Inhibition Of Non-homologous End Joining Research Articles

Disease-Associated Changes In The Repair Efficiency Of Double Strand Breaks Affect Melphalan Sensitivity Of The Bone Marrow Plasma Cells and Correlate With The Clinical Outcome Of Anti-Myeloma Therapy

Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeutic drugs in the treatment of patients with multiple myeloma (MM). There is clear evidence that the formation and subsequent persistence of ICL correlates with its cytotoxicity. Previous studies have established that during ICL repair, replication forks stall at the ICL inducing the formation of a lethal form of DNA damage (DNA double-strand breaks, DSBs), which is repaired mainly by homologous recombination (HR) and non-homologous end joining (NHEJ). In this report, we investigated the molecular mechanisms of therapeutic efficiency and drug resistance to ICL-inducing agents using melphalan as a model.We studied two MM cell lines (melphalan-sensitive RPMI-8226 and melphalan-resistant RPMI-LR5) and 70 MM patients (38 males/32 females; median age 59 years) who underwent high-dose melphalan (HDM) therapy with autologous stem cells transplantation (ASCT) as first line therapy. Patient response status was assessed 100 days after ASCT according to the International Myeloma Working Group Criteria; patients were grouped into responders (≥PR, n=48) and non-responders (<PR, n=22). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples obtained from MM patients, at diagnosis or at least 1 week prior to the treatment with any anti-myeloma drug. In addition, bone marrow plasma cells (BMPCs) were isolated from bone marrow trephine aspiration samples during diagnostic clinical assessment. Primary cells (PBMCs and BMPCs) and MM cell lines were ex vivo treated with melphalan either alone or in combination with RI-1 (selective inhibitor of HR) or NU7026 (selective inhibitor of NHEJ) and the extent of the N-ras-specific ICLs and DSBs (intermediates of ICL repair) were evaluated using a quantitative PCR assay and quantification of γH2AX foci, respectively. The γH2AX foci were viewed under a laser-scanning confocal immunofluorescence microscope and quantitated using Image J software. The induction of the apoptotic pathway by melphalan, using a photometric enzyme-immunoassay, was also studied.Following ex vivo treatment of BMPCs with melphalan, ICLs reached maximal levels within 8h of the melphalan treatment. Thereafter, ICLs levels were reduced with the repair efficiency being significantly higher in non- responders (half-time of damage removal, t1/2 23h) than in responders (t1/2 48h) (P<0.01). Moreover, γ-H2AX foci formation followed the timing of ICL formation and reached maximal levels within 8h. Thereafter, γ-H2AX foci levels declined rapidly, suggesting the resolution of the intermediate DSBs by downstream pathways (HR, NHEJ). Interestingly, the repair efficiency of DSBs in BMPCs was significantly higher in non-responders (t1/2 9h) than in responders (t1/2 12h) (P<0.02). Similar results were obtained using PBMCs.Also, in both BMPCs and PBMCs, the melphalan-induced apoptosis inversely correlated with the repair efficiencies of ICLs and DSBs, with the toxicity being higher in responders than in non-responders (P<0.01). Moreover, RPMI-LR5 cells showed higher repair efficiencies of both ICLs and DSBs and lower toxicity than RPMI-8226 cells. Interestingly, in all cell types analyzed, significant correlation between ICL and DSBs levels was observed (linear regression analysis, R2=0.67, P<0.01).To further elucidate the mechanism of drug-induced DSBs repair, MM cell lines and primary cells (BMPCs and PBMCs) were treated with melphalan in combination with nontoxic doses of RI-1 or NU7026. We found that the combined treatment of melphalan with RI-1 or NU7026 significantly increased the melphalan only-induced phosphorylation of H2AX (suggesting that both HR and NHEJ contribute to the repair of melphalan-induced DSBs), delayed the repair of ICLs and strongly enhanced the cytotoxic activity of melphalan (all P<0.01).Collectively, these results highlight that in BMPCs significant changes in the repair efficiency of DSBs occur in MM patients. These changes affect the removal of the cytotoxic ICLs, modify drug sensitivity of the malignant plasma cells ex vivo, and correlate with the clinical outcome of anti-myeloma therapy. Interestingly, these changes are also reflected in PBMCs. Specific inhibition of HR and/or NHEJ may be useful as an adjunct to melphalan therapy in MM patients. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.

