Nonhomologous DNA End Joining Research Articles

Alteration of N-glycosylation of CDON promotes H2O2-induced DNA damage in H9c2 cardiomyocytes

Protein glycosylation is involved in DNA damage. Recently, DNA damage has been connected with the pathogenesis of heart failure. Cell adhesion associated, oncogene regulated (CDON), considered as an N-linked glycoprotein, is a transmembrane receptor for modulating cardiac function. But the role of CDON and its glycosylation in DNA damage remains unknown. In this study, we found that the knockdown of CDON caused DNA double-strand breaks as indicated by an increase in phosphorylated histone H2AX (γH2AX) protein level, immunofluorescent intensity of γH2AX and tail DNA moment in H9c2 cardiomyocytes. Conversely, overexpression of CDON led to decreasing DNA damage induced by hydrogen peroxide (H2O2) and upregulating the expression of genes related to DNA repair pathways-homologous recombination (HR) and non-homologous end joining (NHEJ). Moreover, we expressed nine predicted N-glycosylation site mutants in H9c2 cells prior to treatment with H2O2. The results showed that mutation of N-glycosylation sites (N99Q, N179Q, and N870Q) increased the accumulation of DNA damage and downregulated the expression of HR-related genes, demonstrating that CDON N-glycosylation on DNA damage is site-specific and these specific N-glycan sites may regulate HR repair-related transcript abundance of genes. Our data highlight that N-glycosylation of CDON is critical to cardiomyocyte DNA lesion. It may uncover the potential strategies targeting DNA damage pathway in heart disease.

Investigation of the association between polymorphisms in DNA repair enzymes and STEMI

STEMI (ST-elevation myocardial infarction), a condition in which DNA damage likely contributes to its pathogenesis, is among the most severe manifestations of coronary artery disease. There are very few publications in this field in the literature. In our study, we examined the association between four polymorphisms in repair enzymes (LIG4 Thr9Ile, XRCC6 promoter C-57G, XPA -4A/G, OGG1 Ser326Cys) involved in three distinct DNA repair mechanisms [NHEJ (non-homologous end joining)], NER (nucleotide excision repair), and BER (base excision repair), and their impact on the risk of STEMI. This study involved 185 patients diagnosed with STEMI and included 155 healthy controls. The genotyping of SNPs was conducted through the PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) method for the following variants: XRCC6 (rs2267437), XPA (rs1800975), LIG4 (rs1805388), and OGG1 (rs1052133). No significant differences were observed in the genotype distributions of the XRCC6 and OGG1 variations between the control and patient groups. On the other hand, our findings indicate that individuals carrying the mutant G allele for XPA polymorphism and the mutant T allele for LIG4 polymorphism are susceptible to STEMI. Our findings demonstrate the significance of NHEJ and NER DNA repair processes in the pathogenesis of STEMI, as evidenced by the observed relationship between LIG4 and XPA polymorphisms.

Repeated radon exposure induced ATM kinase-mediated DNA damage response and protective autophagy in mice and human bronchial epithelial cells.

Radon ( 222 Rn) is a naturally occurring radioactive gas that has been closely linked with the development of lung cancer. In this study, we investigated the radon-induced DNA strand breaks, a critical event in lung carcinogenesis, and the corresponding DNA damage response (DDR) in mice and human bronchial epithelial (BEAS-2B) cells. Biomarkers of DNA double-strand breaks (DSBs), DNA repair response to DSBs, ataxia-telangiectasia mutated (ATM) kinase, autophagy, and a cell apoptosis signaling pathway as well as cell-cycle arrest and the rate of apoptosis were determined in mouse lung and BEAS-2B cells after radon exposure. Repeated radon exposure induced DSBs indicated by the increasing expressions of γ-Histone 2AX (H2AX) protein and H2AX gene in a time and dose-dependent manner. Additionally, a panel of ATM-dependent repair cascades [i.e. non-homologous DNA end joining (NHEJ), cell-cycle arrest and the p38 mitogen activated protein kinase (p38MAPK)/Bax apoptosis signaling pathway] as well as the autophagy process were activated. Inhibition of autophagy by 3-methyladenine pre-treatment partially reversed the expression of NHEJ-related genes induced by radon exposure in BEAS-2B cells. The findings demonstrated that long-term exposure to radon gas induced DNA lesions in the form of DSBs and a series of ATM-dependent DDR pathways. Activation of the ATM-mediated autophagy may provide a protective and pro-survival effect on radon-induced DSBs.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Abstract C150: Targeted inhibition of homologous recombination and nonhomologous end joining in diffuse intrinsic pontine gliomas to prevent tumor recurrence

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of Diffuse Intrinsic Pontine Glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. Based on these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrate that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Since radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of ATM to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease. Citation Format: Ivana Barravecchia, Monika Sharma, Robert Teis, Jeanette Cruz, Rachel Mumby, Elizabeth Ziemke, Carlos Espinoza, Brian Magnuson, Mats Ljungman, Carl Koschmann, Christopher Whitehead, Judith Sebolt-Leopold, Stefanie Galban. Targeted inhibition of homologous recombination and nonhomologous end joining in diffuse intrinsic pontine gliomas to prevent tumor recurrence [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C150.

