Inhibition of ATM kinase activity does not phenocopy ATM protein ...

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Aug 25, 2010 - ATM protein disruption. Implications for the clinical utility of ATM kinase inhibitors. Serah Choi,1 Armin M. Gamper,2 Jason S. White4 and ...
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Cell Cycle 9:20, 4052-4057; October 15, 2010; © 2010 Landes Bioscience

Inhibition of ATM kinase activity does not phenocopy ATM protein disruption Implications for the clinical utility of ATM kinase inhibitors Serah Choi,1 Armin M. Gamper,2 Jason S. White4 and Christopher J. Bakkenist2,3,* Medical Scientist Training Program; Molecular Pharmacology Graduate Program; 2Department of Radiation Oncology; Department of Pharmacology and Chemical Biology; University of Pittsburgh School of Medicine; Hillman Cancer Center; Pittsburgh, PA USA; 4 BioCytics Inc.; Huntersville, NC USA 1 3

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Key words: ataxia telangiectasia, ATM, KU55933, KU60019, double-strand break repair, homologous recombination, sister chromatid exchange Submitted: 08/25/10 Accepted: 08/27/10 Previously published online: www.landesbioscience.com/journals/cc/ article/13471 DOI: 10.4161/cc.9.20.13471 *Correspondence to: Christopher J. Bakkenist; Email: [email protected]

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iallelic mutations in ataxiatelangiectasia mutated (ATM), which encodes for a protein kinase, cause ataxia telangiectasia (A-T). A-T is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation (IR). A-T patients typically lack both detectable ATM protein and ATM kinase activity, and small molecule inhibitors of ATM kinase activity have been developed as strategies to improve radiotherapy for the treatment of cancers. As predicted, inhibition of ATM kinase activity is sufficient to radiosensitize cells. However, we recently showed that inhibition of ATM kinase activity disrupts DNA damage-induced sister chromatid exchange (SCE). This result was unanticipated since SCE is normal in A-T cells that lack detectable ATM protein. In these studies, we showed, for the first time, that the consequences of inhibition of ATM kinase activity and adaptation to ATM protein disruption are distinct. Here, we discuss the mechanistic implications of this finding for the function of ATM at the replication fork and the clinical utility of ATM kinase inhibitors. Introduction

Ataxia telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, predisposition to cancers and clinical radiosensitivity.1 Despite its rare incidence (1 to 2.5 in 100,000),2 the pleiotropic nature of A-T and the pathophysiology underlying its radiosensitive

phenotype have long puzzled the biomedical community. A-T is caused by biallelic mutations in the ATM gene3 (which encodes for the ATM protein kinase) and patients with A-T typically lack detectable ATM protein. Cells derived from A-T patients exhibit defective cell cycle checkpoint responses to ionizing radiation (IR), profound radiosensitivity and high levels of chromosome aberrations, indicating the importance of ATM for the maintenance of chromosome stability and radioprotection.1 Hence, efforts to improve radiotherapy have driven the development of small molecule inhibitors of ATM kinase activity, which would allow the radiosensitivity of A-T patients to be mimicked by cancer cells. Consistent with the A-T phenotype, ATM kinase inhibitors, such as KU55933 and KU60019, radiosensitize cells. However, recently we showed that KU55933 and KU60019 also disrupt DNA damage-induced sister chromatid exchange (SCE), a mechanism of homologous recombination repair (HRR), in cells with functional ATM protein.4 This phenotype is incongruent with that of A-T cells, in which SCE is normal.5-7 Moreover, we showed that KU55933 and KU60019 had no effect on SCE in cells lacking ATM protein, indicating that disruption of SCE is not due to an off-target effect of the inhibitors.4 These data demonstrate that inhibition of ATM kinase activity in a “normal” individual that expresses stable ATM protein can result in a strikingly different phenotype to that of A-T cells or animals that do not express ATM protein.

