Pharmacology of the ATM Inhibitor AZD0156: Potentiation of … · the clinic and to overcome DDR-associated resistance (6). Ataxia-telangiectasia mutated (ATM) kinase belongs to a

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Pharmacology of the ATM Inhibitor AZD0156:Potentiation of Irradiation and Olaparib ResponsesPreclinically A C

Lucy C. Riches1, Antonio G. Trinidad1, Gareth Hughes1, Gemma N. Jones2, Adina M. Hughes1,Andrew G. Thomason1, Paul Gavine1, Andy Cui1, Stephanie Ling3, Jonathan Stott3, Roger Clark3,Samantha Peel3, Pendeep Gill1, Louise M. Goodwin1, Aaron Smith4, Kurt G. Pike5, Bernard Barlaam5,Martin Pass6, Mark J. O’Connor1, Graeme Smith1, and Elaine B. Cadogan1

ABSTRACT◥

AZD0156 is a potent and selective, bioavailable inhibitor ofataxia-telangiectasia mutated (ATM) protein, a signaling kinaseinvolved in the DNA damage response. We present preclinicaldata demonstrating abrogation of irradiation-induced ATMsignaling by low doses of AZD0156, as measured by phosphor-ylation of ATM substrates. AZD0156 is a strong radiosensitizerin vitro, and using a lung xenograft model, we show that systemicdelivery of AZD0156 enhances the tumor growth inhibitoryeffects of radiation treatment in vivo. Because ATM deficiencycontributes to PARP inhibitor sensitivity, preclinically, we eval-

uated the effect of combining AZD0156 with the PARP inhibitorolaparib. Using ATM isogenic FaDu cells, we demonstrate thatAZD0156 impedes the repair of olaparib-induced DNA damage,resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiatedthe effects of olaparib across a panel of lung, gastric, and breastcancer cell lines in vitro, and improved the efficacy of olaparib intwo patient-derived triple-negative breast cancer xenograft mod-els. AZD0156 is currently being evaluated in phase I studies(NCT02588105).

IntroductionCells are subject to ongoing DNA damage resulting from physio-

logic processes (e.g., reactive oxygen species generated through met-abolic processes), and exposure to external agents (e.g. chemicals andradiation), which if left unrepaired may compromise genome integ-rity (1). Consequently, networks of proteins involved in the repair ofDNA damage, chromatin remodeling, and cell-cycle maintenanceensure a coordinated response to limit the detrimental effects of DNAdamage (2, 3). Together these pathways comprise the DNA damageresponse (DDR). During cancer development, activation of oncogenes(e.g., MYC and RAS), may induce DNAdamage, which if coupled with

tumor DDR defects is proposed to drive genomic instability, a corefeature of cancer progression (4, 5). The high burden of DNA damagecan be exploited in cancer therapy using agents that induce DNAdouble-strand breaks (DSB), thereby increasing DNA damage tocytotoxic levels. Developing drugs that target DDR proteins presentsan opportunity to enhance the therapeutic value of such regimens inthe clinic and to overcome DDR-associated resistance (6).

Ataxia-telangiectasia mutated (ATM) kinase belongs to a family ofserine/threonine phosphatidylinositol 3-kinase-like-protein-kinases(PIKK) and propagates an extensive signaling cascade in response toDNA damage (7). Under unstressed conditions, ATM resides pre-dominantly in the nucleus as an inactive dimer and in response to DSBATM is recruited to chromatin by the MRE11-NBS1-RAD50 (MRN)complex where it undergoes auto-phosphorylation, dissociating intocatalytically active monomers (8–10). Through phosphorylation of amultitude of effector proteins including P53, CHK2, KAP1, RAD50,SMC1, MDC1, and H2AX, ATM signal transduction mediates theintra-S-phase, G1–S, and S–G2M checkpoints, and promotes recruit-ment of DNA repair proteins to sites of damage through H2AX andMDC1 (11–16). Because of its key role in DSB signaling, ATM is apromising therapeutic target.

