Alternative Non-homologous End-joining Pathway Research Articles

Alternative NHEJ Pathway Components Are Therapeutic Targets in High-Risk Neuroblastoma

In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets.

Targeting Aberrant Non-Homologous End Joining in Multiple Myeloma: Role of the Classical and Alternative Pathways in Genomic Instability

Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM.To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes.In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2731: PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance

Abstract Estrogen Receptor positive breast cancer is commonly treated with hormonal therapy; however, many patients acquire therapy resistance, a major clinical problem. Recent evidence suggests that the accumulation of chromosomal abnormalities via increased error-prone Alternative-Non-Homologous End Joining (A-NHEJ) DNA repair pathway could contribute to breast cancer therapy resistance. Proline, Glutamic acid, Leucine rich Protein-1 (PELP1), an oncogenic co-regulator for transcription factors, is commonly overexpressed in breast cancer, and its deregulation has been implicated in therapy resistance. Our recent studies identified PELP1 as a novel substrate of the DNA damage response (DDR) kinases. In this study, we discovered that PELP1 regulates the A-NHEJ pathway, and we developed a novel peptide drug that targets PELP1-A-NHEJ axis for the treatment of therapy resistance. Our results suggest that PELP1 is phosphorylated at Ser1033 by DDR kinases and phosphorylated PELP1 co-localizes with the gamma-H2AX foci. Using Homologous Recombination (HR) and NHEJ pathway specific reporter assays, we found that PELP1 knockdown decreases NHEJ, with no apparent effect on the HR pathway. Further, using A-NHEJ specific reporter cell line, we found that PELP1 knockdown decreased the frequency of A-NHEJ, while PELP1 overexpression increased the repair frequency. PELP1's ability to regulate A-NHEJ pathway was further confirmed by sequencing repaired plasmids and by quantitating the degree of end resection. More importantly, metaphase chromosome spreads revealed gross chromosomal abnormalities in PELP1 deregulated breast cancer cells. Mechanistic studies revealed that PELP1 interacts with Mre11, and modulates the degree of end resection at the DNA double strand breaks. Mapping studies identified the C-terminus of PELP1 as the binding site for Mre11. Using yeast based genetic screen composed of 10 million peptides, we identified a unique peptide, PIP3 that binds the C-terminus of PELP1. We developed a stapled peptide of PIP3 (sPIP3), which functioned as a potent cytotoxic agent for breast cancer cells with little activity on normal cells. Further, sPIP3 efficiently inhibited the A-NHEJ pathway and significantly, reduced survival and promoted apoptosis of therapy resistant model cells. Collectively, these findings suggest that PELP1 plays a critical role in the A-NHEJ pathway and sPIP3 represents a novel drug for the treatment of therapy resistant breast cancer. Citation Format: Samaya R. Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2014-2731

Abstract P5-09-12: Novel role of proto-oncogene PELP1 in A-NHEJ pathway of double strand break repair: Implications for treatment of hormonal therapy resistance