DDDR-05. TARGETED INHIBITION OF DNA REPAIR AND SURVIVAL SIGNALING IN DIFFUSE INTRINSIC PONTINE GLIOMAS

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of Diffuse Intrinsic Pontine Glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. Based on these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrate that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Since radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of ATM to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy . Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

DNA damage and repair: underlying mechanisms leading to microcephaly.

DNA-damaging agents and endogenous DNA damage constantly harm genome integrity. Under genotoxic stress conditions, the DNA damage response (DDR) machinery is crucial in repairing lesions and preventing mutations in the basic structure of the DNA. Different repair pathways are implicated in the resolution of such lesions. For instance, the non-homologous DNA end joining and homologous recombination pathways are central cellular mechanisms by which eukaryotic cells maintain genome integrity. However, defects in these pathways are often associated with neurological disorders, indicating the pivotal role of DDR in normal brain development. Moreover, the brain is the most sensitive organ affected by DNA-damaging agents compared to other tissues during the prenatal period. The accumulation of lesions is believed to induce cell death, reduce proliferation and premature differentiation of neural stem and progenitor cells, and reduce brain size (microcephaly). Microcephaly is mainly caused by genetic mutations, especially genes encoding proteins involved in centrosomes and DNA repair pathways. However, it can also be induced by exposure to ionizing radiation and intrauterine infections such as the Zika virus. This review explains mammalian cortical development and the major DNA repair pathways that may lead to microcephaly when impaired. Next, we discuss the mechanisms and possible exposures leading to DNA damage and p53 hyperactivation culminating in microcephaly.

Design and Synthesis of Ketenimine Sulfonamide Conjugates through Multicomponent Reactions; A Combined Cytotoxic Analysis and Computational Exploration.

Multicomponent reactions involving zwitterion generated from dimethyl acetylenedicarboxylate, aryl sulfonamide, and isocyanide to generate sulfonamide-conjugated ketenimines is reported. The synthetic strategy adopted is highly atom economical and stereoselective. Ketenimine sulfonamide analogues are key intermediates for further synthetic conversions to generate a combinatorial library of compounds. Furthermore, sulfonamide compounds are known to possess a broad spectrum of biological applications. All the novel molecules synthesized exhibit the potential to target the nonhomologous DNA end-joining (NHEJ) pathway with cytotoxic ability. Computational studies compliment the in vitro biological assays of the 8 small-molecule inhibitors. DNA double-strand breaks (DSBs) are considered as the most lethal among different DNA damages. NHEJ repairs about 70% of the DSBs generated in cells within mammals. The DNA-dependent protein kinase catalytic subunit is one of the PI3 kinases associated with NHEJ. Compounds DK01-DK08 were investigated for their ability to induce cancer cell death by treating with two leukemic cell lines where NHEJ is high. Results showed that bromoaryl (DK04)- and nitroaryl (DK05)-conjugated molecules showed excellent biological activity, having IC50 values of ∼2 μM in Nalm6 cell lines.

Uncovering the Molecular Drivers of NHEJ DNA Repair-Implicated Missense Variants and Their Functional Consequences.

Variants in non-homologous end joining (NHEJ) DNA repair genes are associated with various human syndromes, including microcephaly, growth delay, Fanconi anemia, and different hereditary cancers. However, very little has been done previously to systematically record the underlying molecular consequences of NHEJ variants and their link to phenotypic outcomes. In this study, a list of over 2983 missense variants of the principal components of the NHEJ system, including DNA Ligase IV, DNA-PKcs, Ku70/80 and XRCC4, reported in the clinical literature, was initially collected. The molecular consequences of variants were evaluated using in silico biophysical tools to quantitatively assess their impact on protein folding, dynamics, stability, and interactions. Cancer-causing and population variants within these NHEJ factors were statistically analyzed to identify molecular drivers. A comprehensive catalog of NHEJ variants from genes known to be mutated in cancer was curated, providing a resource for better understanding their role and molecular mechanisms in diseases. The variant analysis highlighted different molecular drivers among the distinct proteins, where cancer-driving variants in anchor proteins, such as Ku70/80, were more likely to affect key protein-protein interactions, whilst those in the enzymatic components, such as DNA-PKcs, were likely to be found in intolerant regions undergoing purifying selection. We believe that the information acquired in our database will be a powerful resource to better understand the role of non-homologous end-joining DNA repair in genetic disorders, and will serve as a source to inspire other investigations to understand the disease further, vital for the development of improved therapeutic strategies.

