. Figure 4: Representation of the solubility in bacteria of the recombinant mutated BRCT domains. (A) Bars of the HR plot of Figure 3A are colored as a function of the solubility in E. coli of the corresponding mutated BRCT domains. Brown bars mark mutants that are completely insoluble as GST fusion proteins in E. coli. Yellow bars indicate mutants that are partially soluble in bacteria but aggregate during removal of the GST tag and purification. Only mutants corresponding to grey and black bars could be further characterized in vitro by fluorescence. The difference between these two last classes is based on their expression and purification yields, grey meaning low production yield and black WT-like production yield. (B) 3D representation of the position of the residues mutated in VUS characterized by both (1) defective HR capacities and (2) recombinant BRCT domains that could not be produced and purified from bacteria (in brown and yellow/green as in (A)). Figure 5: Evaluation of the impact of missense mutations on the BRCT phosphopeptide-binding capacities. (A) Thermal shifts due to the addition of phosphopeptides at 256 M onto the WT and mutated BRCT domains. Mutations are ordered as a function of the mutated residue number. Likely neutral and neutral mutations are marked by blue LN and N letters, respectively, whereas causal mutations are marked by a red C. Black, brown, grey and yellow, light orange and dark orange bars correspond to the addition of a control peptide (CTRL-P), ACC1-P, CtiP-P, BACH1-P, AB-1P and AB-2P, respectively. Bars boxed in green correspond to a reduction of the thermal shifts by at least a factor 2 (dark and light green when residual binding or no binding was observed, respectively; see (B)). The bars boxed in yellow/green mark a mutation that enhanced binding to CtiP-P, ACC1-P, AB1P and AB-2P but decreased binding to BACH1-P. (B) 3D representation of the positions corresponding to the mutations identified by boxes in (A). The BACH1-P peptide is displayed in red. Positions in dark and light green are in contact or far from the phosphopeptide-binding site, respectively. Position in yellow (1836) interacts with Lys995 from BACH1-P. As the corresponding residue is specific to BACH1 (see (A)), mutation of Glu1836 has different impacts on the binding of BRCA1 to BACH1-P compared to ACC1-P, CtiP-P, AB-1P and AB-2P.

26













Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk. Ambre Petitalot, Elodie Dardillac, Eric Jacquet, et al. Mol Cancer Res Published OnlineFirst September 26, 2018.

Updated version Supplementary Material Author Manuscript

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