BRCA1 C-terminal Research Articles

Using AI-predicted protein structures as a reference to predict loss-of-function activity in tumor suppressor breast cancer genes

BackgroundThe loss-of-function (LOF) classification of most missense variants in tumor suppressor breast cancer genes BRCA1, BRCA2, PALB2, and RAD51C remains unclassified and confounds clinical actionability. Classifying these variants is challenging due to their rarity, leading clinicians to rely on in silico predictive methods. Protein stability changes are associated with function, making stability predictors valuable. Stability predictions upon missense variant perturbations require high-resolution protein structures. However, the availability of these high-resolution structures is lacking. This study explores using generative AI to predict high-resolution protein structures, which can then be analyzed with in silico protein stability prediction methods to assess LOF activity in ordered regions of the protein. This study also determines the appropriate in silico protein stability and dedicated in silico missense prediction methods in dbNSFP v4.7 database to predict LOF activity in ordered regions of these four genes. Functional classifications from homology recombination DNA repair (HDR) assays and variant classifications from the ClinVar database provide a reliable dataset for evaluating the performance of these in silico prediction methods. ResultsComplex AlphaFold2 structures of the BRCA1-C terminal (BRCT) domain and the DNA-binding (DB) domain of BRCA2, analyzed using protein stability tool FoldX predicts LOF activity from missense variants significantly better than experimentally-derived structures in ordered regions. The BRCT domain achieved an Area Under the Curve (AUC)= 0.861 (95 % CI:0.858–0.863) and AUC= 0.842 (95 % CI:0.840–0.845), while the DB domain achieved an AUC= 0.836 (95 % CI:0.8322–0.841), compared to AUC= 0.847 (95 % CI:0.844–0.850) and AUC= 0.835 (95 % CI:0.832–0.837) from the BRCT domain, and AUC= 0.830 (95 % CI:0.821–0.8320) from the DB domain from experimentally-derived structures. Protein stability does not predict LOF activity from missense variants better than dedicated in silico missense predictors. Overall, we find that AlphaMissense ranks highly, with an average AUC= 0.890 (95 % CI 0.886–0.895) from ordered regions across these four cancer genes, compared to all other in silico missense predictors present in the dbNSFP database. ConclusionsThe study reveals that generative AI protein predicted structures can outperform experimentally-derived structures in evaluating LOF activity from predicted protein stability in ordered regions of genes BRCA1, BRCA2, PALB2 and RAD51C. The study also highlights the predictive performance of AlphaMissense as the premier in silico missense prediction method to predict LOF activity from missense variants in these four tumor suppressor breast cancer genes. The code for this study can be downloaded for free on GitHub (https://github.com/rohandavidg/CarePred)

Efficacy of PARP inhibitors in advanced high-grade serous ovarian cancer according to BRCA domain mutations and mutation type.

Preclinical studies have emphasized the potential connection between BRCA specific domains defects and the activity of Poly ADP-ribose polymerase inhibitors (PARPi). Nevertheless, real-world evidence regarding the impact of BRCA domain defects and mutations on PARPi efficacy are limited. The aim of his study was to evaluate the efficacy of PARPi in terms of progression free survival (PFS) according to BRCA domains defects and mutation types. A retrospective analysis was performed among 79 BRCA mutated patients, diagnosed with advanced High-grade serous ovarian carcinoma (HGSOC) who received first- and second-line platinum- based chemotherapy followed by PARPi maintenance treatment. PFS was evaluated according to BRCA1 [Really Interesting Gene (RING), DNA Binding (DBD), Serine Cluster (SCD), BRCA1 C-terminal (BRCT)] and BRCA2 [RAD-51 Domain (RAD-51 BD), DBD] specific domain defects and mutation types [missense (MS), nonsense (NS), frameshift (FS), splicing (S), or large rearrangements (LR)]. After a median follow-up of 51 months, no significant difference in PFS was observed between the BRCA functional domains or mutation types in the BRCA1 and BRCA2 subgroups. Patients with BRCA2 DBD and RAD51-BD defects had the longest (39.8 months) and shortest (24.1 months) median PFS, respectively (p = 0.11). Additionally, patients with BRCA1 DBD defects had the greatest benefit (median PFS = 33.8 months) while those with BRCA1 RING domain mutations experienced the worst outcome (median PFS = 30.9 months (p = 0.43). The efficacy of maintenance treatment with PARPi is independent by BRCA domain defects or mutation types. Patients DBD domain defects experienced numerically longer median PFS compared to those with other BRCA1/2 alterations.