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ATM kinase Activity

ATM kinase Inhibitors

ATM encodes a 350 kDa predominantly nuclear serine/threonine protein kinase. Cells derived from patients with the classical A-T phenotype lack ATM kinase activity as a result of either compound heterozygosity or, less frequently, homozygosity for truncating mutations (frameshift or nonsense mutations). In both cases, the mutations result in an absence of stable ATM protein.8,9 Thus, historically studies investigating the pathophysiology of A-T have, quite appropriately, been performed using cells and animal models that lack ATM kinase activity as a result of a failure to express ATM protein. ATM kinase activity is rapidly stimulated in cells exposed to IR.10-12 We have previously shown that ATM kinase activation is associated with autophosphorylation on serine-1981 and have generated highly sensitive antibodies that recognize ATM solely when phosphorylated on serine-1981.10 With these reagents, we determined that ATM kinase activity is maximal within 15 min following 0.4 Gy IR, at which point over 50% of ATM is phosphorylated.10 Moreover, ATM kinase activity is increased in cells exposed to as little as 0.05 Gy IR and following the introduction of just 2 DSBs per cell.10,13 A considerable body of literature documents the ATM kinase-dependent mobilization, modification and upregulation of proteins critical for the induction of cell cycle checkpoints and apoptosis following IR. Over 1,000 ATM and ATR kinase-dependent phosphorylations have been identified in cells.14,15 ATM kinasedependent phosphorylations have been found to modify proteins involved in DNA replication, DNA repair, cell cycle progression and numerous signaling pathways.14 Despite these efforts, the indispensable ATM kinase-dependent mechanisms that ensure genome stability and cell survival are not well understood. Isolating changes in protein function that are causally related to A-T or its cellular phenotype may be particularly challenging since stress-activated kinases such as ATM have little selective pressure to restrict functionally insignificant phosphorylations.

ATM kinase inhibitors have proven to be instrumental in studies of ATM kinasedependent functions. To date, three selective inhibitors of ATM kinase activity have been identified: KU55933,16 CP466722,17 and KU60019.18 As expected, ATM kinase inhibition using KU55933, CP466722 or KU60019 is sufficient to enhance cellular sensitivity to IR.16-18 We showed that the competitive ATP inhibitors KU55933 and KU60019 can be used as “molecular switches” to selectively and transiently inhibit ATM kinase activity in cells. ATM kinase activity is inhibited in irradiated cells within 15 min of the addition of KU55933 or KU60019 and is restored within 15 min following the removal of either inhibitor.4,13 Thus, the immediate and reversible nature of KU55933- and KU60019-mediated inhibition enables studies that temporally isolate ATM kinase-dependent functions. We showed that transient inhibition of ATM kinase activity for just 1 h following irradiation is sufficient to sensitize cells to ionizing radiation. Surprisingly, the radiosensitization seen when ATM kinase activity was inhibited for just 1 h, from +15 to +75 min following exposure to IR, accounted for over 70% of the total cellular radiosensitization seen when ATM kinase activity was inhibited for 17 h. Furthermore, transient inhibition of ATM kinase activity from +15 to +75 min resulted in significantly more cell death than ATM kinase inhibition from -45 to +15 min following IR.13 These data show that an indispensable ATM kinase-dependent mechanism exists during this 1 h post-IR window that ensures cell survival. To elucidate the mechanism of increased radiosensitivity within this 1 h post-IR window, we investigated the effect of transient ATM kinase inhibition on chromosome aberrations. A previous study in murine B cells had already shown accumulation of chromosome aberrations in cells treated with KU55933 for 2 to 4 days.19 We observed that inhibition of ATM kinase activity for just 1 h, from +15 to +75 min following exposure to IR, doubled the number of chromosome aberrations occurring in late-S- and G2-, but not M-phase, cells.13 Therefore,

