Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption.     

Irreversible chromosome damage accumulates rapidly in the absence of ATM kinase activity

Mutations in the ATM kinase cause the neurodegenerative disorder ataxia telangiectasia (A-T) and affected individuals are exquisitely radiation-sensitive and cancer-prone. Cells derived from A-T individuals contain chromosome aberrations and exhibit profound cellular radiosensitivity. ATM is an apical kinase critical for the activation of cell cycle checkpoints and the induction of apoptosis in irradiated cells. However, defects in these pathways are insufficient to account for the chromosomal instability seen in A-T cells. We show here that the small molecule KU55933 can be used as a “molecular switch” to selectively and transiently inhibit ATM kinase activity in cells. We subsequently show that the cellular radiosensitization seen when ATM kinase activity is inhibited for one hour following exposure to γ-rays, accounts for over 70% of the total cellular radiosensitization seen when ATM kinase activity is inhibited for 17h. Finally, we show that inhibition of ATM kinase activity for one hour following exposure to irradiation doubles the number of chromosome aberrations occurring in late-S- and G2-, but not M-phase, cells. These observations are unexpected and suggest that irreversible chromosome damage accumulates very rapidly when ATM kinase activity is transiently inhibited following irradiation. We propose that we have revealed an essential, yet previously undescribed, role for ATM kinase in suppressing chromosomal instability     

DNA-activated protein kinase functions in a newly observed S phase checkpoint that links histone mRNA abundance with DNA replication

DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression. Here, we expose S phase–arrested cells to the kinase inhibitors caffeine and LY294002. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress–induced histone mRNA down-regulation. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. Histone mRNA decay does not require Chk1/Chk2. Replication stress induces phosphorylation of UPF1 but not hairpin-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase–like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK–mediated phosphorylation of UPF1.     

Participation of ATM in insulin signalling through phosphorylation of eIF-4E-binding protein 1

One of the critical responses to insulin treatment is the stimulation of protein synthesis through induced phosphorylation of the eIF-4E-binding protein 1 (4E-BP1), and the subsequent release of the translation initiation factor, eIF-4E. Here we report that ATM, the protein product of the ATM gene that is mutated in the disease ataxia telangiectasia, phosphorylates 4E-BP1 at Ser 111 in vitro and that insulin treatment induces phosphorylation of 4E-BP1 at Ser 111 in vivo in an ATM-dependent manner. In addition, insulin treatment of cells enhances the specific kinase activity of ATM. Cells lacking ATM kinase activity exhibit a significant decrease in the insulin-induced dissociation of 4E-BP1 from eIF-4E. These results suggest an unexpected role for ATM in an insulin-signalling pathway that controls translation initiation. Through this mechanism, a lack of ATM activity probably contributes to some of the metabolic abnormalities, such as poor growth and insulin resistance, reported in ataxia telangiectasia cells and patients with ataxia telangiectasia.     

Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

ATMIN defines an NBS1-independent pathway of ATM signalling

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.     

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5′ ends of a bridged DSB are juxtaposed such that DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.Key words: ATM, Mre11, MRN complex, DNA degradation, double-strand break repair, microhomology-mediated end joining, PI-3-kinase-like kinases     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

Purification and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive serine/threonine protein kinase

ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.     

ATM and Chk2 kinase target the p53 cofactor Strap

The p53 cofactor Strap (stress responsive activator of p300) is directly targeted by the DNA damage signalling pathway where phosphorylation by ATM (ataxia telangiectasia mutated) kinase facilitates nuclear accumulation. Here, we show that Strap regulation reflects the coordinated interplay between different DNA damage-activated protein kinases, ATM and Chk2 (Checkpoint kinase 2), where phosphorylation by each kinase provides a distinct functional consequence on the activity of Strap. ATM phosphorylation prompts nuclear accumulation, which we show occurs by impeding nuclear export, whereas Chk2 phosphorylation augments protein stability once Strap has attained a nuclear location. These results highlight the various functional roles undertaken by the DNA damage signalling kinases in Strap control and, more generally, shed light on the pathways that contribute to the regulation of the p53 response.     

