Double-strand Break Repair Genes Research Articles

DNA double-strand break repair gene mutation and the risk of papillary thyroid microcarcinoma: a case\u2013control study

ObjectiveTo study the relationship between DNA double-strand break (DSB) repair gene mutations and the risk of papillary thyroid microcarcinoma (PTMC).MethodsOne hundred patients with PTMC or benign thyroid nodules (BTNs) at Henan Cancer Hospital were retrospectively analyzed. The DSB repair capacity of peripheral blood T lymphocytes in the two groups was assessed by flow cytometry. Data were compared using Student’s t-test to evaluate the relationship between DSB repair capacity and the risk of PTMC. Factors influencing DSB repair capacity were analyzed by multivariate logistic regression analysis. The relationship between PTMC and DSB repair capacity was analyzed by univariate analysis. Targeted next-generation DNA sequencing was applied to screen and analyze DSB repair genes related to PTMC.ResultsThe DSB repair capacity was 31.30% in the PTMC group and 44.40% in the BTN group, with that of the former being significantly lower (P < 0.05). Multivariate logistic regression analysis of age, sex, obesity status, radiation and other factors showed that radiation exposure was positively correlated with reduced DSB repair capacity(OR = 3.642; 95% CI 1.484–8.935, P = 0.020). Moreover, univariate analysis showed that a reduction in DSB repair capacity was a risk factor for PTMC(OR = 2.333; 95% CI 1.027–5.300, P = 0.043).Targeted next-generation DNA sequencing was performed on the DSB repair genes discovered, and those that were mutated in association with PTMC were Rad50 and FANCA; Rad51 mutations were related to BTN.ConclusionRadiation exposure is positively associated with induced DSB repair gene mutations, which may cause a reduced capacity for DSB repair and eventually lead to PTMC.

BRCA2 3'-UTR Polymorphism rs15869 Alters Susceptibility to Papillary Thyroid Carcinoma via Binding hsa-mir-1178-3p.

ObjectiveTo investigate the associations of polymorphisms in the following DNA double-strand break repair (DSBR) genes with papillary thyroid carcinoma (PTC) risk (including RAD51 rs11852786, RAD51B rs963917, BRCA1 rs12516 and rs8176318, BRCA2 rs15869, XRCC4 rs2035990 and XRCC5 rs2440).Materials and MethodsA matched case–control study was implemented to examine associations between PTC risk and the above polymorphisms. Subsequently, we evaluated the effects of the potential PTC susceptibility-related variant rs15869 on BRCA2 mRNA secondary structure and BRCA2 expression through bioinformatics analysis and experiment validation. Additionally, luciferase assay was used to identify whether rs15869 polymorphism can substantially affect the binding of hsa-miR-1178-3p to BRCA2 mRNA. Finally, Pearson correlation analysis was performed to determine the correlation between the expression of hsa-miR-1178-3p and BRCA2 mRNA and protein in thyroid tissues harboring rs15869 different genotypes.ResultsBRCA2 rs15869 CC genotype was associated with a higher risk of PTC than its AA genotype. Subsequently, stratified analyses came to the same conclusion in the female or age<50 population. Furthermore, we confirmed that the A-to-C substitution of rs15869 changed BRCA2 mRNA secondary structure and contributed to a decreased BRCA2 expression. Mechanistically, a significantly decreased luciferase activity verified a greater binding between hsa-miR-1178-3p and rs15869 C allele, but not the A allele, which was evidenced by the significant negative correlation between hsa-miR-1178-3p with BRCA2 mRNA and protein levels in thyroid tissues with AC and CC genotype but not AA genotype at rs15869.ConclusionBRCA2 rs15869 is characterized as a potential biomarker associated with PTC risk, highlighting the contribution of the hsa-miR-1178-3p via functional exploration.

Haploinsufficiency in non-homologous end joining factor 1 induces ovarian dysfunction in humans and mice

BackgroundPremature ovarian insufficiency (POI) is a common disease in women that leads to a reduced reproductive lifespan. The aetiology of POI is genetically heterogeneous, with certain double-strand break (DSB) repair...