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it is apparent that ATM kinase activity is required to phosphorylate a substrate(s) in the hour following irradiation to suppress the formation of DNA damage that ultimately manifests as persistent chromosome aberrations (Fig. 1). Moreover, our finding that the number of chromosome aberrations in IMR90 and H460 cells is doubled in late-S- and G2-, but not M-phase, when ATM kinase is inhibited from +15 min to +75 min following exposure to 2 Gy IR, suggests that the persistent DNA damage that accumulates in cells in the absence of ATM kinase activity can in fact be recognized by cell cycle checkpoints, as previously suggested.20 Since ATM kinase activity is restored when KU55933 is removed from cells, the persistent DNA damage may induce an ATM kinase-dependent G2 checkpoint. Thus, our studies were able to dissociate an ATM kinase-dependent DNA repair mechanism from an ATM kinasedependent cell cycle checkpoint mechanism. We investigated whether the increase in chromosome aberrations could be attributed to a defect in homologous recombination repair (HRR), since a defect in HRR can result in an accumulation of chromatid breaks.21,22 Defective HRR is consistent with all known phenotypes of ataxia telangiectasia and A-T cells, from radiosensitivity to chromosome instability and predisposition to malignancy. To assay for HRR, we looked at sister chromatid exchange (SCE), a cytological manifestation of the repair of stalled and collapsed DNA replication forks via a subset of HRR.23 Similarly, we showed that SCE is disrupted in irradiated cells in the absence of ATM kinase activity. Transient inhibition of ATM kinase activity for just 1 h, from +15 to +75 min following exposure to IR, abrogates IR-induced SCE.4 Remarkably, IR-induced SCE is not recovered when cellular ATM kinase activity is restored in cells following a 1 h interval of inhibition. We also showed that ATM kinase inhibition disrupts topoisomerase 1 poison-induced SCE.4 These results were surprising because SCE had previously been shown to be normal in A-T cells.5-7 Indeed, we determined that DNA damage-induced SCE was maintained in A-T cells that express

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Figure 1. Transient kinase inhibition of ATM has long-term consequences, even after restoration of ATM kinase activity (removal of inhibitor). At 12 h post irradiation (IR), we observed a decrease in sister chromatid exchange (SCE), a cytological manifestation of homologous recombination repair of double-strand breaks at the replication fork.4 At 48 h post-IR, there is an increase in chromosome aberrations. Cell proliferation and survival are decreased at ~10 days, as measured by clonogenic assay.13

no ATM protein and that the ATM kinase inhibitors had no effect on DNA damageinduced SCE in A-T cells, indicating that the disruption of DNA damage-induced SCE in cells expressing ATM is not a consequence of an “off-target” effect of either KU55933 or KU60019. These data revealed for the first time that the consequences of inhibition of ATM kinase activity and adaptation to ATM protein disruption are distinct.4 Mechanistic Considerations Currently, we are interested in determining how SCE is disrupted by ATM kinase inhibition but not by ATM protein disruption and the localization of ATM is likely key to this mechanism. Since SCE represents the repair of damaged DNA replication forks,23 we favor a model in which “kinase-inhibited ATM” presents a physical impediment to SCE at DSBs at damaged replication forks. We had considered that ATM’s known functions in cell cycle checkpoints might contribute to the observed defect in SCE but we were unable to detect any defect in either the activation or the recovery of the G2 /M checkpoint when ATM kinase activity was transiently inhibited.4 This was the most significant checkpoint to our analyses since any delay in G2 /M checkpoint recovery following transient ATM kinase inhibition might have prevented cells containing SCEs from entering mitosis, resulting in an apparent lack of SCE. We speculate that kinase-inhibited ATM can localize to DSBs at damaged replication forks. In fact, several mutant ATM proteins that lacked kinase activity against downstream substrates formed IR-induced foci (IRIF) in A-T cells, suggesting that ATM recruitment to DNA damaged-induced foci is independent of

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Figure 2. Inhibition of ATM kinase activity does not phenocopy A-T cells. In cells with functional ATM protein, double-strand breaks (DSBs) at the replication fork can be repaired via sister chromatid exchange (SCE) (A). When ATM kinase activity is inhibited, SCE is disrupted, possibly via a mechanism that immobilizes ATM itself at the break, resulting in a physical and irreversible block to DSB repair (B). A-T cells, which lack detectable ATM protein, are competent for SCE (C). SCE in A-T cells is not disrupted by ATM kinase inhibitors, indicating that the ATM kinase-dependent decrease in SCE in cells with normal ATM is not due to an off-target effect of the inhibitors (D).

