ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A.

Identifying genetic biomarkers of synthetic lethal drug sensitivity effects provides one approach to the development of targeted cancer therapies. Mutations in ARID1A represent one of the most common molecular alterations in human cancer, but therapeutic approaches that target these defects are not yet clinically available. We demonstrate that defects in ARID1A sensitize tumour cells to clinical inhibitors of the DNA damage checkpoint kinase, ATR, both in vitro and in vivo. Mechanistically, ARID1A deficiency results in topoisomerase 2A and cell cycle defects, which cause an increased reliance on ATR checkpoint activity. In ARID1A mutant tumour cells, inhibition of ATR triggers premature mitotic entry, genomic instability and apoptosis. The data presented here provide the pre-clinical and mechanistic rationale for assessing ARID1A defects as a biomarker of single-agent ATR inhibitor response and represents a novel synthetic lethal approach to targeting tumour cells.

Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities.

ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.

Development of pharmacodynamic biomarkers for ATR inhibitors.

BACKGROUND: ATR, which signals DNA damage to S/G2 cell cycle checkpoints and for repair, is an attractive target in cancer therapy. ATR inhibitors are being developed and a pharmacodynamic assay is needed to support clinical studies. METHODS: Phosphorylation of ATR targets, Chk1 and H2AX, was evaluated in MCF7 and K562 cells, human volunteer PBMCs and whole blood by Western blot, immunofluorescence microscopy and flow cytometry after DNA damage. The effect of cell cycle phase, ATR knockdown and inhibition on these phosphorylation events was determined. RESULTS: Hydroxyurea, UV and 4NQO induced Chk1 and H2AX phosphorylation in MCF7 and K562 cells. UV/4NQO activation of ATR was detectable in non-cycling cells. Chk1 phosphorylation was reduced by ATR knockdown and reflects ATR activity for 3 h, H2AX phosphorylation after UV/4NQO is ATR-dependent for 1 h but increasingly ATM and DNA-PK-dependent at later time points. In isolated PBMCs both phospho-targets were detectable after UV/4NQO but in PBMCs from whole blood treated with 4NQO only H2AX was detectable. CONCLUSION: PhosphoChk1 and H2AX are useful biomarkers for ATR inhibition using a variety of immuno-detection methods, but timing may be critical. Importantly, ATR activity is detectable in non-cycling PBMCs allowing them to be used as a surrogate tissue for biomarker measurement. In PBMCs from whole blood treated with 4NQO phosphoH2AX was the most useful biomarker of ATR activity and a clinically viable pharmacodynamic assay for ATR inhibitors has been developed.

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

Structure of human Bloom's syndrome helicase in complex with ADP and duplex DNA.

Bloom's syndrome is an autosomal recessive genome-instability disorder associated with a predisposition to cancer, premature aging and developmental abnormalities. It is caused by mutations that inactivate the DNA helicase activity of the BLM protein or nullify protein expression. The BLM helicase has been implicated in the alternative lengthening of telomeres (ALT) pathway, which is essential for the limitless replication of some cancer cells. This pathway is used by 10-15% of cancers, where inhibitors of BLM are expected to facilitate telomere shortening, leading to apoptosis or senescence. Here, the crystal structure of the human BLM helicase in complex with ADP and a 3'-overhang DNA duplex is reported. In addition to the helicase core, the BLM construct used for crystallization (residues 640-1298) includes the RecQ C-terminal (RQC) and the helicase and ribonuclease D C-terminal (HRDC) domains. Analysis of the structure provides detailed information on the interactions of the protein with DNA and helps to explain the mechanism coupling ATP hydrolysis and DNA unwinding. In addition, mapping of the missense mutations onto the structure provides insights into the molecular basis of Bloom's syndrome.

The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy.

DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR.

Here we report a comprehensive biological characterization of a potent and selective small-molecule inhibitor of the DNA damage response (DDR) kinase ATR. We show a profound synthetic lethal interaction between ATR and the ATM-p53 tumor suppressor pathway in cells treated with DNA-damaging agents and establish ATR inhibition as a way to transform the outcome for patients with cancer treated with ionizing radiation or genotoxic drugs.

Discovery of potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase as potential anticancer agents.

DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in most cancers. This may be attributed to a genome maintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a K(i) of 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC(50) of 0.42 µM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlights the great potential for ATR inhibition as a novel mechanism to dramatically increase the efficacy of many established drugs and ionizing radiation.

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response.

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.

Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM.

The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio- and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3'-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC(50) of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3'-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, gammaH2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.

Activation of the DNA damage response by telomere attrition: a passage to cellular senescence.

Critical telomere shortening induces senescence in many normal human cell types grown in culture. Recent data have revealed that dysfunctional telomeres can resemble certain forms of DNA damage, and point to a role for DNA damage signaling in the establishment and maintenance of telomere-initiated senescence. Here, we review these new observations and highlight potential avenues of future research. We consider the identities of the key DNA damage response factors involved in senescence and discuss a model for the molecular events occurring in pre-senescent cells that ultimately lead to a permanent cell cycle arrest phenotype.

The ATR-p53 pathway is suppressed in noncycling normal and malignant lymphocytes.

Chronic lymphocytic leukaemia (CLL) results from the accumulation of apoptosis-resistant clonal B cells that are arrested in G0/G1, and is heterogeneous with respect to clinical outcome. An aggressive form of the disease is identified by an impaired p53 response to ionizing radiation (IR). This is associated with inactivating mutations of either p53 or ATM, a regulator of p53 activated by IR-induced DNA damage. Since other forms of DNA damage activate p53 via ATR, a kinase closely related to ATM, abnormalities of the ATR-p53 pathway also have the potential to result in p53 dysfunction. We therefore tested cases of CLL for abnormal p53 responses to ultraviolet irradiation (UVC), a known activator of ATR, to screen for additional forms of p53 dysfunction. CLL cells and normal peripheral blood mononuclear cell (PBMC) preparations (predominantly noncycling lymphocytes) were treated with UVC and assessed for p53 responses. In all of the CLL cases and PBMC preparations tested, we were unable to detect p53 accumulation, phosphorylation or transcriptional consequences in response to UVC-induced DNA damage. The most likely explanation for the absence of UVC-induced p53 activation in CLL and normal lymphocytes was that, in contrast to other cell types, the UVC-induced ATR pathway was inactive. This notion was confirmed by showing that ATR protein was absent or undetectable in all of the cases of CLL and normal PBMCs screened. This was an unexpected finding because ATR was thought to be essential for the viability of somatic cells and for normal human and murine embryonic development. An obvious difference between the cell lines used as positive controls for ATR antibodies and the CLL cells/PBMCs was that the former were actively cycling while the latter were quiescent. We therefore hypothesized that the ATR-p53 pathway is selectively downregulated in noncycling lymphocytes. To test this, we induced cycling in the T-cell fraction of PBMC preparations and demonstrated that ATR protein expression was restored. Furthermore, p53 was upregulated and phosphorylated in response to UVC in these cells. Our data support the conclusion that the ATR-p53 pathway is suppressed in noncycling lymphocytes via ATR downregulation. We tentatively suggest that this repressed DNA damage response may have evolved to protect quiescent lymphocytes from the potential for p53-dependent apoptosis in the face of some forms of endurable genotoxic stress. If this is the case, DNA repair and genome stability might be compromised in quiescent lymphocytes with potentially negative consequences.

A DNA damage checkpoint response in telomere-initiated senescence.

Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres--the ends of linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.