Trans-resveratrol imparts disparate effects on transcription of DNA damage sensing/repair pathway genes in euglycemic and hyperglycemic rat testis

Prevention or repair of DNA damage is critical to inhibit carcinogenesis in living organisms. Using quantitative RT2 Profiler™ PCR array, we investigated if trans-resveratrol could modulate the transcription of DNA damage sensing/repair pathway genes in euglycemic and non-obese type 2 diabetic Goto-Kakizaki rat testis. Trans-resveratrol imparted disparate effects on gene expressions. In euglycemic rats, it downregulated 79% and upregulated 2% of genes. However, in diabetic rats, it upregulated only 2% and downregulated 4% of genes. As such, diabetes upregulated 16% and downregulated 4% of genes. Trans-resveratrol normalized the expression of 9 (60%) out of 15 upregulated genes in diabetic rats. In euglycemic rats, trans-resveratrol inhibited ATM/ATR, DNA damage repair, pro-cell cycle progression, and apoptosis signaling genes. However, it increased Cdkn1a and Sumo1, indicating cell cycle arrest, apoptosis, and cytostasis in conjunction with increased DNA double-strand breaks and apoptosis. Diabetes increased DNA damage and apoptosis but did not affect ATM/ATR and double-strand break repair genes, although it increased few single-strand repair genes. Diabetes increased Abl1 and Sirt1, which may be related to apoptosis, but their increase may well suggest the enhanced cell cycle progression and putative carcinogenicity. The transcription of Rad17 and Smc1a increased in diabetic rats indicating G2 phase arrest and increases in a few DNA single-strand breaks repair genes suggesting DNA damage repair. Trans-resveratrol inhibits the cell cycle and causes cell death in euglycemic rat testis but normalizes diabetes-induced genes related to DNA damage and cell cycle control, suggesting its usefulness in maintaining DNA integrity in diabetes.

The DNA damage response network in the treatment of head and neck squamous cell carcinoma

BackgroundWe sought to determine whether DNA damage response (DDR)-related aberrations predict therapeutic benefit in cisplatin-treated head and neck squamous cell carcinoma (HNSCC) patients and how DDR pathways are modulated after treatment with olaparib alone or in combination with cisplatin or durvalumab.Patients and methodsOxidative stress, abasic sites and DDR-related parameters, including endogenous DNA damage, DNA repair mechanisms and apoptosis rates, were evaluated in HNSCC cell lines and peripheral blood mononuclear cells from 46 healthy controls (HC) and 70 HNSCC patients at baseline and following treatment with cisplatin-containing chemoradiation or nivolumab or enrolled in the OPHELIA phase II trial (NCT02882308; olaparib alone, olaparib plus cisplatin, olaparib plus durvalumab).ResultsHNSCC patients at diagnosis exhibited deregulated DDR-related parameters and higher levels of oxidative stress and abasic sites compared with HC (all P < 0.05). Accordingly, nucleotide excision repair (NER; ERCC1, ERCC2/XPD, XPA, XPC) and base excision repair (APEX1, XRCC1) genes were downregulated in patients versus HC whereas double-strand breaks repair (MRE11A, RAD50, RAD51, XRCC2) and mismatch repair (MLH1, MSH2, MSH3) genes were overexpressed. Corresponding results were obtained in cell lines (all P < 0.001). Excellent correlations were observed between individual ex vivo and in vivo/therapeutic results, with cisplatin non-responders showing higher levels of endogenous DNA damage, augmented oxidative stress and abasic sites, increased NER capacities and reduced apoptosis than responders (all P < 0.05). Also, longer progression-free survival correlated with lower NER capacity (P = 0.037) and increased apoptosis (P = 0.029). Interestingly, treatment with olaparib-containing regimens results in the accumulation of cytotoxic DNA damage and exerts an extra antitumor effect by elevating oxidative stress (all P < 0.05). Nivolumab induced no significant changes in the DDR parameters examined.ConclusionsAberrations in DDR signals are implicated in the response to HNSCC chemotherapy and can be exploited as novel therapeutic targets, sensitive/effective non-invasive biomarkers as well as for the design of novel clinical trials.