The catalytic subunit of DNA-dependent protein kinase is downstream of ATM and feeds forward oxidative stress in the selenium-induced senescence response

Selenium induces a senescence response in cells through induction of ataxia–telangiectasia mutated (ATM) and reactive oxygen species (ROS). Although a role of the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs) in DNA double-strand break repair is established, it is unclear how these proteins function in response to selenium-induced oxidative stress and senescence induction. In this study, we demonstrated that pretreating normal human diploid fibroblasts with DNA-PK kinase inhibitor NU 7026 suppressed selenium-induced senescence response. Selenium treatment induced phosphorylation of DNA-PK_cs on Thr-2647 and Ser-2056, the extent of which was decreased in the presence of ATM kinase inhibitor KU 55933 or the antioxidants N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl. In contrast, the selenium-induced phosphorylation of ATM on Ser-1981 was not affected by NU 7026. Cells deficient in DNA-PK_cs or pretreated with NU 7026 or N-acetylcysteine were defective in selenite-induced ROS formation. Taken together, these results indicate a distinct role of DNA-PK_cs, in which this kinase can respond to and feed forward selenium-induced ROS formation and is placed downstream of ATM in the resultant senescence response.     

ATM is involved in cell‐cycle control through the regulation of retinoblastoma protein phosphorylation

Ataxia telangiectasia mutated protein (ATM) is a member of the phosphatidylinositol‐3 kinase (PI3K) family, which has a role in the cellular response to DNA double‐strand breaks (DSBs). In the present study, we evaluated the role of ATM in cell‐cycle control in dopaminergic rat neuroblastoma B65 cells. For this purpose, ATM activity was either inhibited pharmacologically with the specific inhibitor KU‐55933, or the ATM gene was partially silenced by transfection with small interfering RNA (siRNA). Our data indicate that although ATM inhibition did not affect the cell cycle, both treatments specifically decreased the levels of cyclin A and retinoblastoma protein (pRb), phosphorylated at Ser780. Furthermore, ATM inhibition decreased the active form of p53, which is phosphorylated at Ser15, and also decreased Bax and p21 expression. Using H₂O₂ as a positive control of DSBs, caused a rapid pRb phosphorylation, this was prevented by KU‐55933 and siRNA treatment. Collectively, our data demonstrate how a new molecular network on ATM regulates the cell cycle through the control of pRb phosphorylation. These findings support a new target of ATM. J. Cell. Biochem. 110: 210–218, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair

Poly (ADP-ribose) polymerase (PARP-1), ATM and DNA-dependent protein kinase (DNA-PK) are all involved in responding to DNA damage to activate pathways responsible for cellular survival. Here, we demonstrate that PARP-1^−/− cells are sensitive to the ATM inhibitor KU55933 and conversely that AT cells are sensitive to the PARP inhibitor 4-amino-1,8-napthalamide. In addition, PARP-1^−/− cells are shown to be sensitive to the DNA-PK inhibitor NU7026 and DNA-PKcs or Ku80 defective cells shown to be sensitive to PARP inhibitors. We believe PARP inhibition results in an increase in unresolved spontaneous DNA single-strand breaks (SSBs), which collapse replication forks and trigger homologous recombination repair (HRR). We show that ATM is activated following inhibition of PARP. Furthermore, PARP inhibitor-induced HRR is abolished in ATM, but not DNA-PK, inhibited cells. ATM and DNA-PK inhibition together give the same sensitivity to PARP inhibitors as ATM alone, indicating that ATM functions in the same pathways as DNA-PK for survival at collapsed forks, likely in non-homologous end joining (NHEJ). Altogether, we suggest that ATM is activated by PARP inhibitor-induced collapsed replication forks and may function upstream of HRR in the repair of certain types of double-strand breaks (DSBs).     

Loss of ATM kinase activity leads to embryonic lethality in mice

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.     

Inhibition of the cGAS‐STING pathway ameliorates the premature senescence hallmarks of Ataxia‐Telangiectasia brain organoids

Ataxia‐telangiectasia (A‐T) is a genetic disorder caused by the lack of functional ATM kinase. A‐T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A‐T remains elusive. Here, we utilize human pluripotent stem cell‐derived cortical brain organoids to study A‐T neuropathology. Mechanistically, we show that the cGAS‐STING pathway is required for the recognition of micronuclei and induction of a senescence‐associated secretory phenotype (SASP) in A‐T olfactory neurosphere‐derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self‐DNA‐triggered SASP expression in A‐T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A‐T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A‐T and constitutes a novel therapeutic target for treating neuropathology in A‐T patients.     

Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites

Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11–RAD50–NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Autophosphorylated ATM interacts with MDC1 and the latter is required for the prolonged association of ATM to DSBs. Ablation of ATM autophosphorylation or knock-down of MDC1 protein affects the ability of ATM to phosphorylate downstream substrates and confer radioresistance. Together, these data suggest that autophosphorylation at serine 1981 stabilizes ATM at the sites of DSBs, and this is required for a proper DNA damage response.