The value of targeting DDR factors in the clinic is exemplified byPARP inhibitors (e.g., olaparib), for which benefit has been demon-strated in patients with tumors that harbor BRCA mutations (e.g.,ovarian and breast cancers; refs. 17, 18). The activity of PARPinhibitors in BRCAmut tumors exploits the principle of syntheticlethality (19), and preclinical studies indicate that PARP syntheticlethal interactions extend beyond BRCA to DDR proteins includingATM (20–22). Consequently, inhibition of ATM is anticipated topotentiate the effects of PARP inhibitors in the clinic. Indeed, selectiveinhibitors of ATM have been described, such as KU-55933 andKU-60019 (Fig. 1C), and have been shown to sensitize cancer cellsto classicDSB-inducing agents including topoisomerase inhibitors andirradiation (23), in addition to olaparib, in vitro (24–26). These studiesprovide proof-of-concept for the use of selective ATM inhibitors to

1Bioscience, Oncology R&D, AstraZeneca, Cambridge, United Kingdom.2Translational Medicine, Oncology R&D, Oncology, AstraZeneca, Cambridge,United Kingdom. 3Quantitative Biology, Discovery Science, BioPharmaceuticalsR&D, AstraZeneca, Cambridge, United Kingdom. 4DMPK, Oncology R&D,AstraZeneca, Cambridge, United Kingdom. 5Chemistry, Oncology R&D,AstraZeneca, Cambridge, United Kingdom. 6Oncology R&D, AstraZeneca,Cambridge, United Kingdom.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

L.C. Riches, A.G. Trinidad, G. Hughes, G.N. Jones, A.M Hughes, S. Ling, L.M.Goodwin, K.G. Pike, S. Peel, A. Smith, B. Barlaam, M. Pass, M.J. O’Connor, and E.BCadogan are all current AstraZeneca employees.

A. Thomason, P. Gavine, A. Cui, J. Stott, R. Clark, P. Gill, andG. Smith are all formeremployees of AstraZeneca.

Corresponding Author: Elaine B. Cadogan, AstraZeneca, CRUK CambridgeInstitute, Li Ka Shing Centre, Robinson Way, Cambridge, Cambridgeshire CB2ORE, United Kingdom. Phone: 44-74-6940-8838; E-mail:[email protected]

Mol Cancer Ther 2020;19:13–25

doi: 10.1158/1535-7163.MCT-18-1394

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potentiate the activity of DNA-damaging agents, including PARPinhibitors. While compounds such as KU-55933 and KU-60019 haveproved valuable for probing the effects of ATM inhibition in vitro, therelatively modest cellular potency of the compounds (Table 1), com-bined with the reported low aqueous solubility and low oral bioavail-ability, means that these compounds are not considered suitable todeliver meaningful levels of ATM inhibition in the clinic (27).AZD0156 was discovered after chemical optimization of a novel seriesof ATM inhibitors resulting in a highly significant increase in bothpotency and selectivity, while also delivering a compound with goodphysicochemical properties, good preclinical pharmacokinetic pro-files, and an acceptable predicted clinically efficacious dose(ref. 21; Fig. 1C). As a result, AZD0156 was considered a suitablemolecule to explore the effects of ATM inhibition in humans and wassubsequently selected for development as a clinical agent. AZD0156 iscurrently being evaluated in phase I studies (NCT02588105).

Importantly, another potent ATM inhibitor under clinical evalu-ation, AZD1390 has been recently reported to radiosensitize preclin-ical brain tumormodels inducing tumor regression and shows efficientblood–brain barrier penetration in vivo (28).

Herewe present preclinical data characterizing the pharmacology ofAZD0156 with properties considered suitable for clinical develop-ment. Our work presents a comprehensive data package providingmechanistic insights into how AZD0156 potentiates the effects ofolaparib, thus providing supporting evidence for the evaluation of thiscombination in the clinic (NCT02588105).

Materials and MethodsCompounds/chemicals

Compounds (AZD0156 and olaparib) were synthesized internallyat AstraZeneca laboratories (21) and dissolved in 100% DMSO at1 mmol/L (AZD0156) or 10 mmol/L (olaparib) stocks, which wereused for in vitro studies.