Radio-adaptive response and correlation of non-homologous end joining repair gene polymorphisms [XRRC5 (3R/2R/1R/0R), XRCC6(C/G) and XRCC7 (G/T)] in human peripheral blood mononuclear cells exposed to gamma radiation

BackgroundRadio-adaptive response (RAR) is transient phenomena, where cells conditioned with a small dose (priming) of ionizing radiation shows significantly reduced DNA damage with a subsequent high challenging dose. The role of DNA double strand break repair gene polymorphism in RAR is not known. In the present study attempt was made to find out the influence of NHEJ repair gene polymorphisms [a VNTR; XRCC5 (3R/2R/1R/0R); two single nucleotide polymorphisms (SNPs); XRCC6 (C/G) and XRCC7 (G/T)] with DNA damage, repair and mRNA expression in human PBMCs in dose and adaptive response studies. Genomic DNA extracted from venous blood samples of 20 random healthy donors (16 adaptive and 4 non-adaptive) and genotyping of NHEJ repair genes was carried out using PCR amplified length polymorphism.ResultsThe dose response study revealed significant positive correlation of genotypes at XRRC5 (3R/2R/1R/0R), XRCC6(C/G) and XRCC7 (G/T) with DNA damage. Donors having genotypes with 2R allele at XRCC5 showed significant positive correlation with mRNA expression level (0R/2R: r = 0.846, P = 0.034; 1R/2R: r = 0.698, P = 0.0001 and 2R/2R: r = 0.831, P = 0.0001) for dose response. Genotypes C/C and C/G of XRCC6 showed a significant positive correlation (P = 0.0001), whereas, genotype T/T of XRCC7 showed significant negative correlation (r = − 0.376, P = 0.041) with mRNA expression.ConclusionInterestingly, adaptive donors having C/G genotype of XRCC6 showed significantly higher (P < 0.05) mRNA expression level in primed cells suggesting their role in RAR. In addition, NHEJ repair gene polymorphisms play crucial role with radio-sensitivity and RAR in human PBMCs.

Association of XRCC2 rs2040639 with the survival of patients with oral squamous cell carcinoma undergoing concurrent chemoradiotherapy.

To investigate the association between the variants of DNA double-strand break repair genes and the clinical outcomes of patients with oral squamous cell carcinoma (OSCC) undergoing concurrent chemoradiotherapy. Five variants of DNA double-strand break repair genes in samples from 319 patients with OSCC were genotyped using the Sequenom iPLEX MassARRAY system. Kaplan-Meier curves and Cox proportional hazards analysis were used to identify the factors associated with patient survival. The XRCC2 rs2040639 (G3063A) polymorphism in the codominant model was associated with decreased recurrence risk (hazard ratio [HR]=0.55, 95% confidence interval [CI]=0.31-0.98; p=0.042). A marginally significant interaction was observed between XRCC2 rs2040639 and PRKDC rs7003908 in patients carrying the AA and AA genotypes; these patients showed reduced recurrence risk (HR=0.36, 95% CI=0.17-0.79; p=0.010). The A-allele of XRCC2 rs2040639 is a favorable prognostic factor for disease-free survival. Patients with these genotypes may benefit from concurrent chemoradiotherapy. Additional confirmation from studies with larger samples or other ethnic populations is warranted.

LncRNA SLC26A4-AS1 suppresses the MRN complex-mediated DNA repair signaling and thyroid cancer metastasis by destabilizing DDX5.

Lymph node metastasis is the major adverse feature for recurrence and death of thyroid cancer patients. To identify lncRNAs involved in thyroid cancer metastasis, we systemically screened differentially expressed lncRNAs in lymph node metastasis, thyroid cancer, and normal tissues via RNAseq. We found that lncRNA SLC26A4-AS1 was continuously, significantly down-regulated in normal tissues, thyroid cancer, and lymph node metastasis specimens. Low SLC26A4-AS1 levels in tissues were significantly associated with poor prognosis of thyroid cancer patients. LncRNA SLC26A4-AS1 markedly inhibited migration, invasion, and metastasis capability of cancer cells in vitro and in vivo. Intriguingly, SLC26A4-AS1 could simultaneously interact with DDX5 and the E3 ligase TRIM25, which promoting DDX5 degradation through the ubiquitin-proteasome pathway. In particular, SLC26A4-AS1 inhibited expression of multiple DNA double-strand breaks (DSBs) repair genes, especially genes coding proteins in the MRE11/RAS50/NBS1 (MRN) complex. Enhanced interaction between DDX5 and transcriptional factor E2F1 due to silencing of SLC26A4-AS1 promoted binding of the DDX5-E2F1 complex at promoters of the MRN genes and, thus, stimulate the MRN/ATM dependent DSB signaling and thyroid cancer metastasis. Our study uncovered new insights into the biology driving thyroid cancer metastasis and highlights potentials of lncRNAs as future therapeutic targets again cancer metastasis.