Abstract Background: DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier. Deregulation of key components of the DDR pathway such as p53 and ATM is associated with breast cancer progression. Recent studies implicated Alternative Non-homologous End-Joining (A-NHEJ) pathway in hormonal therapy resistance. Expression of proline, glutamic acid, leucine rich protein 1 (PELP1), a proto-oncogene, is an indicator of poor prognosis and therapy resistance in ER+ breast cancer. PELP1 was recently identified as a novel substrate of DNA damage kinases such as ATM. The objective of this study is to determine the role of PELP1 in the regulation of DNA repair and to study its effects on therapy resistance Methods: We have used ER+ therapy sensitive MCF7, MCF7-Aro, ZR75 and therapy resistant MCF7-Tam, MCF7-LTLT, MCF7-HER2 model cells in our assays. UV-laser micro-irradiation, immunofluorescence staining and confocal microscopy were used to examine Ser-1033 phospho-PELP1 localization upon DNA damage. Homologous recombination (HR) reporter cell lines and NHEJ pathway reporter plasmids were used to determine the role of PELP1 in DNA repair pathways. U2OS-EJ2-GFP reporter cell line was used to evaluate the role of PELP1 in A-NHEJ pathway. MEFs lacking PELP1 and PELP1 specific siRNA were used to determine the role of PELP1 in DNA repair. Immunoprecipitation and western blotting were used to determine PELP1 interacting proteins. Yeast based peptide library screen was performed to identify PELP1 binding inhibitory peptides. MTT and clonogenic assays were used to determine the therapeutic effect of PELP1 inhibitor on therapy resistant breast cancer model cells. Results: Results from this study showed that Ser-1033 phospho PELP1 was recruited to the site of DSB and co-localize with γ-H2A.X in breast cancer cells. Using reporter plasmid assays that distinguish various DNA repair pathways, we found that PELP1 regulates the NHEJ pathway and not the HR pathway. Further, using U2OS-EJ2-GFP reporter cell lines, we demonstrated that PELP1 is important for A-NHEJ mediated double strand break repair. Mechanistic studies revealed PELP1 interacts with E3ubiquitin ligase, SNF2 Histone Linker PHD RING Helicase (SHPRH, a yeast homolog of Rad5p). We have developed cell permeable peptide inhibitor (PIP3) that bind PELP1 and interferes with its interactions with SHPRH. Accordingly, treatment of U2OS-EJ2-GFP reporter cell lines with the (PIP3) peptide reduced the A-NHEJ repair frequency. Further, therapy resistant breast cancer cells which are addicted to A-NHEJ pathway were highly sensitive to PIP3 treatment. Conclusions: These data provide novel insights into role of PELP1 in DNA repair, underscore the importance of PELP1 in the A-NHEJ pathway of DNA repair and demonstrates the significance of targeting PELP1 to sensitize therapy resistant breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-12.

Alternative pathways of non-homologous end joining (NHEJ) in genomic instability and cancer

An incompletely defined alternative non-homologous end joining pathway (A-NHEJ) frequently functioning as backup has recently been shown to process DNA double strand breaks (DSBs) when canonical cellular DSB repair activities are somehow compromised. While A-NHEJ offers survival advantage for the damaged cell, its propensity for large sequence alterations at the junction and the relatively frequent joining of unrelated DNA ends generates alterations in the genome that can have severe biological consequences. Indeed, evidence accumulates that A-NHEJ is a driver of the chromosomal rearrangements and the overall genomic instability that underpin the development of certain forms of cancer. Features of A-NHEJ causally related to these unfavorable outcomes are the apparent promiscuity in utilized factors and the delayed repair kinetics. The purpose of this review is to briefly summarize recent advances in our understanding regarding the function of A-NHEJ and to review emerging evidence for its role in oncogenic transformation.

Targeting abnormal DNA double-strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias.

Resistance to imatinib (IM) and other tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. As chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double-strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα, were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib-resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with the sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.

Targeting Abnormal DNA Repair in Therapy-Resistant Breast Cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor-positive (ER+) MCF7 breast cancer cells and progesterone receptor-positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA-protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER(-)/PR(-) tumors had elevated levels of ALT NHEJ and reduced levels of DNA-PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach.

Réparation des cassures double-brin de l’ADN, un mécanisme peut en cacher un autre : la ligature d’extrémités non homologues alternative

DNA double strand breaks are major cytotoxic lesions encountered by the cells. They can be induced by ionizing radiation or endogenous stress and can lead to genetic instability. Two mechanisms compete for the repair of DNA double strand breaks: homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires DNA sequences homology and is initiated by single strand resection. Recently, advances have been made concerning the major steps and proteins involved in resection. NHEJ, in contrast, does not require sequence homology. The existence of a DNA double strand break repair mechanism, independent of KU and ligase IV, the key proteins of the canonical non homologous end joining pathway, has been revealed lately and named alternative non homologous end joining. The hallmarks of this highly mutagenic pathway are deletions at repair junctions and frequent use of distal microhomologies. This mechanism is also initiated by a single strand resection of the break. The aim of this review is firstly to present recent data on single strand resection, and secondly the alternative NHEJ pathway, including a discussion on the fidelity of NHEJ. Based on current knowledge, canonical NHEJ does not appear as an intrinsically mutagenic mechanism, but in contrast, as a conservative one. The structure of broken DNA ends actually dictates the quality repair of the alternative NHEJ and seems the actual responsible for the mutagenesis attributed beforehand to the canonical NHEJ. The existence of this novel DNA double strand breaks repair mechanism needs to be taken into account in the development of radiosensitizing strategies in order to optimise the efficiency of radiotherapy.

DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation

Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

Abstract 5495: ALT NHEJ is a therapeutic target in hormone therapy resistant ER/PR/HER+ and ER/PR/HER- breast cancers

Abstract Approximately 75% of breast cancers are estrogen and/or progesterone positive (ER/PR+) and can be treated in the clinic with anti-hormone therapy; however, resistance is an increasing problem. ER/PR negative (−) breast cancers, on the other hand, have few treatment options. Therefore, novel therapies are required for therapy resistant ER/PR+ and ER/PR- breast cancers. Breast cancers have abnormal responses to the repair of DNA double strand breaks (DSBs) leading to the generation of structural chromosomal abnormalities and genomic instability. DSBs are repaired by at least 2 pathways: homologous recombination (HR) that performs error-free repair and non-homologous end-joining (NHEJ) that is error-prone. An alternative (ALT) NHEJ pathway has been identified that is associated with the generation of chromosomal translocations, deletions and genomic instability. We have recently shown that with respect to non-tumorigenic MCF10A, ER/PR+ MCF7 breast cancer cells express reduced levels of DNA ligase IV, a component of the classic (C) NHEJ pathway, whereas, expression of DNA ligase IIIα, a component of ALT NHEJ, is increased. To determine whether abnormal ALT NHEJ may be a target for therapy in hormone resistant ER/PR+ and ER/PR- patients, we examined steady state levels of C- and ALT-NHEJ proteins in tamoxifen (TAM 1,2,3) and aromatase (LTLT) resistant cell lines derived from MCF7 cells and ER/PR- cells (MDAMB231, SKBR3). Only ER/PR- cells show further reductions in DNA ligase IV and Ku70 compared with MCF10A. Both hormone resistant ER/PR+ and ER/PR- cells show further increases in ALT NHEJ proteins DNA ligase IIIα, poly- (ADP-ribose) polymerase (PARP1) and ALT NHEJ activity as measured by established NHEJ assays compared to controls. Since PARP1 inhibitors have been shown to specifically kill cells with defects in DSB repair and sporadic breast cancer cells have upregulated PARP1 and DNA ligase IIIα, we examined the effect of PARP1 inhibitor ABT888 in ER/PR+ and ER/PR- cells in the presence or absence of a DNA ligase I/III inhibitor. While either inhibitor alone had little effect on cell survival, both therapy resistant ER/PR+ and ER/PR- cells are highly sensitive to a combination of DNA repair inhibitors, acting synergistically. This treatment also decreases ALT NHEJ repair, demonstrating specific targeting of this pathway. Importantly, MCF10A is insensitive to these inhibitors. Finally, siRNA knockdown of C NHEJ protein Ku70 in hormone therapy sensitive ER/PR+ MCF7 increases ALT NHEJ repair activity and sensitivity to the combination of DNA repair inhibitors. Intriguingly, MCF7 cells with increased ALT NHEJ activity also acquire resistance to hormone therapy. Thus, ALT NHEJ appears to be linked to resistance to hormone therapy and is a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2011-5495

Induction and repair of DNA double strand breaks: The increasing spectrum of non-homologous end joining pathways

A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.