End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1

In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.

COM-1 Promotes Homologous Recombination during Caenorhabditis elegans Meiosis by Antagonizing Ku-Mediated Non-Homologous End Joining

Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1–deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41°C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.

An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells,and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Inhibition of Non-Homologous End Joining Repair Impairs Pancreatic Cancer Growth and Enhances Radiation Response

Pancreatic ductal adenocarcinoma (PDAC) is amongst the deadliest of human cancers, due to its late diagnosis as well as its intense resistance to currently available therapeutics. To identify mechanisms as to why PDAC are refractory to DNA damaging cytoxic chemotherapy and radiation, we performed a global interrogation of the DNA damage response of PDAC. We find that PDAC cells generally harbor high levels of spontaneous DNA damage. Inhibition of Non-Homologous End Joining (NHEJ) repair either pharmacologically or by RNAi resulted in a further accumulation of DNA damage, inhibition of growth, and ultimately apoptosis even in the absence of exogenous DNA damaging agents. In response to radiation, PDAC cells rely on the NHEJ pathway to rapidly repair DNA double strand breaks. Mechanistically, when NHEJ is inhibited there is a compensatory increase in Homologous Recombination (HR). Despite this upregulation of HR, DNA damage persists and cells are significantly more sensitive to radiation. Together, these findings support the incorporation of NHEJ inhibition into PDAC therapeutic approaches, either alone, or in combination with DNA damaging therapies such as radiation.

The secret life of Bcl-2: Apoptosis-independent inhibition of DNA repair by Bcl-2 family members

Programmed cell death and DNA repair are two fundamental biological processes that play essential roles in cell fate and genetic transmission. The canonical role of Bcl-2 family members is the regulation of programmed cell death. Strikingly, numerous studies from different laboratories have shown that although Bcl-2 increases cell survival, it also inhibits all DNA repair systems, resulting in genome instability/variability. Bcl-2 affects the mechanistically distinct DNA repair systems via different mechanisms. These effects are generally independent of the regulation of apoptosis, revealing additional roles for Bcl-2. The targets of Bcl-2 include APE1, MSH2, PARP1, Ku70 and the oncosuppressor BRCA1. Targetting BRCA1 should be of particular importance because this might impact many essential cellular processes in which BRCA1 is involved, including homologous recombination (HR), non-homologous end joining (NHEJ), base excision repair, cell-cycle regulation, cell death, ubiquitination, inactivation of the X-chromosome, transcription, and protein translation. Beside the pathological consequences, inhibition of DNA repair by Bcl-2 can be, in contrast, advantageously used in some physiological situations: (1) repression of excessive unschedule HR, thus protecting against the accumulation of toxic HR intermediates and HR-dependent genome rearrangements; (2) inhibition of NHEJ might protect against retrovirus integration; (3) it has been proposed that inhibition of mismatch repair might also favors hypermutation at immunoglobulin genes. Finally, because Bcl-2 affects the maintenance of genome stability, one can suggest Bcl-2 might play a role in molecular evolution. Bcl-2 family members control cell death through complex stochiometric equilibriums. Incorporating DNA repair proteins to such an elaborate network should allow for a fine tuning of the coordinated control of cell viability and genetic stability/instability. Relationships between DNA repair and regulation of cell death represent exciting challenges for future prospects and are essential for the development of promising new strategies against cancer.

