DNA Double-strand Break Repair Genes Research Articles

MYB4, a member of R2R3-subfamily of MYB transcription factor functions as a repressor of key genes involved in flavonoid biosynthesis and repair of UV-B induced DNA double strand breaks in Arabidopsis

Plants accumulate flavonoids as part of UV-B acclimation, while a high level of UV-B irradiation induces DNA damage and leads to genome instability. Here, we show that MYB4, a member of the R2R3-subfamily of MYB transcription factor plays important role in regulating plant response to UV-B exposure through the direct repression of the key genes involved in flavonoids biosynthesis and repair of DNA double-strand breaks (DSBs). Our results demonstrate that MYB4 inhibits seed germination and seedling establishment in Arabidopsis following UV-B exposure. Phenotype analyses of atmyb4-1 single mutant line along with uvr8-6/atmyb4-1, cop1-6/atmyb4-1, and hy5-215/atmyb4-1 double mutants indicate that MYB4 functions downstream of UVR8 mediated signaling pathway and negatively affects UV-B acclimation and cotyledon expansion. Our results indicate that MYB4 acts as transcriptional repressor of two key flavonoid biosynthesis genes, including 4CL and FLS, via directly binding to their promoter, thus reducing flavonoid accumulation. On the other hand, AtMYB4 overexpression leads to higher accumulation level of DSBs along with repressed expression of several key DSB repair genes, including AtATM, AtKU70, AtLIG4, AtXRCC4, AtBRCA1, AtSOG1, AtRAD51, and AtRAD54, respectively. Our results further suggest that MYB4 protein represses the expression of two crucial DSB repair genes, AtKU70 and AtXRCC4 through direct binding with their promoters. Together, our results indicate that MYB4 functions as an important coordinator to regulate plant response to UV-B through transcriptional regulation of key genes involved in flavonoids biosynthesis and repair of UV-B induced DNA damage.

The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

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.

Boric Acid Alters the Expression ofDNA Double Break Repair Genes in MCF-7-Derived Breast Cancer Stem Cells.

Breast cancer pathology ranks second in mortality among women worldwide due to the resistance of cancer stem cells in tumor tissue to radiotherapy and chemotherapy and their effective DNA damage response system (DDR). Targeting the expression of DNA double-strand break (DSB) repair genes in breast cancer stem cells (BC-SCs) is essential for facilitating their elimination with conventional therapies. This study aims to investigate the effects of boric acid (BA) on the expression of DNA DSB repair genes in BC-SCs, which has not been studied in the literature before. BS-SCs were isolated by the MACS method and characterized by flow cytometry. The effects of BA on BC-SCs' DNA DSB repair genes were deciphered by cell viability assay, inverted microscopy, and RT-qPCR. While the expression of the BRCA1 and BRCA2 was upregulated, the expression of the ATM (p < 0.001), RAD51 (p < 0.001), and KU70 (p < 0.001) was downregulated in dose-treated BC-SCs (p < 0.001) to the qPCR results. Consequently, BA affects some of the DNA DSB repair genes of breast cancer stem cells. Findings from this study could provide new insights into the potential therapeutic application of BA in BC-SC elimination and cancer intervention.

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.

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.

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.

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.

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.

Analysis of ionizing radiation induced DNA damage response in human adult stem cells and differentiated neurons

Findings of neurodegenerative features associated with human radiosensitive syndromes such as Ataxia telangiectasia suggest that DNA repair efficiency is crucial for maintaining the functional integrity of central nervous system. To gain a better understanding of ionizing radiation (IR) induced DNA damage response in undifferentiated and differentiated neural cell types and to evaluate the role of ATM in DNA double strand break (DSB) repair, an in vitro human neural cell differentiation model system was utilized in this study. As compared to adult stem cells, differentiated neurons displayed an attenuated DSB repair response (as judged by the persistence of 53BP1 foci) after IR exposure and the attenuation was even more pronounced in stem cells and neurons after suppression of ATM (Ataxia Telangiectasia Mutated) gene product suggesting the importance of ATM for an optimal DSB repair efficiency in human neural cell types. In corroboration with an attenuated DNA damage response, a sharp decline in the expression levels of several DSB repair genes was observed in neurons. Our results suggest that cellular differentiation modulates the expression of several genes thereby compromising the DSB repair fidelity in post mitotic neurons. Further studies are required to verify whether or not ATM mediated exacerbation of DNA repair deficiency in differentiated neurons leads to neurodegeneration.

Targeted Cancer Therapy Based on Acetylation and Deacetylation of Key Proteins Involved in Double-Strand Break Repair.