BRCA functional domains associated with PARP-inhibitors in patients with prostate cancer: The PROGRESS study.

10543 Background: Homologous recombination repair (HRR) defects are driver mutational imprints and actionable biomarkers in prostate cancer (PrC) patients. However, recent research provides evidence of PrC onset also in the context of mismatch repair deficiency (MMRd). In the current report, we investigated whether the position of pathogenic variants (PVs) in the functional domains (FDs) of HRR genes could be associated with reduced or increased sensitivity to PARP-inhibitors (PARPis). We also questioned whether the HRR FDs had a prognostic significance and affected the development of second primary tumor (SPT) in males with a genetic predisposition. Methods: This was a real-world, observational, study including PrC patients undergoing germline, somatic, and liquid biopsy testing between January 2020 and December 2023. Prognostic factors, SPT history, and PARPi benefit were evaluated according to mutated genes ( BRCA1, BRCA2,No- BRCAHRR), PV location within the FDs, and germline (g) or somatic (s) PV. The BRCA1FDs were RING, DNA-binding domain (DBD), BRCA1 C terminus (BRCT), or other location, and the BRCA2 FDs were RAD51-BD, DBD, or other location. Results: A total of 833 metastatic PrC patients, aging 40 to 91, were included in this study; 169 (20.3%) were carriers of germline (n.48, 5.8%) or somatic (n.121, 14.5%) PVs in HRR genes: 17 in BRCA1 (g BRCA1: n.4, 2.4%; s BRCA1: n.13, 7.7% ), 102 in BRCA2 (g BRCA2: n.33, 19.5%; s BRCA2: n.69, 40.8% ), and 53 in no- BRCA HRR genes (gHRR: n.11, 6.5%; sHRR: n.42, 24.8%). CHEK2was the HRR gene most frequently affected by germline PVs (27.3%), while the most frequent somatic PVs were in the ATM (40.5%). Liquid biopsy identified PVs in 33 patients out of 185 tested, including n.10 (5.4%) not detected through somatic and germline testing, considerably expanding the therapeutic window for the use of PARPi. Notably, differences in PFS rates at 48 months were observed when comparing sub-groups according to PV location in the FDs of BRCA2.Surprisingly, when we investigated the SPT in the cohorts of patients with and without germline PVs, the difference was not significant (14.5% vs 12.4%, respectively) and, in the subgroup without germline PVs, the sites of SPT were predominantly colorectal, gastric, bladder or other tumors classically dominated by MMRd and recognized as Lynch Syndrome-spectrum tumors. This observation may result in unexplored forms of hypermutable tumor phenotypes, potentially impacting cancer risk management and increasing therapeutic opportunities. Conclusions: Our results suggest preliminary evidence that not all BRCA2-mutated PrC are equally sensitive to PARPis, and the response could depend on the PV location within FDs. The study of SPT in patients with no-HRR-associated PrC highlights the need to expand the genes in multigene panel testing, including MMR along with HRR genes, to capture unrecognized constitutional predisposition.

Abstract 5581: The DACH1 gene, frequently co-deleted with BRCA2 in prostate cancer, governs PARP inhibitor resistance

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities (1,2). The DACH1 gene, in the 13q21.31-q21.33 region, encodes a winged helix/Forkhead DNA-binding protein that governs cell-fate determination that is deleted in ~ 18% of human PCa, where it is associated with poor prognosis (3). Prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN). Methods: Analysis of DACH1 gene deletion and expression was conducted from public data bases and human prostate cancer samples. Cells derived from Dach1 gene deletion mice and human prostate cancer cell lines with DACH1 overexpression were analyzed for PARP inhibitor responses and mechanisms of PARPi resistance. Transgenic prostate Oncomice were generated in which the Dach1 gene was deleted in the prostate. Results: Herein, DACH1 was shown to be co-deleted with BRCA2 in each of 8 separate cohorts (N=2,186 patients), in ~3-12% of patients. Dach1 gene deletion or knockdown conferred PARP inhibitor resistance (talazoparib > niraparib > olaparib > rucaparib > veliparib). DACH1 enhanced PARP sensitivity through several mechanisms affecting replication stress, PARP1 binding, inhibiting G9a (reducing H3K9me2), inhibiting CHK1p and CDK1p and inducing expression of snoRNA, specifically the snoRNA known to bind PARP. PARP1 has a BRCT (BRCA1 C-terminus 1) homology domain governing PARP1 dimerization and binding to intact DNA in a complex within a nucleosome. DACH1 binding to PARP1 requires the BRCT domain. Conclusions: There is a correlation between DACH1a-/- and PARPi resistance, correlating with PARPi trapping ability. As reduced Dach1a expression may define a subclass of PCa that warrants specific therapies, testing for DACH1a may be warranted.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463–645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

A plant-specific module for homologous recombination repair

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

Exon splicing analysis of intronic variants in multigene cancer panel testing for hereditary breast/ovarian cancer.