Cell cultureAll cell lines were obtained from the ATCC and were authenticated

by short tandem repeat profile and tested negative for Mycoplasmacontamination. Cells were passaged at least twice following thawingand cultured in RPMI media supplemented with 10% FCS and2 mmol/L L-glutamine and maintained under standard cell cultureconditions at 37�C, 5% CO2. Cells were routinely passaged using1� TrypleX to detach cells from the tissue culture vessel. Onlyexponentially growing cells below passage 10 and with a viabilitygreater than 95% as measured by Trypan blue exclusion were usedduring experiments.

In vitro growth inhibition assays; sytox green assayCells were seeded into clear bottom, black 96-well plates at a

preoptimized seeding density and left to adhere overnight. Thefollowing day, cells were dosed with 10� concentrated compound toachieve a 5-point dose response of olaparib and doses of AZD0156 asstated in figure legends. After 5–7 days, depending on cell doubling

time, cells were permeabilized by the addition of saponin and stainedwith sytox green DNA stain. Cell number was quantified using theAcumen.

Colony formation assayExponentially growing cells were seeded into 6-well plates at 1,000

cells/well [FaDu wild-type (WT)] or 2,000 cells/well [FaDu ATMknockout (KO)] and dosed the following daywithAZD0156 (30 nmol/L). After 1 hour, cells were dosed with olaparib (10–0.1 mmol/L) orirradiated at the stated dose using a bench topCellRad X-ray Irradiator(Faxitron). After 7–10 days, once colonies of >50 cells had formed inthe DMSO control wells, cells were fixed and stained. Plates werescanned, and colonies quantified using the Gel Count System (OxfordOptronics Ltd).

Analysis of proliferation assay dataData obtained from the sytox green and colony formation assays

was normalized to DMSO control samples, and dose–response curvesfor olaparib or irradiation þ/� AZD0156 were plotted in GraphPadPrism to generate GI50 values (the concentration of treatment requiredto inhibit cell growth by 50%).

Protein extraction and Western blot analysisFor protein extraction, cells were lysed in NP-40 lysis buffer

(50 mmol/L Tris-HCl pH7, 150 mmol/L NaCl, 1 mmol/L EDTA, and1% NP-40 supplemented with complete protease and phosphataseinhibitor cocktail, Roche). A total of 30 mg protein was separated onSDS-PAGE Bis-Tris 4%–12% or Tris-Acetate 3%–8% for high molec-ular weight proteins and transferred by iBLOT (Invitrogen) for 10minutes at 20 V onto nitrocellulose membranes. Membranes wereblocked in 3% BSA:0.1% Tween 20:TBS and incubated with primaryantibody at 4�C overnight. Membranes were washed in 0.05% T-TBS,incubated with horseradish peroxide (HRP)-coupled secondary anti-bodies (1:4,000) then incubated with ECL Reagent (Pierce), andvisualized using the ODYSSEY CLX system or film. For pharmaco-dynamic studies, the intensity of Western blot analysis signal wasmeasured and normalized to vinculin expression.

The following antibodies were used at 1:1,000 dilution unlessotherwise stated; pATM Ser1981 (Abcam, 1:1,000), pThr68-CHK2(Cell Signaling Technology, 1:500), tCHK2 (ProSCI, 1:500), pSer824-KAP1 (Abcam, 1:1,000), pSer473-KAP1 (BioLegend, 1:500), pSer345-CHK1 (Cell Signaling Technology, 1:500), tCHK1 (Abcam, 1:500),pSer635-Rad50 (Cell Signaling Technology, 1:500), b-Actin (Sigma,1:10,000), PAR (Trevigen, 1:1,000), pDNA-PKcs (developed internallyat AstraZeneca, 1:1,000), tDNA-PKcs (Cell Signaling Technology,1:1,000), and gH2AX (Millipore, 1:500).

Cell-cycle studiesCells were compound treated in 6-well plates for the specified time

(24–72 hours) then harvested and fixed in ice-cold 70% EtOH for1 hour. Cells were washed in PBS and incubated with 5 mg/mL DAPIfor 30 minutes. Samples were acquired on the FACS Aria I, and datawere analyzed in FlowJo to determine the percent of cells in each phaseof the cell cycle based on DNA content.

