DNA repair and cell cycle checkpoint defects in a mouse model of ‘BRCAness’ are partially rescued by 53BP1 deletion

‘BRCAness’ is a term used to describe cancer cells that behave similarly to tumors with BRCA1 or BRCA2 mutations. The BRCAness phenotype is associated with hypersensitivity to chemotherapy agents including PARP inhibitors, which are a promising class of recently-licensed anti-cancer treatments. This hypersensitivity arises because of a deficiency in the homologous recombination (HR) pathway for DNA double-strand break repair. To gain further insight into how genetic modifiers of HR contribute to the BRCAness phenotype, we created a new mouse model of BRCAness by generating mice that are deficient in BLM helicase and the Exo1 exonuclease, which are involved in the early stages of HR. We find that cells lacking BLM and Exo1 exhibit a BRCAness phenotype, with diminished HR, and hypersensitivity to PARP inhibitors. We further tested how 53BP1, an important regulator of HR, affects repair efficiency in our BRCAness model. We find that deletion of 53BP1 can relieve several of the repair deficiencies observed in cells lacking BLM and Exo1, just as it does in cells lacking BRCA1. These results substantiate the importance of BRCAness as a concept for classification of cancer cases, and further clarify the role of 53BP1 in regulation of DNA repair pathway choice in mammalian cells.     

Base damage,local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.     

DNA repair,recombination, and damage signaling

DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.     

TP53 Mutational Status Is a Potential Marker for Risk Stratification in Wilms Tumour with Diffuse Anaplasia

Purpose

The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information.

Patients and Methods

We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling.

Results

From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes.

Conclusion

This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.     

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.     

Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation

Maintenance of genomic integrity in tissue-specific stem cells is critical for tissue homeostasis and the prevention of deleterious diseases such as cancer. Stem cells are subject to DNA damage induced by endogenous replication mishaps or exposure to exogenous agents. The type of DNA lesion and the cell cycle stage will invoke different DNA repair mechanisms depending on the intrinsic DNA repair machinery of a cell. Inappropriate DNA repair in stem cells can lead to cell death, or to the formation and accumulation of genetic alterations that can be transmitted to daughter cells and so is linked to cancer formation. DNA mutational signatures that are associated with DNA repair deficiencies or exposure to carcinogenic agents have been described in cancer. Here we review the most recent findings on DNA repair pathways activated in epithelial tissue stem and progenitor cells and their implications for cancer mutational signatures. We discuss how deep knowledge of early molecular events leading to carcinogenesis provides insights into DNA repair mechanisms operating in tumours and how these could be exploited therapeutically.     

Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.     

Clinical use and mechanisms of resistance for PARP inhibitors in homologous recombination-deficient cancers

Cells are continuously subjected to DNA damaging agents. DNA damages are repaired by one of the many pathways guarding genomic integrity. When one or several DNA damage pathways are rendered inefficient, cells can accumulate mutations, which modify normal cellular pathways, favoring abnormal cell growth. This supports malignant transformation, which can occur when cells acquire resistance to cell cycle checkpoints, apoptosis, or growth inhibition signals. Mutations in genes involved in the repair of DNA double strand breaks (DSBs), such as BRCA1, BRCA2, or PALB2, significantly increase the risk of developing cancer of the breast, ovaries, pancreas, or prostate. Fortunately, the inability of these tumors to repair DNA breaks makes them sensitive to genotoxic chemotherapies, allowing for the development of therapies precisely tailored to individuals’ genetic backgrounds. Unfortunately, as with many anti-cancer agents, drugs used to treat patients carrying a BRCA1 or BRCA2 mutation create a selective pressure, and over time tumors can become drug resistant. Here, we detail the cellular function of tumor suppressors essential in DNA damage repair pathways, present the mechanisms of action of inhibitors used to create synthetic lethality in BRCA carriers, and review the major molecular sources of drug resistance. Finally, we present examples of the many strategies being developed to circumvent drug resistance.     

Cockayne syndrome group B protein regulates fork restart,fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.     

A kinetoplastid BRCA2 interacts with DNA replication protein CDC45

The gene BRCA2, first identified as a breast cancer susceptibility locus in humans, encodes a protein involved in DNA repair in mammalian cells and mutations in this gene confer increased risk of breast cancer. Here we report a functional characterisation of a Trypanosoma brucei BRCA2 (TbBRCA2) orthologue and show that the protein interacts directly with TbRAD51. A further protein-protein interaction screen using TbBRCA2 identified other interacting proteins, including a trypanosome orthologue of CDC45 which is involved in initiation and progression of the replication fork complex during DNA synthesis. Deletion of the TbBRCA2 gene retards cell cycle progression during S-phase as judged by increased incorporation of BrdU and an increased percentage of cells with one nucleus and two kinetoplasts. These results provide insights into the potential role played by BRCA2 in DNA replication and reveal a novel interaction that couples replication and recombination in maintaining integrity of the genome.     