The use of multigene panel testing for patients with a predisposition to breast/ovarian cancer is increasing as the identification of variants is useful for diagnosis and disease management. We identified pathogenic and likely pathogenic (P/LP) variants of high‐and moderate‐risk genes using a 23‐gene germline cancer panel in 518 patients with hereditary breast and ovarian cancers (HBOC). The frequency of P/LP variants was 12.4% (64/518) for high‐ and moderate‐penetrant genes, namely, BRCA2 (5.6%), BRCA1 (3.3%), CHEK2 (1.2%), MUTYH (0.8%), PALB2 (0.8%), MLH1 (0.4%), ATM (0.4%), BRIP1 (0.4%), TP53 (0.2%), and PMS2 (0.2%). Five patients possessed two P/LP variants in BRCA1/2 and other genes. We also compared the results from in silico splicing predictive tools and exon splicing patterns from patient samples by analyzing RT‐PCR product sequences in six P/LP intronic variants and two intronic variants of unknown significance (VUS). Altered transcriptional fragments were detected for P/LP intronic variants in BRCA1, BRIP1, CHEK2, PARB2, and PMS2. Notably, we identified an in‐frame deletion of the BRCA1 C‐terminal (BRCT) domain by exon skipping in BRCA1 c.5152+6T>C—as known VUS—indicating a risk for HBOC. Thus, exon splicing analysis can improve the identification of veiled intronic variants that would aid decision making and determination of hereditary cancer risk.

Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.

p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

Abstract B91: BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts

Abstract Tumor microenvironment plays an indispensable role in cancer development as it can be described as the territory where the interaction takes place between tumor and microenvironment elements such as the endothelial cells, epithelial cells, fibroblasts, immune cells, and ECM proteins. Among these structural stromal components, specifically cancer-associated fibroblasts (CAFs) have attracted substantial attention over recent decades. CAFs in the tumor microenvironment assist the accelerated growth demands of the cancer cells and enhance cancer progression through their paracrine activity by the enhanced secretion of growth factors and cytokines, which also help in remodeling the extracellular matrix (ECM). Studies reveal that CAFs have extended their role in cancer metastasis by accompanying the cancer cells to distant metastatic sites. From our in vitro studies, we have identified that coculturing the CAFs with the conditioned medium (CM) from BRCA1 mutated but not wild-type cancer cells results in the transformation of CAFs to an altered phenotype. Since these transformed CAFs showed increased migrative, invasive, and proliferative abilities, we named them “Metastasis Associated Fibroblasts” (MAF) (Sreelatha et al., 2018). The MAF thus generated showed a significant increase in the expression of the Ezrin, Radixin, and Moesin (ERM) protein complex, which may be attributed to the association of BRCA1 C-terminal (BRCT) domains of BRCA1 with the ERM protein superfamily. This association of BRCA1 with the key regulators of ERM complex can influence cell adhesion and motility, suggesting that BRCA1 may act upon their signaling pathways. To study the effect of BRCA1 mutation and ERM protein expression in CAFs in vivo, BRCA1 mutated breast cancer cells were orthotopically implanted to NOD-SCID mice. CAFs isolated from the tumor were analyzed for the expression of ERM. It was found that there is a significant increase in the expression of ERM protein in the tumors isolated from mice that were implanted with BRCA1 mutated breast cancer cells compared to the mouse group that received BRCA1 wild-type breast cancer cells. Inhibitors to ezrin and CCL5 could effectively reduce the metastatic potential of BRCA1 mutant cancer models. A combination of ezrin inhibitor (NSC 668394) and plumbagin, a plant-derived naphthoquinone that effectively reduces BRCA1 mutant breast cancer cell proliferation, is under investigation. Citation Format: Neetha Rajan Latha, Geetu Rose Varghese, Vishnu Sunil Jaikumar, Arathi Rajan, Revathy Nadhan, Arathy Warrier, Priya Srinivas. BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B91.