Genome-wide identification of DNA double-strand break repair genes and transcriptional modulation in response to benzo[α]pyrene in the monogonont rotifer Brachionus spp.

The DNA repair system has evolved from the common ancestor of all life forms and its function is highly conserved within eukaryotes. In this study, to reveal the role of DNA double-strand break repair (DSB) genes in response to benzo[α]pyrene (B[α]P), we first identified DSB genes in relation to homologous recombination and non-homologous end joining events in four Brachionus rotifer spp.: B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis. In all the Brachionus spp., 39 orthologous genes to human DSB repair genes were identified. Furthermore, three genes in B. koreanus, two genes in B. plicatilis, and one gene in B. calyciflorus and B. rotundiformis were present as duplicated genes, indicating that these genes were diversified over speciation in the genus Brachionus. Moreover, we compared DSB repair genes on the gene structures in four monogonont Brachionus rotifers and the bdelloid rotifer Adineta vaga, which possesses highly efficient DNA repair ability. The transcriptional responses of four monogonont Brachionus rotifers in response to B[α]P exposure showed how B[α]P exposure led to DSBs and subsequently recruited DNA DSB repair pathways in the rotifer B. koreanus. Taken together, this study provides a better understanding of the potential role of DSB repair genes in the monogonont rotifer Brachionus spp. in response to B[α]P.

Mutants in mitochondrial DNA repair genes in Arabidopsis thaliana generated by using CRISPR‐Cas9

The genomes of plant mitochondria exhibit unique mutation rates and structural variability. Compared to their animal counterparts, plant mitochondria have low mutation rates, high recombination rates, and non‐circular genomes. Recent evidence suggests the prevalence of double‐strand break repair (DSBR) in plant mitochondria as one possible cause of these traits. Interestingly, all of the genes required for plant mitochondrial DSBR are located in the nucleus.In order to study DSBR, it is necessary to be able to reliably generate single and double‐mutants for DSBR genes. Using traditional methods, such as T‐DNA insertions, double mutant generation has proven cumbersome due to the occasional difficulty in PCR‐based identification of single mutants, as well as the low map distance between several genes of interest. CRISPR‐induced knockouts allow us to overcome some of these difficulties and generate a greater variety of mutants in a shorter period of time. In this study, we selected five proteins related to the processing of double strand breaks (DSBs) in plant mitochondria for mutagenesis: MSH1, RECA3, RNaseH1, SUV3, and OTP87. We prepared CRISPR Cas9 vectors to produce knockouts for each of these genes in Arabidopsis thaliana. The reliable production of mutants for these 5 genes will allow for in depth study of DSBR in the future. CRISPR Cas9 constructs were designed to produce two guide RNAs for each gene, targeting two different exons in the gene of interest, to give a better chance of successful knockouts, or deletions, between the two sites. These constructs were then transformed into Agrobacterium. Successful transformants were selected using a spectinomycin resistance gene in the construct. We then used a standard floral dip protocol to generate mutant Arabidopsis plants. PCR analysis confirmed Arabidopsis knockout mutations in all five lines.The mutants generated in this study will allow us to investigate several avenues of research on DSBR. We plan to expose plants with knockouts in either one or two DSBR genes to ciprofloxacin, an antibiotic which induces double strand breaks in plant mitochondrial DNA. From there we can observe the mitochondrial genome recombination rates using PCR and Illumina sequencing for each of the mutants.Support or Funding InformationThis work was supported in part by grants from the National Science Foundation to A.C.C. (MCB‐1413152 and MCB‐1933590) and a grant from the UNL UCARE program (26‐0101‐8001‐001)

PRMT5 Cooperates with pICln to Function as a Master Epigenetic Activator of DNA Double-Strand Break Repair Genes.

SummaryDNA double-strand break (DSB) repair is critical for cell survival and genome integrity. Upon recognition of DSBs, repair proteins are transiently upregulated to facilitate repair through homologous recombination (HR) or non-homologous end joining (NHEJ). We present evidence that PRMT5 cooperates with pICln to function as a master epigenetic activator of DNA damage response (DDR) genes involved in HR, NHEJ, and G2 arrest (including RAD51, BRCA1, and BRCA2) to upregulate gene expression upon DNA damage. Contrary to the predominant role of PRMT5 as an epigenetic repressor, our results demonstrate that PRMT5 and pICln can activate gene expression, potentially independent of PRMT5's obligate cofactor MEP50. Targeting PRMT5 or pICln hinders repair of DSBs in multiple cancer cell lines, and both PRMT5 and pICln expression positively correlates with DDR genes across 32 clinical cancer datasets. Thus, targeting PRMT5 or pICln may be explored in combination with radiation or chemotherapy for cancer treatment.

