Double-strand Break Repair Genes Research Articles

Boric Acid Affects the Expression of DNA Double-Strand Break Repair Factors in A549 Cells and A549 Cancer Stem Cells: An In Vitro Study.

DNA double-strand break (DSB) repair genes interact with tumor stemness- and resistance-associated processes in cancer stem cells (CSCs). Therefore, targeting DNA DSB genes in cancer treatment is important for the CSC phenotype. Although the anti-cancer effect of boric acid (BA) has been studied, its effect on DNA DSB is unclear. Moreover, no studies investigate BA's effects on DNA DSB of lung cancer stem cells (LC-SCs). To fill the gap, we aimed to assess the effects of BA on A549 cancer stem cells. CSCs were isolated from human non-small cell lung cancer cells (A549) and characterized by flow cytometry. Different concentrations of BA (at doses ranging from 1 to 100mM) were applied to cancer stem cells. Cytotoxic activities were determined using the cell viability assay (MTT assay) at 24 and 48h. Expression levels ofDNA DSB genes that BRCA1, BRCA2, RAD51, KU70/80, ATM, and XRCC4 were evaluated by RT-qPCR. Additionally, immunofluorescence staining analysis was exploited forcaspase-3 and E-cadherin. ATM expression increased significantly (p < 0.001). No significant change was observed in the expression of other genes. Moreover, BA up-regulated caspase-3 and E-cadherin expression. Consequently, we can say thatBA affects DNA DSB and the apoptotic abilities of LC-SCs.

Exploiting Synthetic Lethality between Germline BRCA1 Haploinsufficiency and PARP Inhibition in JAK2V617F-Positive Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/- Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/- cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/- cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/- cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.

DNA Double-Strand Break Response and Repair Gene Polymorphisms May Influence Therapy Results and Prognosis in Head and Neck Cancer Patients.

Radiotherapy and cisplatin-based chemotherapy belong to the main treatment modalities for head and neck squamous cell carcinoma (HNSCC) and induce cancer cell death by generating DNA damage, including the most severe double-strand breaks (DSBs). Alterations in DSB response and repair genes may affect individual DNA repair capacity and treatment sensitivity, contributing to the therapy resistance and poor prognosis often observed in HNSCC. In this study, we investigated the association of a panel of single-nucleotide polymorphisms (SNPs) in 20 DSB signaling and repair genes with therapy results and prognosis in 505 HNSCC patients treated non-surgically with DNA damage-inducing therapies. In the multivariate analysis, there were a total of 14 variants associated with overall, locoregional recurrence-free or metastasis-free survival. Moreover, we identified 10 of these SNPs as independent predictors of therapy failure and unfavorable prognosis in the whole group or in two treatment subgroups. These were MRE11 rs2155209, XRCC5 rs828907, RAD51 rs1801321, rs12593359, LIG4 rs1805388, CHEK1 rs558351, TP53 rs1042522, ATM rs1801516, XRCC6 rs2267437 and NBN rs2735383. Only CHEK1 rs558351 remained statistically significant after correcting for multiple testing. These results suggest that specific germline variants related to DSB response and repair may be potential genetic modifiers of therapy effects and disease progression in HNSCC treated with radiotherapy and cisplatin-based chemoradiation.

Characterization of DNA Damage Response-Associated Somatic Mutations in Borderline Resectable and Locally Advanced Pancreatic Cancer