In silico analysis for determining the deleterious nonsynonymous single nucleotide polymorphisms of BRCA genes.

Recent advances in DNA sequencing techniques have led to an increase in the identification of single nucleotide polymorphisms (SNPs) in BRCA1 and BRCA2 genes, but no further information regarding the deleterious probability of many of them is available (Variants of Unknown Significance/VUS). As a result, in the current study, different sequence- and structure-based computational tools including SIFT, PolyPhen2, PANTHER, SNPs&GO, FATHMM, SNAP, PhD-SNP, Align-GVGD, and I-Mutant were utilized for determining how resulted BRCA protein is affected by corresponding missense mutations. FoldX was used to estimate mutational effects on the structural stability of BRCA proteins. Variants were considered extremely deleterious only when all tools predicted them to be deleterious. A total of 10 VUSs in BRCA1 (Cys39Ser, Cys64Gly, Phe861Cys, Arg1699Pro, Trp1718Cys, Phe1761Ser, Gly1788Asp, Val1804Gly, Trp1837Gly, and Trp1837Cys) and 12 in BRCA2 (Leu2510Pro, Asp2611Gly, Tyr2660Asp, Leu2686Pro, Leu2688Pro, Tyr2726Cys, Leu2792Pro, Gly2812Glu, Gly2813Glu, Arg2842Cys, Asp3073Gly, and Gly3076Val) were considered as extremely deleterious. Results suggested that deleterious variants were mostly enriched in the N- and C-terminal domain of the BRCA1 and BRCA2 C-terminus. Utilizing evolutionary conservation analysis, we demonstrated that the majority of deleterious SNPs ensue in highly conserved regions of BRCA genes. Furthermore, utilizing FoldX, we demonstrated that alterations in the function of proteins are not always together with stability alterations.

Abstract 1716: Evaluating the functional impacts of the BRCA1-mTORC2 interaction in breast cancer

Abstract Objective: The BRCA1 C-Terminal (BRCT) domain of BRCA1 has been found to interact with three accessory proteins (PRR5, RICTOR, and SIN1) of mTOR complex 2 (mTORC2). BRCA1 BRCT domain dissociates the members of the mTORC2 complex to inhibit the activity of this kinase and block activation of Akt. Most breast cancers exhibit hyperactivation of the PI3K/Akt/mTOR pathway, and we hypothesize that genetic and epigenetic loss of functional BRCA1 contributes this signaling activation event. Therefore, the goal of this project is to study the potential cancer impacts of BRCA1-deficient breast cancers and their response to mTOR inhibition. Methods: Yeast two-hybrid and orthogonal mammalian overexpression systems were used to confirm the interactions of PRR5, RICTOR, and SIN1 with the BRCT domain. MCF-10A breast cells were used to examine BRCA1-γH2AX foci formation under DNA damage conditions with cisplatin and mTOR inhibitors. Three-dimensional MCF-10A spheroids were used to study the effects of breast acini formation with shRNA-mediated repression BRCA1 and RICTOR. Breast cancer cell lines MCF-7, MDA-MB-231, and HCC1937 were used to study the effects of BRCA1 deficiency and response to pan-mTOR and pan-PI3K/mTOR inhibitors. Results: Wild type BRCA1 BRCT domain was found to have stronger interactions with PRR5, RICTOR, and SIN1 than with the M1775R mutated BRCA1 BRCT domain and empty vector control. MCF-10A breast cells treated with rapamycin, which hyperactivates mTORC2, exhibited significantly higher amounts of BRCA1-γH2AX foci formation in comparison to control and pan-mTOR PP242-treated cells. This was comparable to the effect of cisplatin-treated cells. BRCA1-RICTOR double KD rescued the effect of distorted breast cell acini formation displayed by BRCA1 loss that could lead to tumorigenesis. This suggests that inhibition of the mTORC2 complex can be an effective target, especially for cells that lack functional BRCA1. MCF-7 and MDA-MB-231 cells with BRCA1 knockdown were significantly more sensitive to pan-mTOR inhibition than non-targeting scramble control. HCC1937 cells, which have a truncated, non-functional BRCA1 BRCT domain, were more sensitive to pan-PI3K/mTOR inhibition that HCC1937 cells with restored full-length BRCA1. Significance: Breast cancer is the most commonly diagnosed cancer among women in the U.S. BRCA1 is the most characterized gene associated with breast cancer. Genetic alterations in BRCA1 appear in about 5-10% of breast cancer cases, and epigenetic loss of BRCA1 appears in about 10-15% of breast cancer cases. Also, about 70% of breast tumors have hyperactive PI3K/Akt/mTOR pathways. This would suggest that breast cancer is heavily reliant on this oncogenic pathway, and therefore BRCA1-deficient patients may benefit from pan-mTOR cancer therapeutics. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Tyler Ripperger, Nicholas T. Woods. Evaluating the functional impacts of the BRCA1-mTORC2 interaction in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1716.