ATM kinase activity.24 However, we propose that once ATM kinase is recruited to DSBs at damaged replication forks, local ATM kinase activity is required both to phosphorylate substrates that mediate DSB repair and to allow mobilization of ATM itself such that DSB repair can physically proceed (Fig. 2). In such a mechanism it is apparent that ATM kinase activity is required to phosphorylate a substrate(s) and that ATM kinase activity and ATM mobilization are concomitant. A plausible explanation for the abrogation of SCE is that kinase-inhibited ATM physically impedes MRE11/CtIPdependent DNA DSB end resection at the replication fork. The MRE11 complex (MRN complex), which consists of

MRE11, RAD50 and NBS1, is essential for the recruitment of ATM to DSBs through an interaction with the C-terminus of NBS1,25,26 and this binding is required for ATM kinase activation at DSBs.27-29 NBS1 also tethers CtIP and MRE11-RAD50 to coordinate DSB end resection and repair.29 Within this complex, RAD50 coiled-coils are positioned to define the reach of the MRE11 complex to bridge homologous sister chromatids during HRR.30,31 The mammalian CtIP protein functions in DSB end resection and is implicated in restricting the HRR functions of the MRE11 complex to S- and G2-phases of the cell cycle.32-34 Sae2 (the S. cerevisiae homolog of CtIP) and the MRE11 complex cooperate in HRR during the initial

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processing of DSBs to produce short 3' overhangs.35-37 These overhangs are resected via redundant pathways involving Sgs1 and Exo1.35,37 Thus, the NBS1 hub appears suitable to promote integration of repair sensing and effector activities of the MRE11 complex by interface exchange and handoff interactions with multiple partners. Kinase-inhibited ATM may disrupt large-scale MRE11 complex conformational changes, impairing HRR. It is possible that phosphoryations on the MRE11 complex subunits, all known substrates of ATM, are required for ATM mobilization at DSBs. Such a mechanism, in which kinaseinhibited ATM would physically block the repair of replication associated DNA damage by physically impeding HRR, is analogous to the recent finding that 53BP1 inhibits HRR in Brca1-deficient cells by blocking DSB end resection.38 DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome aberrations by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA Ligase 4.38 Loss of 53BP1 in Brca1-mutant cells promotes ATM kinase-dependent processing of DSBs to produce recombinogenic single-stranded DNA competent for error-free HRR.38 In contrast, Lig4 deficiency does not rescue the HRR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements.38 These results indicate that HRR and NHEJ compete to process DSBs that arise during replication. Bunting et al. proposed that a critical function for BRCA1 and perhaps other factors that commit breaks to HRR, might be to remove end-joining proteins such as 53BP1 from replication associated breaks.38 In other words, the key mechanism by which 53BP1 regulates the choice of HRR versus NHEJ could be via inhibition of DSB resection by the physical presence of 53BP1 itself. Thus, it is possible that phosphoryations on 53BP1 and BRCA1, both known substrates of ATM, are also required for ATM mobilization at DSBs. In accordance with this model, we hypothesize that ATM kinase activity is associated with the removal of ATM protein itself and/or NHEJ proteins