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the ProteasomeIn Vivo

Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

Abstract 5720: RK-33, a novel small molecule for lung cancer therapy via inhibition of NHEJ

Abstract Lung cancer is the leading cause of cancer mortality worldwide. More importantly, only about 30% of non-small-cell lung cancer patients respond to current combination therapy, which includes radiation and platinum based compounds. Currently, there are limited molecular targets that can be drugable for lung cancer treatment. Our investigation in cancer biogenesis has identified an RNA helicase, DDX3, being dysregulated in many cancer types including lung cancer. DDX3 is a member of the DEAD-box RNA helicase family and contributes to cancer by promoting proliferation, cell growth, transformation and inhibition of apoptosis. Importantly, knockdown of DDX3 in the highly aggressive lung cancer cell line, A549, abrogated its colony forming abilities. In our efforts to abrogate DDX3 functions in vivo, we synthesized a tricyclic 5:7:5-fused diimidazodiazepine ring (RK-33) to fit into the ATP binding domain of DDX3. Initial results confirmed the binding of RK-33 to DDX3 using a streptavidin pull down assay. Subsequently, we demonstrated the effects of RK-33 in vitro and found a synergy between radiation and RK-33 in lung cancer cells. Also, treatment with RK-33 caused a cell cycle arrest in G1 phase and decreased Cyclin D1 levels in A549 and H1299 lung cancer cells. Furthermore, treatment with RK-33 resulted in decreased radiation induced DNA break repair demonstrated by immunofluorescent staining of A549 lung cancer cells with γH2AX and 53BP1 antibodies. In addition, we showed that the non-homologous end joining (NHEJ) activity in cancer cells was decreased by treatment with RK-33. However, homologues recombination activity was not affected by RK-33. Impaired NHEJ is known to increase radiation sensitivity hence inhibiting NHEJ by RK-33 could improve radiation therapy. Following these encouraging results observed in vitro, we initiated in vivo studies, using a lung cancer model with immune competent mice by expressing Twist1 and Kras selectively in the alveolar type II pneumocytes, which confers lung tissue specificity. Following tumor development, confirmed by micro-CT, the animals were treated with RK-33 (20 mg/kg) and different doses of stereotactic radiation (15 Gy) using a small animal radiation platform. Four weeks following treatment, tumor volumes were determined by micro-CT and H&amp;E staining after necroscopy. A 72% reduction of tumor load was accomplished after treatment with RK-33 and radiation (15 Gy) compared to 28% tumor volume reduction in the radiation alone group. Interestingly, there was less tumor reduction when RK-33 was used in combination with 3 Gy x 10 radiation treatment. No evident toxicity became apparent during the treatment with RK-33. All experiments were done in replicates. Conclusion: These in vitro and in vivo results indicate that RK-33 is a promising and safe radiosensitizer, via inhibition of NHEJ, and could be a promising new drug in combination with stereotactic radiotherapy for lung cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5720. doi:1538-7445.AM2012-5720

Abstract 5081: Inhibiting DNA double strand break repair for alpha radioimmunotherapy

Abstract Metastasis to distant organs is the major cause of mortality in breast cancer patients. Median overall survival remains poor (5 year survival 26.1%) despite recent advances in new targeted biological therapy. Radiolabeled antibody offers an effective approach to deliver cytotoxic radiation doses to eliminate micrometastatic tumors. Alpha particle emitters are promising against small micrometastases because of their high potency and specificity (high linear energy transfer, LET&amp;gt;80 keV/µm, particle range ∼ 80 μm). Alpha particles typically cause DNA double strand breaks (DSBs) when they traverse the cell nucleus. Our experience with alpha particle emitter labeled antibody showed that it significantly prolonged survival in a mouse model of metastatic breast cancer with eventual recurrence at estimated micromet dose of 4.4 Gy. Understanding cellular repair of alpha radiation induced DNA DSBs could help us rationally design more effective strategy by inhibiting components of the DNA DSB repair pathways to sensitize cancer cells to alpha radiation. Under this hypothesis, we evaluated the role of DNA dependent protein kinase catalytic subunit (DNA-PKcs) inhibition on targeted alpha-emitter therapy. Inhibition, with small molecule inhibitor NU7441, of this key enzyme in the non-homologous end joining (NHEJ) pathway sensitizes triple negative breast cancer cells to alpha particle emitter 213Bi labeled anti-EGFR antibody. Immuno-fluorescent staining of γH2AX foci confirms a reduction in the DNA DSB repair rate in these cells. Using a cell-line deficient in the homologous recombination (HR) pathway (BRCA-1 deficient), we also have preliminary evidence suggesting that HR deficiency reduces the incremental effect of NHEJ inhibition but the combination increases overall cell kill. This data suggest inhibiting components of DNA double strand repair pathways is an effective approach to enhance the efficacy of alpha particle emitter labeled monoclonal antibody. 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 5081. doi:10.1158/1538-7445.AM2011-5081