Phosphatase 1 Nuclear Targeting Subunit Mediates Recruitment and Function of Poly (ADP-Ribose) Polymerase 1 in DNA Repair.

PARP, particularly PARP1, plays an essential role in the detection and repair of DNA single-strand breaks and double-strand breaks. PARP1 accumulates at DNA damage sites within seconds after DNA damage to catalyze the massive induction of substrate protein poly ADP-ribosylation (PARylation). However, the molecular mechanisms underlying the recruitment and activation of PARP1 in DNA repair are not fully understood. Here we show that phosphatase 1 nuclear targeting subunit 1 (PNUTS) is a robust binding partner of PARP1. Inhibition of PNUTS led to strong accumulation of endogenous DNA damage and sensitized the cellular response to a wide range of DNA-damaging agents, implicating PNUTS as an essential and multifaceted regulator of DNA repair. Recruitment of PNUTS to laser-induced DNA damage was similar to that of PARP1, and depletion or inhibition of PARP1 abrogated recruitment of PNUTS to sites of DNA damage. Conversely, PNUTS was required for efficient induction of substrate PARylation after DNA damage. PNUTS bound the BRCA1 C-terminal (BRCT) domain of PARP1 and was required for the recruitment of PARP1 to sites of DNA damage. Finally, depletion of PNUTS rendered cancer cells hypersensitive to PARP inhibition. Taken together, our study characterizes PNUTS as an essential partner of PARP1 in DNA repair and a potential drug target in cancer therapy. SIGNIFICANCE: These findings reveal PNUTS as an essential functional partner of PARP1 in DNA repair and suggest its inhibition as a potential therapeutic strategy in conjunction with DNA-damaging agents or PARP inhibitors.See related commentary by Murai and Pommier, p. 2460.

BRCT domains contain an intrinsic post‐translational modification (PTM) recognition code that affects it stability

BRCA1 C‐terminal (BRCT) domains are protein binding modules that relay signals throughout various cellular pathways. The human genome encodes 23 BRCT‐domain containing proteins that are commonly found in tandem and have been characterized as phosphoprotein binding domains with functions throughout the DNA damage response. Emerging evidence suggests these domains bind other post‐translationally modified (PTM) substrates although the details are not defined. Recently, the BRCT domains from BRCA1 were shown to bind an asymmetrically dimethylated arginine (ADMA) p300 substrate to regulate the transcription of the p21 in response to DNA damage. Based on this observation, we hypothesize that the methyl‐specific binding site for the ADMA interaction is distinct from the phospho‐substrate recognition interface. To test this hypothesis, we conducted peptide binding and thermal stability experiments to demonstrate the change in stability upon binding phospho‐versus methylated substrates. Our results show that phosphorylated substrates increase the stability of BRCA1's BRCT domains. Doubly phosphorylated substrates showed the greatest increase in stability while the methylated substrate demonstrated a decrease in stability. To define the BRCT‐ADMA binding interface, we set out to determine the three‐dimensional structure. High‐throughput co‐crystallization of the BRCA1 BRCT protein with a ADMA peptide derived from the p300 substrate identified one condition for crystal growth optimization. In silico modeling of the BRCA1 BRCT structure with the ADMA molecule has highlighted two potential binding sites for the ADMA substrate, which is not conserved throughout the BRCT domain family. Our results indicate that while BRCT domains share a conserved mode of binding phosphorylated substrates, which increases their stability, and their ability to binding methylated substrates may not be conserved. These finding will be critical in understanding how BRCT domains can accommodate binding various modified substrates and regulating signals throughout the DNA damage response.Support or Funding InformationNCI K22 CA166544‐03This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P3-09-02: CRISPR-Cas9 mediated BRCA1 mutation in primary cells: Mutation efficiency and effects