The role of radiation for pancreatic cancer remains controversial, with recent studies showing conflicting results, highlighting the need to develop biomarkers of radiation response. Despite its potential utility in predicting radiosensitivity, the landscape of somatic mutations in borderline resectable pancreatic cancer (BRPC) and locally advanced pancreatic cancer (LAPC), as related to DNA damage response (DDR), has not been well characterized. This study aimed to characterize the frequency of such mutations in a cohort of patients with BRPC/LAPC treated with neoadjuvant chemotherapy and stereotactic body radiotherapy (SBRT). Mutational data was collected from patients with BRPC/LAPC treated at a single institution with neoadjuvant chemotherapy and SBRT, followed by surgical resection from 2016-2021. Chemotherapy consisted of modified FOLFIRINOX or gemcitabine/nab-paclitaxel, and patients were treated with SBRT in 33 Gy in 5 fractions. Genomic data was obtained from either endoscopic biopsy or surgical specimens, and next-generation sequencing was performed either in-house with a Solid Tumor Panel or with FoundationOne CDx. Specific emphasis was placed on the characterization of double-strand DNA break (DSB) repair genes, as this is the type of tumor cell damage traditionally induced by radiation therapy. Genes associated with the two main pathways of DSB repair, non-homologous end joining (NHEJ) and homologous repair (HR), were analyzed. Specific HR pathway mutations assessed were BLM, BRCA1/2, MRE11, NBN, PALB2, RAD50, RAD51B-D, and RAD54L, while PRKDC mutations were assessed for the NHEJ pathway. Mutations in ATM, an important initiator of DDR pathways, were also analyzed. Additionally, the frequency of mutations in TP53, CDKN2A and SMAD4 in patients with concomitant KRAS mutations was assessed. Eighty-five patients were included in the study. Five (5.9%) patients had mutations in the NHEJ pathway of the PRKDC gene. Twenty (23.5%) patients had mutations in the HR pathway, including BRCA2 (10/85; 11.8%), PALB2 (5/85; 5.9%), BRCA1 (3/85; 3.5%), and RAD50 (1/85; 1.2%). Six (7.1%) patients had mutations in ATM. No patients were found to have mutations in BLM, RAD51B-D, RAD54L, or NBN. Amongst patients with KRAS mutations (72/85), concomitant mutations were observed in TP53 (47/85; 55.3%), CDKN2A (16/85; 18.8%), and SMAD4 (9/85; 10.6%). Herein, we characterized the frequency of somatic mutations associated with DSB repair genes in patients with BRPC/LAPC. Data analysis on outcomes related to radiation response in patients with mutations in DDR pathways is ongoing, but will likely also benefit from multi-institutional efforts to increase the power to answer this question.

Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR-edited cells.

Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. Normal human cells (RPE1), genetically modified to introduce defects in DNA double-strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE-LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12keV/µm) and alpha particles (129keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild-type cells to repair-deficient cells, and RBE values were calculated as the ratio of the MID of X-ray and particle irradiated cells. 53BP1 foci were used to quantify radiation-induced DSBs and their repair following irradiation. Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM-/- SER=2.0 and Lig4-/- SER=1.8), with genes associated with HR having smaller effects (BRCA1-/- SER=1.2). Wild-type cells showed RBEs of 1.1, 1.3, 5.0 for low- and high-LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR-defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ-defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR-defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.

Retrospective study of transcriptomic profiling identifies Thai triple-negative breast cancer patients who may benefit from immune checkpoint and PARP inhibitors.

Triple-negative breast cancer (TNBC) is a rare and aggressive breast cancer subtype. Unlike the estrogen receptor-positive subtype, whose recurrence risk can be predicted by gene expression-based signature, TNBC is more heterogeneous, with diverse drug sensitivity levels to standard regimens. This study explored the benefit of gene expression-based profiling for classifying the molecular subtypes of Thai TNBC patients. The nCounter-based Breast 360 gene expression was used to classify Thai TNBC retrospective cohort subgroups. Their expression profiles were then compared against the previously established TNBC classification system. The differential characteristics of the tumor microenvironment and DNA damage repair signatures across subgroups were also explored. Thai TNBC cohort could be classified into four main subgroups, corresponding to the LAR, BL-2, and M subtypes based on Lehmann's TNBC classification. The PAM50 gene set classified most samples as basal-like subtypes except for Group 1. Group 1 exhibited similar enrichment of the metabolic and hormone response pathways to the LAR subtype. Group 2 shared pathway activation with the BL-2 subtype. Group 3 showed an increase in the EMT pathway, similar to the M subtype. Group 4 showed no correlation with Lehmann's TNBC. The tumor microenvironment (TME) analysis showed high TME cell abundance with increased expression of immune blockade genes in Group 2. Group 4 exhibited low TME cell abundance and reduced immune blockade gene expressions. We also observed distinct signatures of the DNA double-strand break repair genes in Group 1. Our study reported unique characteristics between the four TNBC subgroups and showed the potential use of immune checkpoint and PARP inhibitors in subsets of Thai TNBC patients. Our findings warrant further clinical investigation to validate TNBC's sensitivity to these regimens.