Targeting DNA Repair and Survival Signaling in Diffuse Intrinsic Pontine Gliomas to Prevent Tumor Recurrence.

Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

Disruption of the Schizosaccharomyces japonicus lig4 Disturbs Several Cellular Processes and Leads to a Pleiotropic Phenotype.

Gene targeting is a commonly used method to reveal the function of genes. Although it is an attractive tool for molecular studies, it can frequently be a challenge because its efficiency can be low and it requires the screening of a large number of transformants. Generally, these problems originate from the elevated level of ectopic integration caused by non-homologous DNA end joining (NHEJ). To eliminate this problem, NHEJ-related genes are frequently deleted or disrupted. Although these manipulations can improve gene targeting, the phenotype of the mutant strains raised the question of whether mutations have side effects. The aim of this study was to disrupt the lig4 gene in the dimorphic fission yeast, S. japonicus, and investigate the phenotypic changes of the mutant strain. The mutant cells have shown various phenotypic changes, such as increased sporulation on complete medium, decreased hyphal growth, faster chronological aging, and higher sensitivity to heat shock, UV light, and caffeine. In addition, higher flocculation capacity has been observed, especially at lower sugar concentrations. These changes were supported by transcriptional profiling. Many genes belonging to metabolic and transport processes, cell division, or signaling had altered mRNA levels compared to the control strain. Although the disruption improved the gene targeting, we assume that the lig4 inactivation can cause unexpected physiological side effects, and we have to be very careful with the manipulations of the NHEJ-related genes. To reveal the exact mechanisms behind these changes, further investigations are required.

The flexible and iterative steps within the NHEJ pathway

Cellular and biochemical studies of nonhomologous DNA end joining (NHEJ) have long established that nuclease and polymerase action are necessary for the repair of a very large fraction of naturally-arising double-strand breaks (DSBs). This conclusion is derived from NHEJ studies ranging from yeast to humans and all genetically-tractable model organisms. Biochemical models derived from recent real-time and structural studies have yet to incorporate physical space or timing for DNA end processing. In real-time single molecule FRET (smFRET) studies, we analyzed NHEJ synapsis of DNA ends in a defined biochemical system. We described a Flexible Synapsis (FS) state in which the DNA ends were in proximity via only Ku and XRCC4:DNA ligase 4 (X4L4), and in an orientation that would not yet permit ligation until base pairing between one or more nucleotides of microhomology (MH) occurred, thereby allowing an in-line Close Synapsis (CS) state. If no MH was achievable, then XLF was critical for ligation. Neither FS or CS required DNA-PKcs, unless Artemis activation was necessary to permit local resection and subsequent base pairing between the two DNA ends being joined. Here we conjecture on possible 3D configurations for this FS state, which would spatially accommodate the nuclease and polymerase processing steps in an iterative manner. The FS model permits repeated attempts at ligation of at least one strand at the DSB after each round of nuclease or polymerase action. In addition to activation of Artemis, other possible roles for DNA-PKcs are discussed.

Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

Ku-DNA binding inhibitors modulate the DNA damage response in response to DNA double-strand breaks.

The DNA-dependent protein kinase (DNA-PK) plays a critical role in the DNA damage response (DDR) and non-homologous end joining (NHEJ) double-strand break (DSB) repair pathways. Consequently, DNA-PK is a validated therapeutic target for cancer treatment in certain DNA repair-deficient cancers and in combination with ionizing radiation (IR). We have previously reported the discovery and development of a novel class of DNA-PK inhibitors with a unique mechanism of action, blocking the Ku 70/80 heterodimer interaction with DNA. These Ku-DNA binding inhibitors (Ku-DBi's) display nanomolar activity in vitro, inhibit cellular DNA-PK, NHEJ-catalyzed DSB repair and sensitize non-small cell lung cancer (NSCLC) cells to DSB-inducing agents. In this study, we demonstrate that chemical inhibition of the Ku-DNA interaction potentiates the cellular effects of bleomycin and IR via p53 phosphorylation through the activation of the ATM pathway. This response is concomitant with a reduction of DNA-PK catalytic subunit (DNA-PKcs) autophosphorylation at S2056 and a time-dependent increase in H2AX phosphorylation at S139. These results are consistent with Ku-DBi's abrogating DNA-PKcs autophosphorylation to impact DSB repair and DDR signaling through a novel mechanism of action, and thus represent a promising anticancer therapeutic strategy in combination with DNA DSB-inducing agents.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.