Base Damage Immediately Upstream from Double-Strand Break Ends is a More Severe Impediment to Nonhomologous End Joining than Blocked 3′-Termini

Radiation-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions that are typically repaired by nonhomologous end joining (NHEJ) in human cells. Our previous work indicated that the highly cytotoxic DSBs formed by (125)I decay possess base damage clustered within 8 to 10 bases of the break and 3'-phosphate (P) and 3'-OH ends. This study examined the effect of such structures on NHEJ in in vitro assays employing either (125)I decay-induced DSB linearized plasmid DNA or structurally defined duplex oligonucleotides. Duplex oligonucleotides that possess either a 3'-P or 3'-phosphoglycolate (PG) or a ligatable 3'-OH end with either an AP site or an 8-oxo-dG 1 nucleotide upstream (-1n) from the 3'-terminus have been examined for reparability. Moderate to severe end-joining inhibition was observed for modified DSB ends or 8-oxo-dG upstream from a 3'-OH end. In contrast, abolition of end joining was observed with duplexes possessing an AP site upstream from a ligatable 3'-OH end or for a lesion combination involving 3'-P plus an upstream 8-oxo-dG. In addition, base mismatches at the -1n position were also strong inhibitors of NHEJ in this system, suggesting that destabilization of the DSB terminus as a result of base loss or improper base pairing may play a role in the inhibitory effects of these structures. Furthermore, we provide data indicating that DSB end joining is likely to occur prior to removal or repair of base lesions proximal to the DSB terminus. Our results show that base damage or base loss near a DSB end may be a severe block to NHEJ and that complex combinations of lesions presented in the context of a DSB may be more inhibitory than the individual lesions alone. In contrast, blocked DSB 3'-ends alone are only modestly inhibitory to NHEJ. Finally, DNA ligase activity is implicated as being responsible for these effects.

Dicentric breakage at telomere fusions

Nonhomologous end-joining (NHEJ) inhibition at telomeres ensures that native chromosome ends do not fuse together. But the occurrence and consequences of rare telomere fusions are not well understood. It is notably unclear whether a telomere fusion could be processed to restore telomere ends. Here we address the behavior of individual dicentrics formed by telomere fusion in the yeast Saccharomyces cerevisiae. Our approach was to first stabilize and amplify fusions between two chromosomes by temporarily inactivating one centromere. Next we analyzed dicentric breakage following centromere reactivation. Unexpectedly, dicentrics often break at the telomere fusions during progression through mitosis, a process that restores the parental chromosomes. This unforeseen result suggests a rescue pathway able to process telomere fusions and to back up NHEJ inhibition at telomeres.

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G(1), consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G(2), specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3' overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G(2) through end resection and induction of NHEJ in G(1) through ATM-dependent signaling.

Trapping Rap1 at the telomere to prevent chromosome end fusions

The prevention of nonhomologous end‐joining (NHEJ) reactions between chromosome ends is crucial for maintaining genome stability. Although this protective function is known to be fulfilled by a core of conserved telomeric proteins that are collectively known as Shelterin, its mechanistic details remain a mystery. In this issue, Sarthy et al (2009) lend fresh insight by developing an ingenious method to dissect the role of hRAP1 in preventing telomeric NHEJ independently of other Shelterin components.

DNA-Ligase IV and DNA-Protein Kinase Play a Critical Role in Deficient Caspases Activation in Apoptosis-resistant Cancer Cells by Using Doxorubicin

Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

Multiple pathways inhibit NHEJ at telomeres

The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit.