Caspase-3/7 assayFaDu cells were seeded in 96-well plates (2,000/well) and the

following day compound andNucView caspase Glo reagent was added(following themanufacturer's instructions, Essen BioSciences). Imageswere captured in the green fluorescent and phase contrast channelsevery 4 hours, on the IncuCyte Zoom (Essen BioScience). Images were

Table 1. Comparison of ATM inhibitor properties in cell assays.

TargetKU559933 IC50

(mmol/L)KU60019 IC50

(mmol/L)AZD0156 IC50

(mmol/L)

ATM (pATMS1981) 1.13 (N ¼ 222) 0.15 (N ¼ 223) 0.00058 (N ¼ 16)

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Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS14




subsequently analyzed using the Incucyte Zoom software to report thearea of apoptosis-positive cells in the green fluorescent channel relativeto cell confluence, as determined in the phase contrast channel.

Immunofluorescence (gH2AX)FaDu cells cultured in clear bottom, black 96-well plates (2,000 cells/

well) were compound treated with AZD0156 for 1 hour prior toOlaparib or radiation treatment. At the stated time, cells were fixedin 3.7% paraformaldehyde for 20 minutes, washed in PBS, andpermeabilized with 0.5% Triton-X100 for 5 minutes. Cells wereblocked in 3% BSA, incubated with gH2AX antibody (Millipore cloneJBW301; 1:500) at 4�C overnight, then washed with tween, andincubated with Alexa-488–conjugated secondary antibody (1:500;Invitrogen Molecule Probes) for 45 minutes. Cells were stained withHoechst prior to imaging on the Cell Insight at 20 � magnification.The number of nuclei foci was measured using the Cell Insight spotdetector application. Only foci within the nuclear region as defined byHoechst staining were classified. A minimum of 100 cells wereanalyzed per well.

Comet assayCells were treated with compound in 6-well plates and samples

processed at 48 hours. Single-cell suspensions were mixed with lowmelting agarose and transferred onto 20-well slides (Trevigen) thenlysed in Trevigen lysis solution overnight. Samples were incubatedin alkaline solution (pH 13) for 20 minutes and electrophoresed inthe same buffer for 25 minutes at 21 V. Slides were fixed in 100%EtOH for 20 minutes then stained with 1� SyBr Gold (Invitrogen;30 minutes). Washed slides were imaged using wide-field micros-copy at 10 �magnification and a minimum of 100 cells were scoredacross duplicate samples using the Comet IV Software (Perspec-tive). Data are represented as percent of DNA in the tail (% tailintensity–TI).

Xenograft-targeted irradiation studyNCI-H441 cells for in vivo xenograft implant were cultured in

RPMI1640 with 10% v/v FCS and 1% v/v L-glutamine at 37�C, 7.5%CO2. Cells were implanted subcutaneously in serum-free media with50% Matrigel at 5 � 106 per mouse. Male nude mice (Harlan UK) atgreater than 18 g had tumor size monitored twice weekly by bilateralcaliper measurements. Treatments were started when tumor volumesreached an average of approximately 0.26 cm3. Targeted irradiation of2 Gy was delivered over 2minutes daily over the first 5 days. AZD0156(10 mg/kg) was given orally for the duration of the study. This studywas run in the United Kingdom in accordance with UK Home Officelegislation, the Animal Scientific Procedures Act 1986, and withAstraZeneca Global Bioethics policy. Experimental details are outlinedin Home Office project license 40/3451.

Patient-derived xenograft studiesTriple-negative breast cancer (TNBC) HBCx-10 and HBCx-9 (29)

patient-derived xenograft studies were carried out at XenTech, Francein accordance with French regulatory legislation concerning theprotection of laboratory animals. Female athymic nude mice (HarlanFrance) greater than 18 g were implanted with HBCx-10 or HBCx-9tumor derived from a primary ductal adenocarcinoma. Donor micewere sacrificed to provide tumor fragments, which were surgicallyimplanted subcutaneously. Tumor size wasmonitored twice weekly bybilateral caliper measurements. Mice body weights were recorded atthe same time. For efficacy studies, treatment started when tumorvolume averaged approximately 0.15 cm3 and for pharmacodynamic

studies treatment started at approximately 0.5 cm3. In efficacy andpharmacodynamic studies, control mice were dosed orally with vehi-cle, AZD0156, or olaparib alone or in combination using differentdosing schedules.

