ATR Kinase Inhibitor Research Articles

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress

The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo.

ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.

Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma.

BackgroundATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.MethodsWe undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.ResultsNo defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.ConclusionsAnalyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1649-3) contains supplementary material, which is available to authorized users.

Development of a Continuous Flow Sulfoxide Imidation Protocol Using Azide Sources under Superacidic Conditions

The development of a continuous flow sulfoxide imidation protocol for a pharmaceutically relevant target molecule is described. Sulfoxide imidation is a key step in the preparation of certain ATR kinase inhibitors. Reactions with NaN3 or TMSN3 and concentrated sulfuric acid under literature conditions provided low conversions and poor selectivities. In contrast, reactions employing fuming sulfuric acid afforded the target sulfoximine with a selectivity of ∼90% after a reaction time of only 10–15 min at 50 °C. The imidation reaction using TMSN3 as reagent was successfully performed in a flow reactor utilizing CH2Cl2/H2SO4 biphasic conditions. The mixture was subsequently quenched in-line with H2O. Phase separation, neutralization, and re-extraction with an organic solvent furnished the product in excellent purity and good yields, albeit with loss of chirality.

Activation of NF-κB by human papillomavirus 16 E1 limits E1-dependent viral replication through degradation of E1.

NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Abstract B91: Combined ATM and ATR kinase inhibition selectively kills p53-mutated non-small cell lung cancer (NSCLC) cells.

Abstract Introduction: Cancer cells frequently exhibit defects in components of the DNA-damage response (DDR) and may as a consequence become highly dependent on remaining DDR pathways to survive DNA damage. Targeting such remaining DDR pathways to confer synthetic lethality has become a valuable new strategy for personalised cancer therapy, and an attractive target for this approach is the central DDR kinase Ataxia Telangiectasia and Rad3 related (ATR). Several small molecule inhibitors of ATR are now entering clinical development. However, the challenges remain to identify patient subgroups that might benefit from ATR inhibitors, and to integrate these agents with standard therapies. Two important components of the DDR, Ataxia Telangiectasia Mutated (ATM) and p53, are commonly mutated and inactivated in lung cancer. In this study, we therefore evaluate the potential of ATR kinase inhibition for the treatment of ATM- or p53-deficient human NSCLC, both as single agent and in combination with ionizing radiation (IR). Methods: We used proliferation and clonogenic survival assays to measure the sensitivity of a panel of NSCLC cell lines to the selective ATR kinase inhibitor VE-821. Inhibition of ATR mediated signalling was assessed by immunoblotting. Immunofluorescence and chromosome analysis were used to assess the induction of DNA damage following treatment of cells with either VE-821 alone, or in combination with the selective ATM inhibitor KU55933. FACS analysis was used to measure cell cycle phase distribution and DNA content. Results: NSCLC cells deficient in both ATM and p53 were highly sensitive to ATR inhibition (IC50 0.7 µM), but there was no clear association between sensitivity and mutation status of either ATM or p53 alone (IC50 3.9 - 8.9 µM). We observed similar relative sensitivities in clonogenic survival assays and in combination with IR. This increased cytotoxicity of ATR inhibition in a combined ATM- and p53-deficient background was associated with formation of giant micronucleated cells and evidence of mitotic catastrophe within 48 h of treatment. Furthermore, we found a marked increase in VE-821-induced chromosomal damage in cells deficient in ATM and p53, compared with cells deficient in either gene alone. We confirmed this dependency on p53 mutation status and ATM function for sensitivity to ATR inhibition by treating p53-mutated NSCLC cells with a combination of VE-821 and KU55933. In this setting, inhibition of ATM markedly sensitised tumour cells to VE-821 treatment, as shown by a reduction in clonogenic survival and an increase in chromosomal damage. Conclusion: Our data suggest that in NSCLC the functional status of both ATM and p53 determines the cellular response to ATR inhibition and that combined ATM and ATR kinase inhibition selectively kills p53 mutant cells. A combination of ATM and ATR inhibitors may therefore have a broad utility for the treatment of p53 mutated NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B91. Citation Format: Anika M. Weber, Sivan M. Bokobza, Aoife M. Devery, Anderson J. Ryan. Combined ATM and ATR kinase inhibition selectively kills p53-mutated non-small cell lung cancer (NSCLC) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B91.

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage

The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60)

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4h after irradiation by the dose of 6Gy. Within 24h after gamma-irradiation with a dose of 3Gy, the cells accumulated in the G2 phase (67%) and the number of cells in S phase decreased. KU55933 (10μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144h interval after irradiation with 3Gy, there was a considerable induction of apoptosis in the VE-821 group (10μM). The repair of the radiation damage, as observed 72h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77% of cells, respectively, were free of DSBs), whereas in the group incubated with 10μM VE-821, there were only 61% of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

Molecular dynamics-based self-organizing molecular field analysis on 3-amino-6-arylpyrazines as the ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase inhibitors

The ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase is one of the apical regulators in the DNA damaging response signaling pathways. Inhibition of ATR kinase may greatly potentiates the cyto-toxicity by DNA damaging agents to the tumor cells while, have minimum effect on normal cells. In this article, the impact of ligand conformation on the three-dimensional quantitative structure–activity relationship (3D-QSAR) model of a series of 3-amino-6-arylpyrazines as ATR kinase inhibitors was investigated. We employed molecular dynamics (MDs) simulations to get the dynamic active conformations (DACs) of the compounds in the ATP-binding site of ATR kinase. As a result, the model based on the DACs extracted from the first nanosecond MD simulation is superior to that using static active conformations from docking with r 2 = 0.917; $$q_{\text{LOO}}^{2}$$ = 0.880; standard error of estimate [SEE] = 0.259; F = 347.29; $$r_{\text{pred}}^{2}$$ = 0.923; SEEpred = 0.301. Our results highlight the importance of incorporating DACs of ligand using MD simulation in 3D-QSAR studies. This study may also provide useful information to rationalize the design of novel ATR kinase inhibitors.