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from replication-associated breaks. For instance, ATM kinase-dependent MRE11/CtIP-mediated DSB resection39-42 and/or MRE11-mediated nucleosome displacement43 might facilitate the generation of nucleosome-free ssDNA regions that would be incompatible with continued 53BP1 chromatin binding but accessible for recruitment of HRR proteins. Thus, ATM kinase activity may commit replication-associated DNA damage to HRR through the displacement of NHEJ factors. It is possible that kinase inhibition of ATM protein localized at sites of replication-associated DSBs prevents the displacement of ATM protein itself from these sites, leaving behind rather large physical impediments to repair. Furthermore, the fact that SCE is not recovered when the ATM kinase inhibitor is removed and ATM kinase activity is restored in cells4 suggests that there are temporal restrictions to this process (possibly due to loss of substrate availability upon the restoration of ATM kinase activity), which when disrupted results in irreversible consequences. Significance Our data shows that it can be misleading to predict a drug’s effects solely on the basis of animal and cell models in which a protein’s function is disrupted through genetic deletion. While attempting to mimic a disease phenotype, instead we found that inhibition of ATM kinase activity does not phenocopy A-T cells with respect to SCE.4 How does this affect the rational use of ATM kinase inhibitors in the clinic? Many cancer cells acquire a defect in a DNA repair pathway early during tumor development, which can promote additional mutations that are necessary for most normal cells to become transformed. As a result, the multiplicity of DNA repair pathways that function in normal cells is often not available in cancer cells and thus cells are crippled when these residual repair pathways are inhibited. We propose that inhibitors of ATM kinase activity could be effective in eliminating cancer cells that express functional ATM protein and also have compromised DNA repair pathways. These cells are expected to have higher basal levels of replicative

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stress and are likely sensitive to ATM kinase inhibitors, since they are expected to be more reliant on ATM kinase-mediated SCE for DNA repair. Cancer therapies that target a residual DNA repair pathway have significant promise44-46 since the therapeutic index (ratio of the toxic dose to the effective dose of a drug) can be potentially very high. In essence, inhibitors of ATM kinase activity have utility in cancer cells with somatic mutations in genes that are synthetic lethal with ATM kinase activity. Two genes are synthetic lethal if a mutation in either gene alone is compatible with viability but mutations in both lead to cell death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation kills cancer cells while sparing normal cells.47-49 In the case of ATM kinase inhibitors, we are interested in synthetic lethal interactions between a gene and pharmacological inhibition. The paradigm for such an interaction is Poly(ADP-Ribose) polymerase (PARP) inhibition in BRCA1-/- and BRCA2-/tumors. PARP functions in the base excision repair of DNA single-strand breaks (SSBs) and PARP inhibitors cause an accumulation of unrepaired SSBs that induce toxic double-strand breaks via collapsed DNA replication forks.50 PARP inhibitors selectively kill BRCA-/- cancer cells, while sparing BRCA+/+ and BRCA+/normal cells44,45 and show promise in the clinic for the treatment of patients with cancers associated with BRCA1 or BRCA2 mutations.46 Several studies have already documented synthetic lethal interactions with inhibition of ATM kinase activity. KU55933 has been shown to kill pancreatic tumor cell lines deficient in the Fanconi anemia (FA) pathway.51 The FA pathway, which functions in the repair of interstrand crosslinks, is mutated in >10% of pancreatic cancers52 and >15% of lung cancers have inactivated FANCF.53 Thus, ATM kinase inhibitors may be effective in the treatment of these tumor types. In addition, KU55933 has been shown to kill p53-deficient lung cancer cell lines when treated with doxorubicin (64, 65),54,55 demonstrating a synthetic lethal interaction between ATM kinase signaling and p53 when cells are challenged with a

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DNA damaging agent. Future studies are needed to further elucidate the mechanisms underlying these synthetic lethal interactions. Nevertheless, these exciting findings highlight the therapeutic value of ATM kinase inhibitors, especially given the high frequency of p53 or/and FA pathway mutations in cancers.52,53,56 Furthermore, it will be interesting to see what new synthetic lethal relationships with ATM kinase inhibitors are uncovered in the future and how they change the way we envisage the use of these compounds. Acknowledgements