Abstract Background: Germline mutations in Breast Cancer Associated (BRCA) 1 or 2 genes confer an increased risk of the development of breast and ovarian cancer. Germline mutation is followed by somatic loss of heterozygosity (LOH) resulting in biallelic inactivation. BRCA1 is involved in multiple homeostatic functions including control of chromatin organization, gene transcription, protein stability and cell division. Recent studies have demonstrated heterogeneity in LOH within and between premalignant and malignant breast tissues of BRCA1 mutation carriers. We hypothesize that LOH does not have a unitary effect on phenotype but differs by the function that is abrogated. Methods: To test our hypothesis, we adopted CRISPR-Cas9 gene editing technology. The guide RNAs for targeting the exon sequence in the RING finger, nuclear export signal (NES), nuclear localization signal (NLS) and BRCA1 C Terminus (BRCT) domain/motif of BRCA1 were designed and synthesized. MCF10A cells were transfected with a complex of guide RNA and Cas9 protein (RNP) to cause in/del mutation. The mutation was analyzed by both T7E1 assay, and an innovative and more precise method developed in our lab that utilizes linked nucleic acids (LNA) and qPCR. Proliferation and apoptosis assays were performed using the transfected cells. Organoids prepared from BRCA1 mutation carriers also were transfected with RNPs and the mutation burden determined. Results: Since single cell clones of the transfected MCF10A cells could not be selected and expanded, a pool of transfected cells was used for the analyses. T7E1 assay and qPCR analysis using LNAs demonstrated the presence of the mutations. A standard curve was created to enable the calculation of the mutation burden. IncuCyte analysis revealed increased proliferation and apoptosis, induced by irradiation, in cells with the mutation in Exon 10, where the extent of increase varied from 11% to 48% depending on the degree of mutation. In contrast, cells with the mutation in Exon 5 displayed diminished proliferation with no change in apoptosis. That mutations in exon10 and 5 have distinct biological effects when compared to the mutations in other exons is intriguing, and modification of binding proteins will be investigated. Organoids generated from BRCA1 mutation carriers (primary and nonmalignant cells) were able to be successfully transfected using the NEON electroporation system. Mutations were introduced by the CRISPR-Cas9 system and their extent quantified by our LNA-mediated qPCR method. Conclusions: CRISPR-Cas9-mediated gene editing of BRCA1 in MCF10A resulted in a change in the proliferation rate and the extent of apoptosis that is dependent on the location of the de novo mutation within the gene. The development of a novel method, LNA-mediated qPCR, provides quantitative information regarding the mutations that may be used to correlate mutation burden with biological functional change. Successful establishment of this BRCA1 tumorigenesis model has provided us with a method to test other putative tumor suppressors. Citation Format: Choi MR, Yadav S, Shidfar A, Khan SA, Clare SE. CRISPR-Cas9 mediated BRCA1 mutation in primary cells: Mutation efficiency and effects [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-02.

Human DNA ligase IV is able to use NAD+ as an alternative adenylation donor for DNA ends ligation.

All the eukaryotic DNA ligases are known to use adenosine triphosphate (ATP) for DNA ligation. Here, we report that human DNA ligase IV, a key enzyme in DNA double-strand break (DSB) repair, is able to use NAD+ as a substrate for double-stranded DNA ligation. In the in vitro ligation assays, we show that the recombinant Ligase IV can use both ATP and NAD+ for DNA ligation. For NAD+-mediated ligation, the BRCA1 C-terminal (BRCT) domain of Ligase IV recognizes NAD+ and facilitates the adenylation of Ligase IV, the first step of ligation. Although XRCC4, the functional partner of Ligase IV, is not required for the NAD+-mediated adenylation, it regulates the transfer of AMP moiety from Ligase IV to the DNA end. Moreover, cancer-associated mutation in the BRCT domain of Ligase IV disrupts the interaction with NAD+, thus abolishes the NAD+-mediated adenylation of Ligase IV and DSB ligation. Disrupting the NAD+ recognition site in the BRCT domain impairs non-homologous end joining (NHEJ) in cell. Taken together, our study reveals that in addition to ATP, Ligase IV may use NAD+ as an alternative adenylation donor for NHEJ repair and maintaining genomic stability.

Trans-activation-based risk assessment of BRCA1 BRCT variants with unknown clinical significance

BackgroundDeleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants.ResultsTwelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3).ConclusionsThe modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.

Abstract 340: CTDP1 regulates FANCI activation and DNA repair

Abstract Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway. Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions. Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif. Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Dragana Lagundzin, Nicholas T. Woods. CTDP1 regulates FANCI activation and DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 340.

Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling

SummaryIntracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.