XRCC1 632 as a candidate for cancer predisposition via a complex interaction with genetic variants of base excision repair and double strand break repair genes.

Aim: The DNA repair system safeguards integrity of DNA. Genetic alterations force the improper repair which in conjugation with other factors ultimately results in carcinogenesis. Materials & methods: PCR-restriction fragment length polymorphismwas used for genotyping, which was followed by statistical analysis using logistic regression analysis, multifactor dimensionality reduction and classification and regression analysis tree, elaborating the association with lung cancer subjects. Results: Combination of XRCC1 632 and OGG1326 showcased a high risk of eightfold (odds ratio: 7.92;95% CI: 2.68-23.4; p=0.0002; false discovery rate (FDR)p=0.002). Similarly, XRCC1 632 and MUTYH 324 (odds ratio: 5.07; 95% CI: 2.6-9.67; p<0.0001; FDRp=0.002) had a high risk. Multifactor dimensionality reduction analysis revealed five factor model as the best model with prediction error of 0.37 (p=0.02). Conclusion: There was a clear indication that high order interactions were major role players in the study.

Functional deficiency of DNA repair gene EXO5 results in androgen-induced genomic instability and prostate tumorigenesis.

Germline mutations of DNA double-strand break (DSB) response and repair genes that drive tumorigenesis could be a major cause of prostate cancer (PCa) heritability. In this study, we demonstrated the role of novel exonuclease 5 (EXO5) gene in androgen-induced double strand breaks repair via homology-directed repair pathway and prostate tumorigenesis. Using whole-exome sequencing of samples from 20 PCa families, with three or more siblings diagnosed with metastatic PCa, we identified mutations in 31 genes involved in DSB response and repair. Among them, the L151P mutation in the exonuclease 5 (EXO5) gene was present in all affected siblings in three PCa families. We found two other EXO5 SNPs significantly associated with risk of PCa in cases-controls study from databases of genotype and phenotype (dbGaP), which are in linkage disequilibrium (D' = 1) with Exo5 L151P found in PCa family. The L151 residue is conserved across diverse species and its mutation is deleterious for protein functions, as demonstrated by our bioinformatics analyses. The L151P mutation impairs the DNA repair function of EXO5 due to loss of nuclease activity, as well as failure of nuclear localization. CRISPR elimination of EXO5 in a PCa cell line impaired homology-directed recombination repair (HDR) and caused androgen-induced genomic instability, as indicated by frequent occurrence of the oncogenic fusion transcript TMPRSS2-ERG. Genetic and functional validation of the EXO5 mutations indicated that EXO5 is a risk gene for prostate tumorigenesis, likely due to its functions in HDR.

Chromate exposure induces DNA hypermethylation of the mismatch repair gene MLH1 in lung cancer.

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.

Racial Disparity in the Genomic Basis of Radiosensitivity – An Exploration of Whole-Transcriptome Sequencing Data via a Machine-Learning Approach

There is emerging evidence for the existence of intrinsic biological differences in cancer responsiveness to therapeutic radiation between African-American (AA) men compared to non-Hispanic White (NHW) men (Spratt et al. [abstract ASTRO 2018]). The basis of this racial difference in radiosensitivity is likely based on variations in the expression of genes encoding for radiation response pathways. For example, decreased double strand break repair gene expression is associated increased radiosensitivity in somatic and cancer cell lines. We hypothesized that a racial difference in the expression levels of genes participating in radiation response pathways could be identified via a machine learning approach. We extracted the gene expression level data of 7,470 patients from the Genomic Data Commons Pan-Cancer database who had race identified as AA (n=802) or NHW (n=6,668). For each patient, the expression levels of 741 genes that are known to be involved in radiation response pathways were selected for subsequent analysis. An ensemble of five machine learning methods (support vector machine (SVM), linear discriminant analysis (LDA), gradient boosted machine (GBM), Bayesian generalized linear model (BGLM), and sample mean (SM)) was trained on 80% of the data to predict for race based on this 741-gene expression panel. Out-of-sample error was estimated using 5-fold cross validation. The trained ensemble model was used to predict on the remaining 20% of the data. Performance of the ensemble model was evaluated via area under the curve (AUC) of the receiver operating characteristic curve. The mean squared error for the SVM, LDA, GBM, BGLM, and SM methods were 0.071, 0.075, 0.081, 0.076, and 0.096 respectively. The ensemble model achieved a mean square error of 0.068. Prediction by the ensemble model yielded an AUC of 0.861 (95% CI 0.844-0.878). Expression levels of radiation response pathway genes can be used to accurately identify race via an ensemble of machine learning models. This supports the emerging evidence that race may be associated with radiosensitivity via intrinsic biologic differences in gene expression levels. Further studies are warranted to investigate whether these gene expression differences translate to clinically detectable variation in radiosensitivity and tumor control among different patient populations.