Pharmacodynamic analysisFor pharmacodynamic studies, tumor tissue from the HBCx-10

model was homogenized in lysis buffer [20 mmol/L Tris (pH 7.5),137 mmol/L sodium chloride, 10% glycerol, 1% SDS, 1%NP-40,50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate(activated)] supplemented with phosphatase and protease inhibitors,using a FastPrep Machine (MP Biomedicals) for 3� 30-second cyclesat 6.5 m/s. Samples were sonicated for 15 seconds at 50% amplitudeand incubated on ice for 30 minutes. The supernatant was collected bycentrifugation and lysates analyzed byWestern blotting for pATMandgH2AX expression.

PARylation was analyzed using the HT PARP in vivo Pharmaco-dynamic Assay II ELISA 2nd generation ELISA Kit (Trevigen catalogno. 4520-096-K; Trevigen Inc.). Protein (1 mg/mL in Lysis buffer) wasincubated at 100�C for 5 minutes, diluted to 40 ng/mL in kit diluentbuffer and incubated for 16 hours at 4�C on precoated ELISA plates.After washing in PBSTween (0.1%), secondary antibodywas added for1 hour at room temperature, and plates were washed with PBS Tween(0.1%) and detection performed using HRP/PeroxyGlow reagent.Luminescence was quantified using a Tecan Safire Microplate Reader(Tecan Group Ltd) at 540 nm and PAR concentrations were estimatedfrom comparisons with standard curves.

Plasma analysisEach plasma sample (25 mL) was prepared using an appropriate

dilution factor and compared against an 11-point standard calibrationcurve (1–10,000 nmol/L) prepared in DMSO and spiked into blankplasma. Acetonitrile (100 mL) was added with the internal standard,followed by centrifugation at 3,000 rpm for 10 minutes. Supernatant(50 mL) was then diluted in 300 mL water and analyzed via UPLC-MS/MS (Supplementary Tables S1 and S2).

ResultsAZD0156 is a potent inhibitor of ATM, with an IC50 of 0.58 nmol/L

in cell assays, asmeasured by inhibition of ATMauto-phosphorylationat serine 1981, at 1 hour following radiation treatment in HT29 cells(Table 1;Fig. 1C). Selectivity ofAZD0156was confirmed in cell assays,which measure the activity of related kinases including ATR, mTOR,and PI3K-alpha. AZD0156 was >1,000-fold more selective for ATM inthese assays (21).

AZD0156 inhibits ATM signaling and potentiates the effects ofirradiation

ATM undergoes rapid auto-phosphorylation in response to irra-diation (9) and so we employed irradiation to validate AZD0156activity preclinically, using isogenic FaDuATMproficient (FaDuWT)andATM triple knockout (FaDuKO) cell lines. The FaDu-KO cell linewas generated at AstraZeneca using Zinc finger nuclease technology.Cellswere pretreatedwith increasing doses ofAZD0156 (1–30nmol/L)for 1 hour prior to radiation (5 Gy). In FaDu WT cells, AZD0156inhibited irradiation-induced ATM signaling in a dose-dependentmanner asmeasured by auto-phosphorylation on serine 1981 of ATM,and phosphorylation of ATM substrates including KAP1, RAD50, andCHK2 (Fig. 1A). AZD0156 did not modulate DNA-PKcs phosphor-ylation following irradiation, indicating that AZD0156 selectively

ATM Inhibitor AZD0156 Potentiates IR and Olaparib Response

AACRJournals.org Mol Cancer Ther; 19(1) January 2020 15




abrogates ATM signaling (Fig. 1A). As expected, no phosphorylationof ATM or its substrates were observed in the FaDu KO cells. Tofurther confirm inhibition of ATM activity by AZD0156, gH2AX fociformation, a universal biomarker ofDSB anddirect target ofATM(30),was measured by immunofluorescence. Irradiation (5 Gy) aloneresulted in a 20% increase in the number of FaDu WT cells with>5 nuclear gH2AX foci cells at 4 hours, which was reduced to less than2% when pretreated with 30 nmol/L AZD0156 (Fig. 1B). Thisindicates that H2AX phosphorylation was strongly inhibited in theabsence of ATM function following irradiation.