Cells expressing FLT3/ITD mutations exhibit elevated repair errors generated through alternative NHEJ pathways: implications for genomic instability and therapy

AbstractThe internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor found in acute myeloid leukemia patients are associated with poor prognosis. Although DNA double-strand breaks (DSBs) are mainly repaired by the DNA-PK–dependent nonhomologous end-joining (NHEJ) pathway in normal mammalian cells, an alternative and less well-defined NHEJ pathway, characterized by microhomology at the repair junctions, play a role in the generation of deletions and translocations leading to cancer progression. Here we report that in FLT3/ITD-expressing cell lines and bone marrow mononuclear cells from FLT3/ITD knock-in mice, end-joining of DSBs occurs at microhomologous sequences resulting in a high frequency of DNA deletions. Strikingly, levels of Ku proteins, key components of the main NHEJ pathway, are decreased in FLT3/ITD+ cell lines and murine FLT3/ITD bone marrow mononuclear cells. Concomitantly, levels of DNA ligase IIIα, a component of ALT NHEJ, are increased in FLT3/ITD-expressing cells. Cells treated with a FLT3 inhibitor demonstrate decreased DNA ligase IIIα and a reduction in DNA deletions, suggesting that FLT3 signaling regulates the pathways by which DSBs are repaired. Thus, therapy to inhibit FLT3/ITD signaling and/or DNA ligase IIIα may lead to repair that reduces repair errors and genomic instability.

An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway

Chromosomal translocations arise from the misjoining of DNA breaks, but the identity of the DNA repair factors and activities involved in their formation has been elusive. Here we show that depletion of CtIP, a DNA end-resection factor, results in a substantial decrease in chromosomal translocation frequency in mouse cells. Moreover, microhomology usage, a signature of the alternative nonhomologous end-joining pathway (alt-NHEJ), is significantly lower in translocation breakpoint junctions recovered from CtIP-depleted cells than in those from wild-type cells. Thus, we directly demonstrate that CtIP-mediated alt-NHEJ has a primary role in translocation formation. CtIP depletion in Ku70(-/-) cells reduces translocation frequency without affecting microhomology, indicating that Ku70-dependent NHEJ generates a fraction of translocations in wild-type cells. Translocations from both wild-type and Ku70(-/-) cells have smaller deletions on the participating chromosomes when CtIP is depleted, implicating the end-resection activity of CtIP in translocation formation.

The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres

Repair of DNA double-stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non-homologous end-joining (NHEJ) or error-free homologous recombination. NHEJ precedes either by a classic, Lig4-dependent process (C-NHEJ) or an alternative, Lig4-independent one (A-NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere-binding proteins are recognized as DSBs. In this report, we studied which end-joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end-to-end chromosome fusions mediated by the C-NHEJ pathway. In contrast, removal of Tpp1-Pot1a/b initiated robust chromosome fusions that are mediated by A-NHEJ. C-NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A-NHEJ is a major pathway to process dysfunctional telomeres.

Abstract 688: Acetylation of poly-ADP-ribose polymerase and Ku70 by histone deacetylase inhibitors promote abnormal binding to DNA double strand breaks and decrease repair efficiency in leukemia cells

Abstract Histone Deacetylase inhibitors (HDACi) affect gene expression through acetylation of histone and non-histone proteins which could alter their cellular activity. We have previously shown that HDACi trigger DNA damage responses in leukemia cells, and actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist longer compared with normal cells. The mechanism by which this DNA damage occurs is still unknown. Non Homologous End Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. We have recently shown that NHEJ activity is abnormal in myeloid leukemias in that classical NHEJ that is Ku and DNA-PKcs dependent, is down regulated, and compensatory repair of DSBs occurs via alternative (Alt) NHEJ pathways involving Poly-ADP-Ribose Polymerase-1 (PARP-1) and DNA LigaseIIIα. Alt NHEJ is known to generate large deletions and translocations in cancers and leukemias. To determine whether HDACi may decrease the capacity of leukemic cells to repair DSBs by direct acetylation of NHEJ proteins, K562 and HL60 leukemia cell lines were treated with Trichostatin A (TSA, 300nM for 1 and 6 hours) followed by immunoprecipitation and western blot analysis for acetylated NHEJ proteins. TSA treatment of K562 and HL60 show differential acetylation of classical NHEJ protein Ku70 and Alt NHEJ protein PARP-1, compared with controls. To investigate whether TSA treatment alters the binding of Ku70 and PARP-1 to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with DRNeo that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after TSA treatment while Ku70 is not. To determine whether DSB repair activity is altered after HDACi treatment, we performed a plasmid based NHEJ repair assay in our leukemia cell lines after treatment with TSA. Both leukemia cell lines demonstrate a significant decrease in the capacity to repair DSBs following HDACi treatment. The same result can be obtained by the use of another HDACi entinostat. These data suggest that HDACi treatment leads to an increased presence of PARP-1 at DSBs, preventing subsequent critical repair steps in leukemia cells. Importantly, AML cells from patients treated with entinostat in vivo show significantly increased co-localization of PARP-1 and γH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Overall, our results suggest that HDACi result in both a physical and functional alteration of proteins participating in both classical and Alt NHEJ, leading to a loss of binding of Ku70 and an abnormal persistence of PARP-1 at DSBs. Our data supports the idea that acetylated PARP-1 prevents repair of DSBs by Alt NHEJ, leading to a persistence of DSBs in leukemia cells treated with HDACi. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 688.

Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends

Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA double-strand breaks1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or AP sites)2. At single strand breaks, 5' terminal abasic sites are excised by pol β's 5'dRP lyase activity3,4,5,6: we show here in vitro and in cells that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'dRP/AP lyase activity (Figure 1a). Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping distinguish this pathway from otherwise robust alternate NHEJ pathways. Surprisingly, the NHEJ factor Ku can be identified as an effective 5'dRP/AP lyase. Similar to other lyases7, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff base covalent intermediate with the abasic site. We demonstrate using cell extracts that Ku is essential for efficient removal of AP sites near double-strand breaks and, consistent with this result, joining of such breaks is specifically reduced in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that processed ends; our data supports an unexpected direct role for Ku in end processing steps as well.

B-Lymphocyte Development Is Impaired by FLT3/ITD Signaling in Knock-in Mice Because of Defective Non-Homologous End-Joining.

Abstract 184Constitutive activation of FLT3 by internal tandem duplication (ITD) mutations is one of the most common molecular alterations found in up to 35% of acute myeloid leukemia (AML) and 3% of acute lymphoid leukemia (ALL). Its roles in leukemic transformation of normal hematopoietic stem/ progenitor cells (HSPCs) are not yet fully understood. We have generated a FLT3/ITD knock-in mouse model in which mice heterozygous for a FLT3/ITD mutation develop myeloid proliferation and a block in B-lymphocyte development at the Pro-B stage. To investigate the mechanisms for this block in lymphoid development, we studied V(D)J recombination in the B-lineage cells from the bone marrow (BM) of FLT3/ITD mice. The ligation-mediated PCR assay showed that Pro-B cells from FLT3/ITD mice accumulated high levels of signal-end recombination intermediates, generated by DNA cleavage at the DJH recombination signal sequences (RSSs), with a concomitant decrease in the frequency of completed DJH rearrangements when compared to wild-type littermate controls. These observations are consistent with decreased repair of double strand breaks (DSBs) introduced by the V(D)J recombinase in B cell progenitors from FLT3/ITD mice, resulting in impaired development beyond the Pro-B stage.The classical Ku and DNA-PK-dependent non-homologous end-joining (NHEJ) pathway is required for rejoining DSBs during V(D)J recombination. In vivo NHEJ assays demonstrate that BM cells, including Pro-B cells, with FLT3/ITD mutations have a lower efficiency and decreased accuracy of repair of DSBs, suggesting a defect in the classical NHEJ pathway. Quantitative RT-PCR and Western blotting analysis of Pro-B cells from FLT3/ITD mice demonstrate greatly reduced expression of Ku70/Ku86 and Ligase IV, key components of the the classical NHEJ pathway, confirming the idea that Classical NHEJ is defective. We and others have recently demonstrated that alternative (ALT) NHEJ pathway(s) compensate for deficiencies in classical NHEJ. Components of this pathway include DNA ligase III/XRCC1 and poly (ADP) ribose polymerase 1 (PARP1). Indeed, we show by Western blotting analysis that steady state levels of ALT NHEJ components DNA ligase III and PARP1 are increased in FLT3/ITD cells, compared with wild-type controls.These data suggest that during the process of DJH recombination in Pro-B cells from FLT3/ITD mice, impairment of the classical NHEJ pathway decreases the ability of cells to complete post-cleavage DSB ligation, resulting in failure to complete DJH recombination with a subsequent block of Pro-B cell maturation. With the classical NHEJ pathway impaired, BM cells from mice with FLT3/ITD mutations appear to repair DSBs through the highly error-prone ALT NHEJ pathway. The resultant increased genomic instability might underlie an important mechanism for the leukemic transformation of normal HSPCs by FLT3/ITD mutations and explain the poor prognosis of AML patients with this mutation. Disclosures:No relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Promote Abnormal Binding of DNA Repair Proteins to DNA Double Strand Breaks: Consequences for DNA Repair and Genomic Instability?.