Enhanced cytotoxic and genotoxic effects of gadolinium-doped ZnO nanoparticles on irradiated lung cancer cells at megavoltage radiation energies

The purpose of this study was to investigate the radiation dose enhancement effects of gadolinium-doped zinc oxide nanoparticles (Gd-doped ZnO NPs) under the megavoltage (MV) X-ray irradiation. ZnO NPs have preferred photocatalytic properties under UV light for cancer killing. UV light has limited applications in cancer treatment and it is necessary to use X-ray photons with MV energies. In order to increase the absorption of radiation and also to enhance the imaging visualization capabilities of ZnO NPs, gadolinium (Gd) as a high atomic number element was selected for doping into the structure of ZnO NPs. Gd-doped ZnO NPs were synthesized by a chemical precipitation method and characterized by transmission electron microscopy, powder X-ray diffraction, ultraviolet-visible spectroscopy, and energy-dispersive X-ray techniques. Cellular uptake was assessed by TEM and inductively coupled plasma mass spectrometry. NPs cytotoxicity was analyzed by MTT assay and radiation dose enhancement was measured by clonogenic survival assay. Apoptosis induction, cell cycle progression, micronucleus formation and expression of DNA double-strand break repair genes of XRCC2 and XRCC4 were determined by flow cytometry, micronucleus assay, and quantitative real-time polymerase chain reaction. CT and MR imaging were used to analyze the image visualization capabilities of NPs. NPs characterization showed that highly pure crystalline Gd-doped ZnO NPs with a narrow size distribution and grain size of 9 nm were synthesized. Gd-doped ZnO NPs were distributed in the cells and showed dose-dependent toxicity. Combination of Gd-doped ZnO NPs with 6 MV X-rays induced dose-dependent radiosensitivity with sensitizer enhancement ratios (SER) of 1.47 and 1.61 for 10 and 20 μg/mL NPs concentrations. Cancer cells blocked in G1, apoptosis rates, and micronuclei formation was enhanced and inversely, the DNA repair efficiency was impaired by down regulation of the mRNA levels of XRCC2 and XRCC4 genes. Gd-doped ZnO NPs enhanced the contrasts of CT and MR images of cancer cells. Overall, the results of this study provide detailed biological insights on the dose enhancement of Gd-doped ZnO NPs at MV radiations, which would contribute to the further development of this potent theranostic platform for clinical applications.

A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.

The Ethnic-Specific Spectrum of Germline Nucleotide Variants in DNA Damage Response and Repair Genes in Hereditary Breast and Ovarian Cancer Patients of Tatar Descent.

The Russian population consists of more than 100 ethnic groups, presenting a unique opportunity for the identification of hereditary pathogenic mutations. To gain insight into the landscape of heredity pathogenic variants, we employed targeted next-generation sequencing to analyze the germline mutation load in the DNA damage response and repair genes of hereditary breast and ovary cancer syndrome (HBOCS) patients of Tatar ethnicity, which represents ~4% of the total Russian population. Several pathogenic mutations were identified in DNA double-strand break repair genes, and the spectrum of these markers in Tatar patients varied from that previously reported for patients of Slavic ancestry. The CDK12 gene encodes cyclin-dependent kinase 12, the key transcriptional regulator of the genes involved in DNA damage response and repair. CDK12 analysis in a cohort of HBOCS patients of Tatar decent identified a c.1047-2A>G nucleotide variant in the CDK12 gene in 8 of the 106 cases (7.6%). The c.1047-2A>G nucleotide variant was identified in 1 of the 93 (1.1%) HBOCS patients with mixed or unknown ethnicity and in 1 of the 238 (0.42%) healthy control patients of mixed ethnicity (Tatars and non-Tatars) (p = 0.0066, OR = 11.18, CI 95% = 1.53–492.95, Tatar and non-Tatar patients vs. healthy controls). In a group of mixed ethnicity patients from Tatarstan, with sporadic breast and/or ovarian cancer, this nucleotide variant was detected in 2 out of 93 (2.2%) cases. In a cohort of participants of Slavic descent from Moscow, comprising of 95 HBOCS patients, 80 patients with sporadic breast and/or ovarian cancer, and 372 healthy controls, this nucleotide variant was absent. Our study demonstrates a strong predisposition for the CDK12 c.1047-2A>G nucleotide variant in HBOCS in patients of Tatar ethnicity and identifies CDK12 as a novel gene involved in HBOCS susceptibility.