We next determined whether AZD0156 would radiosensitize FaDuWT cells using the colony formation assay. Pretreatment of FaDuWTcells with 30 nmol/L AZD0156 for 1 hour prior to irradiation, whichstrongly inhibited ATM signaling (Fig. 1A), effectively reduced clo-nogenic survival at all doses of radiation employed here (0.2–2Gy; Fig. 2A). No significant radiosensitization was seen in the FaDuKO cells (Fig. 2A) indicating that radiosensitization is mediatedthrough ATM inhibition. Notably, AZD0156 treatment in the FaDuWT cells achieved radiosensitivity comparable with that observed inFaDu KO cells, which are intrinsically more sensitive to irradiation(Fig. 2A), indicating that inhibition by AZD0156 phenocopies theATM knockout model. Our data is consistent with the established roleof ATM signaling in response to radiation and validates AZD0156 as apotent inhibitor of ATM activity in vitro.

To investigate the potential for AZD0156 to combine with irradi-ation in a disease relevant setting, the non–small cell lung adenocar-cinoma cell line NCI-H441 was treated with 3, 10, and 30 nmol/LAZD0156 prior to 2 Gy radiation. Consistent with data generated inthe FaDu WT cell line, pretreatment of cells with AZD0156 reducedcolony formation 24-fold compared with irradiation treatment alone(Fig. 2B). We next determined whether this radiosensitization wouldtranslate to the corresponding in vivo xenograft model. Irradiationtreatment alone, administered during the first 5 days of the study toinduce DNA damage, inhibited tumor growth in the NCI-H4441xenograft model, which was enhanced by the addition of AZD0156(Fig. 2C).

AZD0156 impairs olaparib-induced activation of ATM andpotentiates the activity of olaparib in FaDu ATM-proficient cells

In addition to in vitro studies employing early ATM compounds,siRNA approaches and ATM KO models have demonstrated thatATM deficiency drives PARP inhibitor sensitivity (20, 25, 31), andtherefore we investigated the ability of AZD0156 to potentiate olaparibtreatment in vitro. Activation of ATM was observed following 2 and6 hours olaparib treatment (1–3 mmol/L), as measured by auto-phosphorylation of ATM and induction of pCHK2-T68, which wasreduced by pretreatment with AZD0156 (30 nmol/L; Fig. 3A). Toestablish whether AZD0156 could sensitize cells to olaparib treatment,we employed the colony formation assay to measure cell survival.AZD0156 (30 nmol/L) potentiated the effects of olaparib in FaDuWTcells, however no effect was observed in the FaDu KO cell line(Fig. 3B). This result confirmed that the combination effect wasmediated through inhibition of ATM (Fig. 3B). We noted that inthis assay, the FaDu KO cells were intrinsically more sensitive toolaparib treatment alone, reinforcing the notion that ATM deficiencypotentiates the effects of olaparib treatment (Fig. 3B). Further mech-anistic studies were conducted in the FaDu ATM-proficient cell line.

Mechanistically, olaparib inhibits single-strand break repair andtraps PARP onto DNA, creating complexes that interfere with DNAreplication and manifest as DSBs (32). Therefore, olaparib-inducedDNA damage was anticipated to occur predominantly in S-phase. We