DNA double-strand breaks (DSBs) play an important role in promoting genomic instability and cell death. The precise repair of DSBs is essential for maintaining genome integrity during cancer progression, and inducing genomic instability or blocking DNA repair is an important mechanism through which chemo/radiotherapies exert killing effects on cancer cells. The two main pathways that facilitate the repair of DSBs in cancer cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). Accumulating data suggest that the acetylation and deacetylation of DSB repair proteins regulate the initiation and progression of the cellular response to DNA DSBs, which may further affect the chemosensitivity or radiosensitivity of cancer cells. Here, we focus on the role of acetylation/deacetylation in the regulation of ataxia-telangiectasia mutated, Rad51, and 53BP1 in the HR pathway, as well as the relevant roles of PARP1 and Ku70 in NHEJ. Notably, several histone deacetylase (HDAC) inhibitors targeting HR or NHEJ have been demonstrated to enhance chemo/radiosensitivity in preclinical studies. This review highlights the essential role of acetylation/deacetylation in the regulation of DSB repair proteins, suggesting that HDAC inhibitors targeting the HR or NHEJ pathways that downregulate DNA DSB repair genes may be worthwhile cancer therapeutic agents.

NBS1 I171V variant underlies individual differences in chromosomal radiosensitivity within human populations

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.

Mapping the genetic landscape of DNA double-strand break repair

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

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

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.

DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML

Poly (ADP-ribose) polymerase inhibitors (PARPis) have shown clinical efficacy in breast and ovarian cancers with inherited mutations in BRCA genes that abrogate homologous recombination (HR) DNA double-strand break (DSB) repair activity. However, to date, PARPishave not had success in sporadic breast and ovarian cancers, or other cancers, such as acute myeloid leukemia (AML), with intact BRCA genes. We have recently reported that triple-negative breast cancer (TNBC) and AML are sensitive to low doses of the potent PARPiTalazoparib in combination with DNA methyl transferase inhibitors (DNMTi), decitabine (DAC) or azacitidine (AZA), in vitro and in vivo. In these cancer models, the DNMTi/PARPicombination increases amplitude and retention of PARP1 directly at laser-induced DNA damage sites, increasing DNA DSBs, promoting synergistic tumor cytotoxicity, blunting cell self-renewal, and leading to strong anti-tumor responses. These results have led to a Phase I/II Clinical trial of DNMTi/PARPi combination therapy in patients with relapsed/refractory AML that is underway.In identifying the molecular pathways that may underlie efficacy of this inhibitor combination in sporadic TNBC, ovarian cancer and AML, we find that low doses of DNMTisreprogram the cancer genome, in part by changing gene expression patterns of DSB repair genes, potentially creating a HR defect (HRD) or “BRCAness” effect. Expression array (Agilent Human 44K v2) analysis was performed on RNA extracted from multiple BRCA-intact sporadic cell lines from AML (including, Kasumi-1, KG-1 and MOLM-14), TNBC (including, MDA-MB-231, SUM159) and ovarian cancer (including, A2780) on days 1, 3, 5 and 7 and 10 post DNMTi(DAC 10nM or AZA 500nM) treatment. Hallmark, KEGG and reactome gene ontologies were utilized. Only gene sets with log fold change (FDR) >.250 were considered significant and genes demonstrating 2-fold or greater change with drug treatment were selected. While multiple DNA repair pathway genes were found both up- and down- regulated post DNMTi treatment, one or more HR genes, were consistently downregulated in all cell lines examined. QPCR of mRNA and/or immunoblotting of HR proteins, in particular RAD51, BRCA1, BRCA2, or HR-related genes, FEN1 or FANCD2, showed significantly decreased expression with DNMTi treatment (p<0.05) in TNBC, ovarian and AML cell lines, validating array data. Functional assays in TNBC cell lines, using a stably expressed HR repair reporter (DRGFP) also confirmed decreased HR activity with DNMTi treatment. Additionally, in response to DNA damage with ionizing radiation (IR, 4Gy), RAD51 foci were reduced in AZA-treated TNBC and ovarian cancer cell lines, compared to untreated controls. Significantly increased triradial chromosomes were also observed in TNBC in the presence of AZA when exposed to DNA cross-linking agents mitomycin C (MMC, 50ng/ml) or cisplatin (2μM) treatment, confirming functional down-regulation of the HR-related FANCD2 pathway.The above HRD-like or BRCAness phenotype with DNMTi treatment translated to sensitivity to PARPis in primary bone marrow mononuclear cells (BMMNC) from AML patients. Thus, in 6 of 8 (75%) AML samples, HR gene expression by QPCR of RNA and HR functional activity by immunostaining for RAD51 foci, following DAC treatment (10nM, 72 hrs), DNMTi treatment resulted in a significant decrease in HR or HR-related gene expression, including RAD51, BRCA1, BRCA2, and decreased levels of RAD51 foci (p<0.05). All 6 AML samples were also significantly more sensitive to DAC/talazoparib combination treatment (p<0.05) in colony forming assays, compared with single agent treatments. AML samples included those with MLL translocations that are reported to have high HR activity and resistance to PARPis. Studies are underway to elucidate the molecular characteristics of the minority of AML patient samples (N=2) that did not generate a BRCAness effect with DAC treatment. In conclusion, our data suggest that one of the mechanisms underlying DNMTi/PARPi therapy efficacy in AML is DNMTi-induced BRCAness. In vitro testing for BRCAness with DNMTitreatment could be a biomarker to predict sensitivity to DNMTi/PARPis and inform selection of AML patients for future clinical trials of this inhibitor combination. DisclosuresCivin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; 3DBioWork Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kingsbury:3DBioWorks: Consultancy, Equity Ownership.