Nonhomologous End Joining Is Essential for Cellular Resistance to the Novel Antitumor Agent, β-Lapachone

Commonly used antitumor agents, such as DNA topoisomerase I/II poisons, kill cancer cells by creating nonrepairable DNA double-strand breaks (DSBs). To repair DSBs, error-free homologous recombination (HR), and/or error-prone nonhomologous end joining (NHEJ) are activated. These processes involve the phosphatidylinositol 3'-kinase-related kinase family of serine/threonine enzymes: ataxia telangiectasia mutated (ATM), ATM- and Rad3-related for HR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/radiation resistance and increased genomic instability. beta-Lapachone (beta-lap; also known as ARQ 501), currently in phase II clinical trials for the treatment of pancreatic cancer, causes a novel caspase- and p53-independent cell death in cancer cells overexpressing NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 catalyzes a futile oxidoreduction of beta-lap leading to reactive oxygen species generation, DNA breaks, gamma-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that beta-lap exposure results in NQO1-dependent activation of the MRE11-Rad50-Nbs-1 complex. In addition, ATM serine 1981, DNA-PKcs threonine 2609, and Chk1 serine 345 phosphorylation were noted; indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated beta-lap lethality. These studies give insight into the mechanism by which beta-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of beta-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels.

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

Targeting DNA-PK and ATM Increases Sensitivity to Chlorambucil and Fludarabine in Chemoresistant and p53-Defective B-CLL Cells.

A subgroup of B-cell chronic lymphocytic leukemia (CLL) patients develop a more aggressive form of the disease characterised by ZAP70 expression and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are frequent in these patients and confer chemo-resistance. Overexpression of DNA-dependent protein kinase (DNA-PK) correlates with clinical resistance to Chlorambucil (CLB). Thus, alterations in DNA damage signaling pathways are associated with drug resistance. We previously demonstrated that inhibition of DNA-PK sensitizes leukemia cell lines to DNA topoisomerase II poisons and ionising radiation (IR) by inhibition of non-homologous end joining of DNA double strand breaks (DSB). Loss of ATM function compromises the complementary DSB repair pathway, homologous recombination. We hypothesised that repair of DNA damage induced by CLB and Fludarabine may be mediated in part via DNA-PK and ATM and that inhibiting these enzymes would sensitize CLL cells to these agents.NU7441 and KU55933 are potent small molecule inhibitors of DNA-PK and ATM respectively, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). We used these inhibitors in ex-vivo drug combination experiments on CLL lymphocytes derived from a cohort of 60 patients. Lymphocytes were treated with CLB, Fludarabine, Cyclophosphamide, Mitoxantrone or Etoposide in the presence or absence of NU7441 (1μM) or KU55933 (10μM). We observed a concentration-dependent decrease in viability in response to these drugs (XTT assay), and in 70% of cases this decrease was potentiated in the presence of NU7441 (e.g. CLB 2–20 fold potentiation, Fludarabine 2–14 fold). Drug induced caspase 3/7 activity was further increased by NU7441, indicating that DNA-PK inhibition increased cell death via apoptosis. Early data shows that KU55933 also sensitizes tumors to these agents. Increased drug-sensitivity by NU7441 or KU-55933 was independent of disease stage, progression or CD38 status.We quantified γH2AX foci formation (a surrogate marker for DSB) after drug treatment. Fludarabine induced γH2AX foci formation (7 tumors), and this novel observation suggests that Fludarabine induces DSB in quiescent CLL cells. NU7441 increased the number of fludarabine-induced γH2AX foci, implicating DNA-PK as a mediator of DSB repair following Fludarabine treatment. To investigate the significance of p53 functionality on sensitization by inhibitors, we stratified 14 samples from the cohort according to ability to upregulate p21 after IR treatment (FACS analysis). 6/10 cases that displayed impaired p21 upregulation (associated with defects in p53 or ATM function) were sensitized to either CLB or fludarabine by NU7441. 4/4 cases with normal p21 response were sensitized by NU7441. These data are key, since they suggest that NU7441 may have efficacy in cases with p53 dysfunction, a group of tumors representing poor prognosis, drug-resistant patients. Current studies are characterising the cohort (11q,17p deletions) and measuring kinetics of DNA DSB formation and repair. Future studies will further investigate effects of targeting ATM in CLL, and targeting DNA-PK in ATM-defective tumors. Use of DNA-PK and ATM inhibitors represents a targeted and novel therapeutic approach for poor prognosis CLL.