Dysregulation of DNA repair genes in Fuchs endothelial corneal dystrophy

Fuchs Endothelial Corneal Dystrophy (FECD), a late-onset oxidative stress disorder, is the most common cause of corneal endothelial degeneration and is genetically associated with CTG repeat expansion in Transcription Factor 4 (TCF4). We previously reported accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage in FECD. Specifically, mtDNA damage was a prominent finding in development of disease in the ultraviolet-A (UVA) induced FECD mouse model. We hypothesize that an aberrant DNA repair may contribute to the increased DNA damage seen in FECD. We analyzed differential expression profiles of 84 DNA repair genes by real-time PCR arrays using Human DNA Repair RT-Profiler plates using cDNA extracted from Descemet's membrane-corneal endothelium (DM-CE) obtained from FECD patients with expanded (>40) or non-expanded (<40) intronic CTG repeats in TCF4 gene and from age-matched normal donors. Change in mRNA expression of <0.5- or >2.0-fold in FECD relative to normal was set as cutoff for down- or upregulation. Downregulated mitochondrial genes were further validated using the UVA-based mouse model of FECD. FECD specimens exhibited downregulation of 9 genes and upregulation of 8 genes belonging to the four major DNA repair pathways, namely, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double strand break (DSB) repair, compared to normal donors. MMR gene MSH2 and BER gene POLB were preferentially upregulated in expanded FECD. BER genes LIG3 and NEIL2, DSB repair genes PARP3 and TOP3A, NER gene XPC, and unclassified pathway gene TREX1, were downregulated in both expanded and non-expanded FECD. MtDNA repair genes, Lig3, Neil2, and Top3a, were also downregulated in the UVA-based mouse model of FECD. Our findings identify impaired DNA repair pathways that may play an important role in DNA damage due to oxidative stress as well as genetic predisposition noted in FECD.

Abstract 6263: Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma

Abstract Background: Glioblastoma (GBM) patients face a dismal prognosis, with median survival of ~15 months and 5-year survival rate of 6.8%. Standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, all GBM patients succumb to their disease. Glioma stem cells (GSCs), a GBM subpopulation, possess enhanced DNA repair that contributes to chemoradiotherapy resistance. The epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM and emerging studies show KDM1A is implicated in DNA damage response. This study aims to determine the significance of KDM1A to promote DNA repair in GSCs. We hypothesize KDM1A inhibition augments therapeutic efficacy of TMZ via attenuation of DNA repair pathways. Methods: Patient-derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination KDM1A knockdown or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&amp;Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR), and non-homologous end-joining (NHEJ) reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ was determined using orthotopic murine GBM models. Results: GBM patient data sets showed KDM1A is elevated in recurrent GBM versus primary tumors. KDM1A knockdown, knockout, or pharmacological inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&amp;Tag-seq studies showed KDM1A is enriched at the promoters of DNA repair genes. RNA-seq studies confirmed that KDM1A inhibition reduced expression of DNA repair-related genes. RT-qPCR and Western blotting validated downregulation of DNA double-strand break repair genes. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay demonstrated increased TMZ-mediated DNA damage in NCD38-treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of mice bearing orthotopic GBM tumors. Conclusions: Our results provide compelling evidence KDM1A is essential for DNA repair in GSCs and KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination therapy of KDM1A inhibitor NCD38 with TMZ is a potential novel therapeutic strategy to improve GBM outcomes. Citation Format: Salvador Alejo, Bridgitte Palacios, Prabhakar Pitta Venkata, He Yi, Wenjing Li, Jessica Johnson, Sridharan Jayamohan, Weixing Zhao, Siyuan Zheng, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6263.