RAD50 germline mutations are associated with poor survival in BRCA1/2-negative breast cancer patients.

RAD50 is a highly conserved DNA double-strand break (DSB) repair gene. However, the associations between RAD50 germline mutations and the survival and risk of breast cancer have not been fully elucidated. Here, we aimed to investigate the clinical impact of RAD50 germline mutations in a large cohort of unselected breast cancer patients. In our study, RAD50 germline mutations were determined using next-generation sequencing in 7657 consecutive unselected breast cancer patients without BRCA1/2 mutations. We also screened for RAD50 recurrent mutations (L719fs, K994fs, and H1269fs) in 5000 healthy controls using Sanger sequencing. We found that 26 out of 7,657 (0.34%) patients had RAD50 pathogenic mutations, and 16 patients carried one of the three recurrent mutations (L719fs, n=6 cases; K994fs, n=5 cases; and H1269fs, n=5 cases); the recurrent mutation rate was 0.21%. The frequency of the three recurrent mutations in the 5,000 healthy controls was 0.18% (9/5,000). These mutations did not confer an increased risk of breast cancer in the studied patients [odds ratios (OR), 1.16; 95% confidence interval (CI), 0.51-2.63; p=0.72]. Nevertheless, multivariate analysis revealed that RAD50 pathogenic mutations were an independent unfavourable predictor of recurrence-free survival (RFS) [adjusted hazard ratio (HR) 2.66; 95% CI, 1.18-5.98; p=0.018] and disease-specific survival (DSS; adjusted HR 4.36; 95% CI, 1.58-12.03; p=0.004) in the entire study cohort. Our study suggested that RAD50 germline mutations are not associated with an increased risk of breast cancer, but patients with RAD50 germline mutations have unfavourable survival compared to patients without these mutations.

Abstract 482: Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalence in southern China and Southeast Asia. It is well-known to be associated with latent Epstein-Barr virus (EBV) infection. By using small RNA sequencing, we previously established a comprehensive miRNA profile in a panel of NPC samples and revealed that the EBV-encoded miRNAs derived from BamHI-A rightward transcripts (miR-BARTs) are abundantly expressed. The important roles of miR-BARTs in cancer development have been reported extensively. In the present study, we identified multiple putative binding sites of miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p on the 3'-UTR of a critical DNA double-strand break (DSB) repair gene, Ataxia-Telangiectasia-Mutant (ATM). Notably, the expression of these 4 miR-BARTs represented more than 10% of all EBV-encoded miRNAs in NPC cells while downregulation of ATM expression was observed in our local primary NPC samples in both qRT-PCR and immunohistochemical (IHC) staining analysis. In addition, the abilities of miR-BARTs to downregulate ATM expression were demonstrated in the transient transfection experiments. By manipulating the miR-BARTs expression in epithelial cell lines, we further revealed that these four viral miRNAs could inhibit both ionizing radiation (IR)-induced ATM kinase activity and BZLF1-induced viral lytic reactivation. In conclusion, our findings suggest that miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p work co-operatively to modulate DNA damage response and to maintain viral latency, contributing to the NPC tumorigenesis. Acknowledgement: Theme-based Research Scheme (T12-401/13-R) and GRF (14104415 and 14138016), Research Grant Council, Hong Kong. Citation Format: Raymond W. Lung, Po-Man Hau, Joanna H. Tong, Ka-Hei Lam, Anthony W. Chan, Kevin Y. Yip, George S. Tsao, Kwok-Wai Lo, Ka-Fai To. Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma [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 482.