ATR Inhibition Broadly Sensitizes Ovarian Cancer Cells to Chemotherapy Independent of BRCA Status

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

Abstract 2639: RanGAP1·SUMO1 hyperphosphorylation (RGSH) and mitotic arrest by ON 01910.Na, okadaic acid or tubulin agents

Abstract INTRODUCTION: RanGAP1·SUMO1 is phosphorylated before nuclear envelope breakdown when cells enter mitosis. Previous work from our laboratory showed that a new anticancer antimitotic agent, ON 01910.Na (abbreviated 1910), a benzyl styryl sulfone analog, induced RGSH in all four human tumor cell lines tested. RGSH was not observed in the presence of 1911, inactive analog of 1910. However, RGSH was observed in the presence of tubulin depolymerizing agent nocodazole. Tubulin polymerization assay revealed that, unlike nocodazole, neither 1910 nor 1911 had effects on tubulin polymerization in vitro. In this study, we examined whether other tubulin agents induce RGSH and whether known mitotic phosphatases are involved. METHODS and RESULTS: We used two cell lines, MOLT-3 ALL and DU145 prostate cancer cell lines. We examined RGSH in the presence of known tubulin toxins including paclitaxel, vinblastine, vincristine, colchicine and nocodazole at a concentration of 1 µM in 4 h and 24 h. Doxorubicin and camptothecin served as negative controls. Following treatment with drugs, cells were analyzed by Western blotting. Drug concentrations that inhibited cell growth by 50% (IC50) or 90% (IC90) were determined in 3-day drug exposure experiments. All tubulin agents in the study were capable of inducing RGSH after 4h and 24 h exposure in both cell lines. The drug-induced RGSH was inhibited by caffeine, an ATM and ATR kinase inhibitor, and roscovitine, a Cdk kinase inhibitor. Okadaic acid (OA), known primarily as PP1 and PP2A phosphatase inhibitor, was also found to be capable of RGSH, therefore making it plausible by analogy that 1910 is RanGAP1·SUMO1 phosphatase inhibitor. The concentrations of 1910, OA or tubulin agents sufficient to elicit RGSH in 24 h, also caused 50 to 90% growth inhibition, respectively. 1910, as well as all tubulin agents in the study, induced phosphorylation of the phosphatase Cdc25C (aka “mitotic” Cdc25C) and down-regulation of interphase form of Cdc25C. CONCLUSION: 1910 and selected tubulin agents caused RGSH at IC50 – IC90 concentrations. Similar to tubulin agents, 1910 produced Cdc25C phosphorylation and down-regulation of its interphase form. OA inhibition of PP1 phosphatase and activation of RGSH suggest that 1910 could also serve as a phosphatase inhibitor. 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 2639. doi:10.1158/1538-7445.AM2011-2639

Cell cycle arrest triggered by conjugated eicosapentaenoic acid occurs through several mechanisms including G1 checkpoint activation by induced RPA and ATR expression

Background Conjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). Methods Human colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis. Results The levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells. General significance cEPA could inhibit the activity of replicative pols, such as pols α, δ and ɛ, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells.

ATR-Chk1 Axis Protects BCR/ABL Leukemia Cells from the Lethal Effect of DNA Double-Strand Breaks

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (γ-H2AX). γ-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Modulation of XPA Nuclear Accumulation by ATR Dependent Checkpoint Pathways in Response to UV Irradiation

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progression to allow time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses initiated by UV irradiation. Here we demonstrate that the NER factor XPA accumulated in the nucleus following UV irradiation, and the event occurred in an ATR-dependent manner. Treatment of cells with ATR kinase inhibitors and transfection of cells with siRNA targeted to ATR both compromised the UV-induced XPA nuclear translocation. XPA nuclear accumulation was also disrupted in ATR-deficient cells as well as in cells expressing ATR-kinase dead, as observed by immunofluorescent microscopy and western blot. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggest that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

ATR-Chk1 Axis Is Activated, but the Function of Chk1 Is Disrupted in BCR/ABL Leukemia Cells Responding to DNA Damage.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.

Regulation of Cellular and SV40 Virus Origins of Replication by Chk1-dependent Intrinsic and UVC Radiation-induced Checkpoints

DNA replication is inhibited by DNA damage through cis effects on replication fork progression and trans effects associated with checkpoints. In this study, we employed a combined pulse labeling and neutral-neutral two-dimensional gel-based approach to compare the effects of a DNA damaging agent frequently employed to invoke checkpoints, UVC radiation, on the replication of cellular and simian virus 40 (SV40) chromosomes in intact cells. UVC radiation induced similar inhibitory effects on the initiation and elongation phases of cellular and SV40 DNA replication. The initiation-inhibitory effects occurred independently of p53 and were abrogated by the ATM and ATR kinase inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of cellular origins was also abrogated by the expression of a dominant-negative Chk1 mutant. These results indicate that UVC induces a Chk1- and ATR or ATM-dependent checkpoint that targets both cellular and SV40 viral replication origins. Loss of Chk1 and ATR or ATM function also stimulated initiation of cellular and viral DNA replication in the absence of UVC radiation, revealing the existence of a novel intrinsic checkpoint that targets both cellular and SV40 viral origins of replication in the absence of DNA damage or stalled DNA replication forks. This checkpoint inhibits the replication in early S phase cells of a region of the repetitive rDNA locus that replicates in late S phase. The ability to detect these checkpoints using the well characterized SV40 model system should facilitate analysis of the molecular basis for these effects.