hypothesized that the synergy observed between olaparib andAZD0156 may be in part due to abrogation of checkpoint signalingby AZD0156, because ATM contributes to S-phase checkpoint sig-naling (33). To investigate this, we measured the impact of AZD0156–olaparib combinations on the cell cycle by flow cytometry. At 24 hoursfollowing treatment, olaparib treatment alone had a modest effect oncell-cycle parameters compared with control samples, as measured byDNA content (Fig. 3C). In combination with AZD0156 (30 nmol/L),however, greater than 50% of cells were in G2–M-phase at 24 hours,compared with 20% of cells in the corresponding control group(Fig. 3C). This data are consistent with the growth inhibitory effectobserved using the colony formation assay (Fig. 3B). Cell-cycleparameters were also measured at 48 and 72 hours to investigatelonger term effects of olaparib (1 mmol/L)–AZD0156 combinations onthe cell cycle. A time-dependent decrease in the G2–M-phase popu-lationwas accompanied by an increase in the sub-G1 peak, indicative ofcell death and at 72 hours we noted the appearance of cells with >2NDNA content (Fig. 3C and D). The latter finding is similar to datareported in malignant lymphocyte cells, in which concomitant PARPand ATM inhibition, by olaparib and KU55933, respectively, resultedin a delayed G2 transition and an increase in DNA content in some celllines tested (34).

AZD0156 impairs olaparib-induced DNA damage in FaDu WTcells resulting in cell death

Our cell-cycle observations support the hypothesis that when ATMis inhibited, cells containing olaparib-induced DNA damage inS-phase continue through the cell cycle, and the presence of unrepairedDNA damage activates the G2–Mcheckpoint. Because combination ofKU55933 and olaparib was previously reported to exacerbate gH2AXin CAL51 cells (24), we measured the formation of gH2AX foci, auniversal biomarker of DSB, by immunofluorescence. In FaDu WTcells, olaparib treatment (1 and 3 mmol/L) resulted in an elevatedproportion of cells with >5 gH2AX foci at 48 hours, (Fig. 4A),indicative of DSB, presumably ensuing from unrepaired single-strand breaks or collapsed replication forks. Combining olaparib(3 mmol/L) with 30 nmol/L AZD0156 resulted in a significant increase(2.67-fold) in the percent of gH2AX-positive cells at 48 hours com-pared with olaparib treatment alone (Fig. 4A). Although H2AX is asubstrate of ATM, in this scenario, it is feasible that prolongedinhibition of ATM (48 hours) may result in activation of other kinases,for example ATR or DNA-PKcs, which can also phosphorylateH2AX (35). To explore whether checkpoint signaling was affected,we measured CHK1 phosphorylation at serine 345. After 48 hours oftreatment, pCHK1 was elevated in the combination treated cells abovemonotherapy treatment (Fig. 4A). CHK1 is a substrate of ATR, andcontributes to the G2 checkpoint signaling, which may indicateincreased activation ofATRwhenATM is inhibited. Despite activationof gH2AX in the absence of ATM, the profound growth inhibitoryeffect observed between olaparib and AZD0156 (Fig. 3B) suggests thatother DDR kinases cannot fully compensate in FaDu cells. Rather, thepresence of gH2AX foci following combination treatment likelyrepresents persistent DSB due to inefficient repair of olaparib-induced damage when ATM is inhibited. This is similar to findingsin ATM-deficient lymphoid cells following prolonged olaparibtreatment (36).

From images captured to measure gH2AX foci formation, we notedan increase in the proportion of cells containing micronuclei (Sup-plementary Fig. S1), a characteristic of DNA damage. To confirm thepresence of DNA damage, we performed the alkaline comet assay inFaDu WT cells. Following 30 nmol/L AZD0156 treatment alone, we

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Figure 1.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation.A, FaDuWT and KO cells were pretreatedwith increasing doses of AZD0156 for 1 hour priorto receiving 5 Gy irradiation (IR). After 1 hour, whole-cell lysates were generated, and phosphorylation of ATM substrates and DNA-PKcs was measured byWesternblotting. B, FaDu WT cells were pretreated with 30 nmol/L AZD0156 or DMSO for 1 hour prior to irradiation (5 Gy). After 4 hours, cells were fixed and stained withgH2AXantibody. Imageswere captured on theCell Insight, and the percent of cellswith greater than five nuclear foci, asmeasuredbyHoechst staining,was recorded.Data represent themean of two independent experiments conducted in triplicate� SEM. Images depict gH2AX foci in green and nuclear staining by Hoechst in blue.C, Chemical structure of AZD0156, KU-55933, and KU-60019.






observed amodest but consistent increase in tail intensity, indicative ofDNA damage (Fig. 4B). In combination with 3 mmol/L olaparib, themean tail intensity of comets was elevated 1.48-fold above 3 mmol/Lolaparib treatment alone, and we noted a large heterogeneity in tailintensity, with a small proportion of cells with a tail intensity greaterthan 50% (Fig. 4B). This likely reflects the mode of action of olaparib,in which replicating cells are expected to be more susceptible to theeffects of PARP inhibition.