301 Inhibition of lysine-specific histone demethylase 1A (KDM1A/LSD1) attenuates DNA double strand break repair and enhances efficacy of temozolomide in glioblastoma

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&amp;Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&amp;Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma.

Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults. To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines. Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability. These studies of the genomic, transcriptomic and chemical biology landscape represent a resource 'atlas' for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCSis found.

Relationship between polymorphisms in homologous recombination repair genes RAD51 G172T、XRCC2 & XRCC3 and risk of breast cancer: A meta-analysis.

Genetic variability in DNA double-strand break repair genes such as RAD51 gene and its paralogs XRCC2、XRCC3 may contribute to the occurrence and progression of breast cancer. To obtain a complete evaluation of the above association, we performed a meta-analysis of published studies. Electronic databases, including PubMed, EMBASE, Web of Science, and Cochrane Library, were comprehensively searched from inception to September 2022. The Newcastle-Ottawa Scale (NOS) checklist was used to assess all included non-randomized studies. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by STATA 16.0 to assess the strength of the association between single nucleotide polymorphisms (SNPs) in these genes and breast cancer risk. Subsequently, the heterogeneity between studies, sensitivity, and publication bias were performed. We downloaded data from The Cancer Genome Atlas (TCGA) and used univariate and multivariate Cox proportional hazard regression (CPH) models to validate the prognostic value of these related genes in the R software. The combined results showed that there was a significant correlation between the G172T polymorphism and the susceptibility to breast cancer in the homozygote model (OR= 1.841, 95% CI=1.06-3.21, P=0.03). Furthermore, ethnic analysis showed that SNP was associated with the risk of breast cancer in Arab populations in homozygous models (OR=3.52, 95% CI=1.13-11.0, P= 0.003). For the XRCC2 R188H polymorphism, no significant association was observed. Regarding polymorphism in XRCC3 T241M, a significantly increased cancer risk was only observed in the allelic genetic model (OR=1.05, 95% CI= 1.00-1.11, P=0.04). In conclusion, this meta-analysis suggests that Rad51 G172T polymorphism is likely associated with an increased risk of breast cancer, significantly in the Arab population. The relationship between the XRCC2 R188H polymorphism and breast cancer was not obvious. And T241M in XRCC3 may be associated with breast cancer risk, especially in the Asian population.

Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity.

The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, antiproliferation, and proapoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ-resistance mechanisms, we performed IFNγ-sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.

DNA Repair Mechanisms are Activated in Circulating Lymphocytes of Hospitalized Covid-19 Patients.

Reactive oxygen species (ROS) are an important part of the inflammatory response during infection but can also promote DNA damage. Due to the sustained inflammation in severe Covid-19, we hypothesized that hospitalized Covid-19 patients would be characterized by increased levels of oxidative DNA damage and dysregulation of the DNA repair machinery. Levels of the oxidative DNA lesion 8-oxoG and levels of base excision repair (BER) proteins were measured in peripheral blood mononuclear cells (PBMC) from patients (8-oxoG, n = 22; BER, n = 17) and healthy controls (n = 10) (Cohort 1). Gene expression related to DNA repair was investigated in two independent cohorts of hospitalized Covid-19 patients (Cohort 1; 15 patents and 5 controls, Cohort 2; 15 patients and 6 controls), and by publicly available datasets. Patients and healthy controls showed comparable amounts of oxidative DNA damage as assessed by 8-oxoG while levels of several BER proteins were increased in Covid-19 patients, indicating enhanced DNA repair in acute Covid-19 disease. Furthermore, gene expression analysis demonstrated regulation of genes involved in BER and double strand break repair (DSBR) in PBMC of Covid-19 patients and expression level of several DSBR genes correlated with the degree of respiratory failure. Finally, by re-analyzing publicly available data, we found that the pathway Hallmark DNA repair was significantly more regulated in circulating immune cells during Covid-19compared to influenza virus infection, bacterial pneumonia or acute respiratory infection due to seasonal coronavirus. Although beneficial by protecting against DNA damage, long-term activation of the DNA repair machinery could also contribute to persistent inflammation, potentially through mechanisms such as the induction of cellular senescence. However, further studies that also include measurements of additional markers of DNA damage are required to determine the role and precise molecular mechanisms for DNA repair in SARS-CoV-2 infection.