Mutation Analysis of BRCA1, BRCA2, PALB2 and BRD7 in a Hospital-Based Series of German Patients with Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive form of breast carcinoma with a poor prognosis. Recent evidence suggests that some patients with TNBC harbour germ-line mutations in DNA repair genes which may render their tumours susceptible to novel therapies such as treatment with PARP inhibitors. In the present study, we have investigated a hospital-based series of 40 German patients with TNBC for the presence of germ-line mutations in BRCA1, BRCA2, PALB2, and BRD7 genes. Microfluidic array PCR and next-generation sequencing was used for BRCA1 and BRCA2 analysis while conventional high-resolution melting and Sanger sequencing was applied to study the coding regions of PALB2 and BRD7, respectively. Truncating mutations in BRCA1 were found in six patients, and truncating mutations in BRCA2 and PALB2 were detected in one patient each, whereas no truncating mutation was identified in BRD7. One patient was a double heterozygote for the PALB2 mutation, c.758insT, and a BRCA1 mutation, c.927delA. Our results confirm in a hospital-based setting that a substantial proportion of German TNBC patients (17.5%) harbour germ-line mutations in genes involved in homology-directed DNA repair, with a preponderance of BRCA1 mutations. Triple-negative breast cancer should be considered as an additional criterion for future genetic counselling and diagnostic sequencing.     

DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2

Germline mutations affecting a single allele of BRCA2 increase susceptibility to breast and ovarian cancer, whilst germline inheritance of certain bi-allelic mutations causes a Fanconi anaemia-like syndrome. Here, we review current knowledge of the BRCA2 protein, focussing on recent studies that provide mechanistic insight into its biological function in regulating DNA recombination reactions mediated by the RAD51 recombinase. We argue that the chromosomal instability and cancer predisposition provoked by BRCA2 inactivation are a consequence of the failure to re-start stalled DNA replication, and to repair DNA double-strand breaks, through error-free pathways that depend on homologous pairing between DNA strands.     

RECQL: a new breast cancer susceptibility gene

Identifying and characterizing novel genetic risk factors for BRCA1/2 negative breast cancers is highly relevant for early diagnosis and development of a management plan. Mutations in a number of DNA repair genes have been associated with genomic instability and development of breast and various other cancers. Whole exome sequencing efforts by 2 groups have led to the discovery in distinct populations of multiple breast cancer susceptibility mutations in RECQL, a gene that encodes a DNA helicase involved in homologous recombination repair and response to replication stress. RECQL pathogenic mutations were identified that truncated or disrupted the RECQL protein or introduced missense mutations in its helicase domain. RECQL mutations may serve as a useful biomarker for breast cancer. Targeting RECQL associated tumors with novel DNA repair inhibitors may provide a new strategy for anti-cancer therapy.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

BRCAness profile of sporadic ovarian cancer predicts disease recurrence

Background

The consequences of defective homologous recombination (HR) are not understood in sporadic ovarian cancer, nor have the potential role of HR proteins other than BRCA1 and BRCA2 been clearly defined. However, it is clear that defects in HR and other DNA repair pathways are important to the effectiveness of current therapies. We hypothesize that a subset of sporadic ovarian carcinomas may harbor anomalies in HR pathways, and that a BRCAness profile (defects in HR or other DNA repair pathways) could influence response rate and survival after treatment with platinum drugs. Clinical availability of a BRCAness profile in patients and/or tumors should improve treatment outcomes.

Objective

To define the BRCAness profile of sporadic ovarian carcinoma and determine whether BRCA1, PARP, FANCD2, PTEN, H2AX, ATM, and P53 protein expression correlates with response to treatment, disease recurrence, and recurrence-free survival.

Materials and Methods

Protein microarray analysis of ovarian cancer tissue was used to determine protein expression levels for defined DNA repair proteins. Correlation with clinical and pathologic parameters in 186 patients with advanced stage III–IV and grade 3 ovarian cancer was analyzed using Chi square, Kaplan-Meier method, Cox proportional hazard model, and cumulative incidence function.

Results

High PARP, FANCD2 and BRCA1 expressions were significantly correlated with each other; however, elevated p53 expression was associated only with high PARP and FANCD2. Of all patients, 9% recurred within the first year. Among early recurring patients, 41% had high levels of PARP, FANCD2 and P53, compared to 19.5% of patients without early recurrence (p = 0.04). Women with high levels of PARP, FANCD2 and/or P53 had first year cumulative cancer incidence of 17% compared with 7% for the other groups (P = 0.03).

Conclusions

Patients with concomitantly high levels of PARP, FANCD2 and P53 protein expression are at increased risk of early ovarian cancer recurrence and platinum resistance.