Work in C.J.B.’s laboratory is supported by the Frieda G. and Saul F. Shapira BRCA Cancer Research Program (David C. Whitcomb), the Lung Cancer Research Foundation (CJB), the Lung Cancer SPORE grant P50 CA090440 (Jill Siegfried), the Breast Cancer Research Foundation (Nancy E. Davidson) and the Department of Radiation Oncology (CJB). S.C. was supported by the Predoctoral Training in Pharmaceutical Sciences T32 GM008424 (Donald B. DeFranco). References 1. McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep 2004; 5:772-6. 2. Palau F, Espinos C. Autosomal recessive cerebellar ataxias. Orphanet J Rare Dis 2006; 1:47. 3. Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995; 268:1749-53. 4. White JS, Choi S, Bakkenist CJ. Transient ATM kinase inhibition disrupts DNA damage-induced sister chromatid exchange. Sci Signal 2010; 3:44. 5. Bartram CR, Koske-Westphal T, Passarge E. Chromatid exchanges in ataxia telangiectasia, Bloom syndrome, Werner syndrome and xeroderma pigmentosum. Ann Hum Genet 1976; 40:79-86. 6. Galloway SM. Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes. Mutat Res 1977; 45:343-9. 7. Galloway SM, Evans HJ. Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia. Cytogenet Cell Genet 1975; 15:17-29. 8. Gilad S, Khosravi R, Shkedy D, Uziel T, Ziv Y, Savitsky K, et al. Predominance of null mutations in ataxia-telangiectasia. Hum Mol Genet 1996; 5:433-9. 9. Lakin ND, Weber P, Stankovic T, Rottinghaus ST, Taylor AM, Jackson SP. Analysis of the ATM protein in wild-type and ataxia telangiectasia cells. Oncogene 1996; 13:2707-16. 10. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003; 421:499-506.

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11. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998; 281:1674-7. 12. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998; 281:1677-9. 13. White JS, Choi S, Bakkenist CJ. Irreversible chromosome damage accumulates rapidly in the absence of ATM kinase activity. Cell Cycle 2008; 7:1277-84. 14. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, Luo J, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007; 316:1160-6. 15. Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 2010; 3:3. 16. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI, et al. Identification and characterization of a novel and specific inhibitor of the ataxiatelangiectasia mutated kinase ATM. Cancer Res 2004; 64:9152-9. 17. Rainey MD, Charlton ME, Stanton RV, Kastan MB. Transient inhibition of ATM kinase is sufficient to enhance cellular sensitivity to ionizing radiation. Cancer Res 2008; 68:7466-74. 18. Golding SE, Rosenberg E, Valerie N, Hussaini I, Frigerio M, Cockcroft XF, et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling and inhibits migration and invasion. Mol Cancer Ther 2009; 8:2894-902. 19. Callen E, Jankovic M, Difilippantonio S, Daniel JA, Chen HT, Celeste A, et al. ATM prevents the persistence and propagation of chromosome breaks in lymphocytes. Cell 2007; 130:63-75. 20. Deckbar D, Birraux J, Krempler A, Tchouandong L, Beucher A, Walker S, et al. Chromosome breakage after G2 checkpoint release. J Cell Biol 2007; 176:749-55. 21. Andreassen PR, Ho GP, D’Andrea AD. DNA damage responses and their many interactions with the replication fork. Carcinogenesis 2006; 27:883-92. 22. Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H, et al. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 1998; 17:598-608. 23. Wilson DM, 3rd, Thompson LH. Molecular mechanisms of sister-chromatid exchange. Mutat Res 2007; 616:11-23. 24. Barone G, Groom A, Reiman A, Srinivasan V, Byrd PJ, Taylor AM. Modeling ATM mutant proteins from missense changes confirms retained kinase activity. Hum Mutat 2009; 30:1222-30. 25. Falck J, Coates J, Jackson SP. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 2005; 434:605-11. 26. You Z, Chahwan C, Bailis J, Hunter T, Russell P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol Cell Biol 2005; 25:5363-79. 27. Carson CT, Schwartz RA, Stracker TH, Lilley CE, Lee DV, Weitzman MD. The Mre11 complex is required for ATM activation and the G2 /M checkpoint. EMBO J 2003; 22:6610-20. 28. Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J 2003; 22:5612-21. 29. Williams RS, Dodson GE, Limbo O, Yamada Y, Williams JS, Guenther G, et al. Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair. Cell 2009; 139:87-99.