085 Senescent dendritic cells drive ROS-induced DNA damage in CTCL

Progressive DNA damage and worsening aneuploidy are associated with the development of highly aggressive CTCL. We immunostained MF skin lesions for the phosphorylated DNA double-strand break repair genes γH2AX and 53BP1 and phosphorylated/activated ATM (pATM) and found that ongoing double-strand DNA breaks were frequent within the malignant T cells even in clinically stable MF skin lesions. IL-6, a product of senescent cells, is the second most highly expressed cytokine in MF. Immunostaining demonstrated the senescence marker p21Cip1 was increased in a stage specific manner in MF and localized to OX40L+CD40L+ dendritic cells. These DC also expressed thioredoxin (TXN), indicating production of reactive oxygen species (ROS) and were found clustered closely together with malignant and benign T cells. ROS production by senescent cells is known to drive ROS-induced DNA damage in neighboring cells. We therefore immunostained T cells near senescent DC for evidence of oxidative stress (TXN) and double-strand DNA breaks (γH2AX). We found a stage specific increase in T cells experiencing oxidative stress and ongoing DNA damage in T cells neighboring senescent DC. Malignant T cells were primarily affected but some CD7+ benign infiltrating T cells showed evidence of DNA damage in later stage MF (IIA, IIB). Our results suggest that ROS produced by senescent DC induces DNA damage in T cells and may drive T cell transformation and tumor progression. These studies suggest a possible role for senolytic therapies in the treatment of CTCL.

Correlation of polymorphisms of DNA double-strand break repair genes XRCC5, LIG4 and glioma

To assess the association of DNA double-strand break repair genes XRCC5 and LIG4 with glioma. 126 patients with glioma (case group) and 120 healthy volunteers (control group) were enrolled. The polymorphisms of XRCC5 loci rs828704 and rs9288516, LIG4 loci rs3093737, rs3093739 and rs10131 were detected, and their association with the susceptibility to glioma was analyzed. Hardy-Weinberg test showed that XRCC5 loci rs828704 and rs9288516, LIG4 loci rs3093737, rs3093739 and rs10131 were in equilibrium in both groups (P>0.05). The frequency of A allele of XRCC5 gene rs9288516 locus and T allele of LIG4 gene rs10131 locus in the case group was higher than that in the control group (P<0.05). XRCC5 rs9288516 and LIG4 rs10131 were associated with the susceptibility to glioma under both recessive and additive models (P<0.05), while LIG4 rs3093739 was associated with susceptibility to glioma under the recessive model (P<0.05). XRCC5 rs9288516 and LIG4 rs10131 are associated with the susceptibility to glioma. Above finding may provide a reference for the prevention and treatment of glioma.

Interactions between miRNAs and Double-Strand Breaks DNA Repair Genes, Pursuing a Fine-Tuning of Repair.