Abstract 186Histone Deacetylase inhibitors (HDi) affect gene expression through induction of histone acetylation and lead also to the acetylation of many other proteins which could affect their cellular activity. We have previously shown that HDi trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses, but actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist for long periods of time compared with normal cells (Gaymes TJ. and al., Mol Cancer Res. 2006). This raises the hypothesis that HDi regulate the capacity of leukemic cells to repair DSBs, and the explanation for the increased and persistent DNA damage in leukemic cells may be that HDi directly acetylates proteins involved in DSB repair, thus decreasing repair activity. Non Homologous End-Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. While normal cells use NHEJ that is Ku and DNA-PKcs dependent, an alternative (Alt) NHEJ pathway (DNA-PKcs and Ku independent) involving Poly-ADP-Ribose Polymerase-1 (PARP-1), Werner syndrome helicase (WRN) and DNA LigaseIIIa proteins, has been identified and is responsible for deletions and translocations in cancer. We have recently reported that myeloid leukemia cells repair DSBs using this Alt NHEJ pathway (Sallmyr A. and al., Blood, 2008). Here we show that HDi treatment by Trichostatin A (300nM) results in differential acetylation of main NHEJ protein Ku70 in acute leukemia K562 cell line. In addition, PARP-1, active in several repair pathways, including single strand break repair and Alt NHEJ is also hyperacetylated in K562 cells after 1 and 6 hours of Trichostatin A treatment compared with control treatment. To investigate whether Trichostatin A treatment alters the binding of DNA repair proteins to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with the DRNeo construct that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after 1 hour of Trichostatin A treatment, compared with controls. Preliminary CHIP analysis for the protein XRCC1, necessary for the final step of Alt NHEJ repair, shows that it is decreased at the DSB site. Importantly, AML patients treated with the HDi MS-275 in vivo show significantly increased colocalization of PARP-1 and gH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Altogether, these data suggest that HDi treatment leads to an increased presence of PARP-1 at DSBs, and that this may prevent subsequent critical repair steps, providing a possible explanation for the persistence of DNA damage. Finally, to determine whether DSB repair activity is indeed decreased with HDi treatment, we used an in vivo NHEJ repair assay in K562 and HL60 acute leukemia cell lines before and after treatment with Trichostatin A for 1 hour. Both leukemia cell lines demonstrate a significant decrease in the capacity of the cells to repair DSBs following Trichostatin A treatment. These results suggest that HDi result in both a physical and functional alteration of proteins participating in DNA repair pathways, leading to a decrease in NHEJ activity. The decrease in Alt NHEJ activity may have implications for genomic instability, diminishing abnormal repair following HDi treatment. Disclosures:No relevant conflicts of interest to declare.

Multiple functions of MRN in end-joining pathways during isotype class switching

SummaryThe Mre11/Rad50/NBS1 (MRN) complex plays many roles in response to DNA double strand breaks (DSBs), but its functions in repair by non homologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in Class Switch Recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end we have engineered mice that lack the entire MRN complex in B lymphocytes, or possess an intact complex harboring mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a striking defect in CSR, impacting both the Classic and Alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the Classical and Alternative NHEJ pathways.