30. Hopfner KP, Craig L, Moncalian G, Zinkel RA, Usui T, Owen BA, et al. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 2002; 418:562-6. 31. Moreno-Herrero F, de Jager M, Dekker NH, Kanaar R, Wyman C, Dekker C. Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/ Nbs1 upon binding DNA. Nature 2005; 437:440-3. 32. Lengsfeld BM, Rattray AJ, Bhaskara V, Ghirlando R, Paull TT. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/ Xrs2 complex. Mol Cell 2007; 28:638-51. 33. Limbo O, Chahwan C, Yamada Y, de Bruin RA, Wittenberg C, Russell P. Ctp1 is a cell cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination. Mol Cell 2007; 28:134-46. 34. Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J, et al. Human CtIP promotes DNA end resection. Nature 2007; 450:509-14. 35. Mimitou EP, Symington LS. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 2008; 455:770-4. 36. Trujillo KM, Yuan SS, Lee EY, Sung P. Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11 and p95. J Biol Chem 1998; 273:21447-50. 37. Zhu Z, Chung WH, Shim EY, Lee SE, Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 2008; 134:981-94. 38. Bunting SF, Callen E, Wong N, Chen HT, Polato F, Gunn A, et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 2010; 141:243-54. 39. Cuadrado M, Martinez-Pastor B, Murga M, Toledo LI, Gutierrez-Martinez P, Lopez E, et al. ATM regulates ATR chromatin loading in response to DNA double-strand breaks. J Exp Med 2006; 203:297303. 40. Dodson GE, Tibbetts RS. DNA replication stressinduced phosphorylation of cyclic AMP response element-binding protein mediated by ATM. J Biol Chem 2006; 281:1692-7. 41. Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J, et al. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol 2006; 8:37-45. 42. Myers JS, Cortez D. Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J Biol Chem 2006; 281:9346-50. 43. Tsukuda T, Fleming AB, Nickoloff JA, Osley MA. Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature 2005; 438:379-83. 44. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Specific killing of BRCA2deficient tumours with inhibitors of poly(ADPribose) polymerase. Nature 2005; 434:913-7. 45. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005; 434:917-21. 46. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, MerguiRoelvink M, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009; 361:123-34. 47. Friend SH, Oliff A. Emerging uses for genomic information in drug discovery. N Engl J Med 1998; 338:125-6. 48. Kaelin WG Jr. Choosing anticancer drug targets in the postgenomic era. J Clin Invest 1999; 104:1503-6. 49. Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH. Integrating genetic approaches into the discovery of anticancer drugs. Science 1997; 278:1064-8.

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50. Schultz N, Lopez E, Saleh-Gohari N, Helleday T. Poly(ADP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination. Nucleic Acids Res 2003; 31:4959-64. 51. Kennedy RD, Chen CC, Stuckert P, Archila EM, De la Vega MA, Moreau LA, et al. Fanconi anemia pathway-deficient tumor cells are hypersensitive to inhibition of ataxia telangiectasia mutated. J Clin Invest 2007; 117:1440-9. 52. van der Heijden MS, Brody JR, Gallmeier E, Cunningham SC, Dezentje DA, Shen D, et al. Functional defects in the fanconi anemia pathway in pancreatic cancer cells. Am J Pathol 2004; 165:651-7.

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53. Marsit CJ, Liu M, Nelson HH, Posner M, Suzuki M, Kelsey KT. Inactivation of the Fanconi anemia/ BRCA pathway in lung and oral cancers: implications for treatment and survival. Oncogene 2004; 23:1000-4. 54. Jiang H, Reinhardt HC, Bartkova J, Tommiska J, Blomqvist C, Nevanlinna H, et al. The combined status of ATM and p53 link tumor development with therapeutic response. Genes Dev 2009; 23:1895-909.

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55. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007; 11:175-89. 56. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307-10.

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