The repair of DNA damage is a crucial process for the correct maintenance of genetic information, thus, allowing the proper functioning of cells. Among the different types of lesions occurring in DNA, double-strand breaks (DSBs) are considered the most harmful type of lesion, which can result in significant loss of genetic information, leading to diseases, such as cancer. DSB repair occurs through two main mechanisms, called non-homologous end joining (NHEJ) and homologous recombination repair (HRR). There is evidence showing that miRNAs play an important role in the regulation of genes acting in NHEJ and HRR mechanisms, either through direct complementary binding to mRNA targets, thus, repressing translation, or by targeting other genes involved in the transcription and activity of DSB repair genes. Therefore, alteration of miRNA expression has an impact on the ability of cells to repair DSBs, which, in turn, affects cancer therapy sensitivity. This latter gives account of the importance of miRNAs as regulators of NHEJ and HRR and places them as a promising target to improve cancer therapy. Here, we review recent reports demonstrating an association between miRNAs and genes involved in NHEJ and HRR. We employed the Web of Science search query TS (“gene official symbol/gene aliases*” AND “miRNA/microRNA/miR-”) and focused on articles published in the last decade, between 2010 and 2021. We also performed a data analysis to represent miRNA–mRNA validated interactions from TarBase v.8, in order to offer an updated overview about the role of miRNAs as regulators of DSB repair.

O-211 The DNA double-strand break repair genes expression exhibits significant changes in the postnatal mouse testes from early to aged terms

Abstract Study question How does the DNA double-strand break (DSB) repair genes, Rad51, Rpa70, Ku80, and Xrcc4, expression change in the postnatal mouse testes from early to aged terms. Summary answer The Rad51, Rpa70, and Ku80 genes expression decreased in the postnatal mouse testes from early to aged terms. What is known already The DSB levels occurring in the spermatogenic cells during spermatogenesis increase during biological aging in men. DSBs can be repaired by specific repair mechanisms including homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. While RAD51 and RPA70 are the main components of HR process, KU80 and XRCC4 play important roles in NHEJ pathway. As is known, gH2AX is a commonly used biomarker for determining the DSBs. Study design, size, duration The potential reasons of DSB levels in the spermatogenic cells during aging is not fully addressed yet. In this study, we aimed to analyze the expression of the Rad51, Rpa70, Ku80 and Xrcc4 genes at mRNA and protein levels in the postnatal mouse testes from early to aged terms. Participants/materials, setting, methods We comprised five groups based on the testicular histology, consisting of early (1- and 2-week-old), young (3- and 4-week-old), adult (5- and 6-week-old), late-adult (16-, 18- and 20-week-old), and aged (48-, 50- and 52-week-old). DSB repair genes expression at mRNA and protein levels were determined using qRT-PCR and immunohistochemistry techniques, respectively. The data were evaluated by using one-way ANOVA and Tukey post hoc test. P&amp;lt;0.05 was considered statistically significant. Main results and the role of chance The Rad51, Rpa70, Ku80, and Xrcc4 mRNA levels significantly decreased in the aged group when compared to the young, adult and late-adult groups (P&amp;lt;0.05). gH2AX was intensively localized in the nucleus of primary spermatocytes of postnatal testes, and its levels either in the total or seminiferous tubules or germinal epithelial cells involving primary spermatocytes, round spermatids, elongating spermatids, elongated spermatids, and Sertoli Cells were higher in the aged group than the remained groups (P&amp;lt;0.05). The DSB repair proteins were detected in the spermatogenic cells, in which pachytene spermatocytes showed stronger intensity. The levels of RAD51 and RP70 proteins implicating in HR pathway were lower in the seminiferous tubules of the aged group when compared to the adult and late-adult groups (P&amp;lt;0.05). Moreover, the total and seminiferous tubules analysis revealed that KU80 levels decreased in the aged group in comparison to the remaining groups except for early group, as observed for the spermatogonia, primary spermatocytes and round spermatids of the aged group (P&amp;lt;0.05). Although there were no significant differences found in the total and seminiferous tubule analysis for XRCC4 protein, its levels decreased in the round spermatids and elongating spermatids of the aged group compared to the adult and late-adult groups. Limitations, reasons for caution The limitation of this study is that we did not isolate spermatogenic cell types from aged mice to compare adult ones. Wider implications of the findings The increase of DSB in the spermatogenic cells of aged mice may derive from reduced levels of RAD51, RPA70 and KU80 proteins playing roles in DSB repair. Further researches are required to determine the molecular mechanisms resulting in decrease of these protein levels. Trial registration number not applicable

Somatic mutations of DNA damage repair (DDR) genes in patients with epithelial ovarian cancer

Objectives: Gene mutations are proved to be associated in epithelial ovarian cancer. The majority of researches focus on BRCA1/2 in ovarian serous carcinoma. The incidence of ovarian clear cell carcinoma and endometrioid carcinoma are estimated to be 15% respectively in East Asia, especially in Japan and Taiwan, which is far more prevalent than in the Western countries. However, the somatic gene mutations of DNA damage repair genes in ovarian clear cell carcinoma and endometrioid carcinoma is unclear. Methods: We investigated the somatic mutations of epithelial ovarian cancer patients using a panel containing DNA damage repair genes by next generation sequencing method. Totally 172 epithelial ovarian carcinoma patients, including 69 serous carcinoma, 39 endometrioid carcinoma and 64 clear cell carcinoma, were enrolled. Results: Totally 78 patients with DDR somatic gene mutations were noted, including 48 (69.6%) in serous carcinoma, 13 (33.3%) in endometrioid carcinoma and 17 (26.6%) in clear cell carcinoma. In 69 serous carcinoma patients, gene mutations were 39 TP53 (56.5%), 4 BRCA2 (5.8%), 4 RAD51C (5.8%), 3 MUTYH (4.3%), 3 ERCC8 (4.3%), 1 BRCA1 (1.4%), 1 BRIP1 (1.4%), 1 FANCG (1.4%), 1 RAD51D (1.4%), 1 MLH3 (1.4%), 1 MSH6 (1.4%), 1 OGG1 (1.4%), 1 XRCC6 (1.4%) and 1 ATM (1.4%). In 39 endometrioid carcinoma, gene mutations were 6 TP53 (15.4%), 5 ATM (12.8%), 3 MSH2 (7.7%), 2 MUTYH (5.1%), 2 MLH3 (5.1%), 2 RAD50 (5.1%), 1 BRCA2 (2.6%), 1 MSH6 (2.6%), 1 FANCC (2.6%), 1 FANCM (2.6%), 1 MRE11 (2.6%), 1 MLH1 (2.6%) and 1 MSH3 (2.6%). In 64 clear cell carcinoma, gene mutations were 6 MUTYH (9.4%), 3 TP53 (4.7%), 2 BRCA2 (3.1%), 2 ERCC8 (3.1%), 1 BRCA1 (1.6%), 1 FANCI (1.6%), 1 RAD50 (1.6%), 1 MLH1 (1.6%), 1 MLH3 (1.6%), 1 MSH6 (1.6%), 1 XRCC4 (1.6%), 1 POLD1 (1.6%), 1 CHEK2 (1.6%) and 1 BARD1 (1.6%). In homologous recombination repair genes, the percentage of somatic mutations was 15.9%, 7.7% and 6.3% for serous, endometrioid and clear cell carcinoma (p=0.154, chi-square test). In mismatch repair genes, the percentage of somatic mutations was 2.9%, 15.4% and 4.7% for serous, endometrioid and clear cell carcinoma respectively (p=0.003, chi-square test). In double strand break repair genes, the percentage of somatic mutations was 17.4%, 7.7% and 6.3% for serous, endometrioid and clear cell carcinoma (p=0.092, chi-square test). In single strand break repair genes, the percentage of somatic mutations was 13.0%, 20.5% and 15.6% for serous, endometrioid and clear cell carcinoma (p=0.591, chi-square test). In cell cycle regulation genes, the percentage of somatic mutations was 58.0%, 23.1% and 6.3% for serous, endometrioid and clear cell carcinoma (p Conclusions: The pattern of somatic gene mutations of DNA damage repair genes differs in the serous, endometrioid and clear cell carcinoma patients. Our findings could provide the basis of precision medicine strategy for ovarian cancer treatment.

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.