Having observed an increase in the sub-G1 population of cellstreated with olaparib and AZD0156 combination by flow cytometryanalysis (Fig. 3D), which is indicative of cell death, we investigatedwhether this proceeded through apoptosis. Using the caspase3/7

NucView assay we measured the kinetics of apoptosis using theIncuCyte. A relatively small increase in cleaved caspase3/7 wasobserved with olaparib or AZD0156 treatment alone, indicating thatfew cells underwent apoptosis in these treatment groups (Fig. 4C). Incombination, however, the cleaved caspase3/7 signal was elevated4-fold with 30 nmol/L AZD0156þ 1 mmol/L olaparib compared withcorresponding single-agent control samples at 96 hours (Fig. 4C). Thisdemonstrates that concomitant inhibition of ATM and PARP drivesFaDu WT cells into apoptosis.

Our data suggests that AZD0156 creates a DDR-deficient pheno-type that exacerbates the effects of olaparib in FaDu ATM proficientcells and impedes the repair of olaparib-inducedDNAdamage in vitro.

Figure 2.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation. A, FaDu WT and KO cells were pretreated þ/� 30 nmol/L AZD0156 prior to irradiation.After 7 to 10 days, colonies were scored. Data are represented as the mean of two independent experiments conducted in triplicate � SD for FaDu WT cells and asingle experiment conducted in triplicate for FaDu KO cells. B, NCI-H4441 lung cells were pretreated with increasing doses of AZD0156 for 1 hour prior to receiving2 Gy irradiation. After 14 days, colonies were scored. Data are represented as the mean of two independent experiments conducted in duplicate � SD. C, NCI-H441non–small cell lung cancer xenograft grown subcutaneouslywas treatedwith 5 daysof targeted irradiation (2Gyover 2minutes daily) combinedwith 38 daysof oncedaily oral dosing AZD0156 10 mg/kg, AZD0156 administered 1 hour prior to irradiation (IR; initial group sizes n ¼ 9–12). PO, orally; QD, every day.

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Figure 3.

AZD0156 impairs olaparib-induced activation of ATM and potentiates the activity of olaparib in vitro.A,Cells were treatedwith olaparibþ/� 30 nmol/L AZD0156 for2 to 6 hours. pATM-S1981 and pCHK2-T68 were measured as markers of ATM signaling byWestern blotting. B, Cells were treated with 30 nmol/L AZD0156 (red) orDMSO (black) and increasing doses of olaparib. After 7 to 10 days, colonies were scored and the surviving fraction plotted relative to DMSO control cells. Datarepresent the mean of two independent repeats run in triplicate � SD. C, FaDu WT and KO cells treated with olaparib þ/� AZD0156 were processed for flowcytometry at 24, 48, or 72 hours. For 24-hour samples, cell-cycle phase was determined on the basis of DNA content (blue, G1; yellow, S; red, G2M; and black, sub-G1).D, Cell-cycle histograms are shown for FaDu WT cells treated with olaparib þ/� AZD0156 at 24, 48, or 72 hours.






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As such, AZD0156 presents an opportunity to create a contextualDDR-deficient phenotype to sensitize cancer cells to PARP inhibitors.

Potentiation of olaparib by AZD0156 across a panel of cancercell lines

Previously published data has indicated that cancer cell linesincluding colorectal, mantle cell lymphoma, and gastric cancer cellscan be sensitized to the effects of olaparib in vitro using the early ATMinhibitor KU59933 (25, 37). To determine whether the combination

effect betweenAZD0156 and olaparib extended beyond FaDu cells andto build upon published findings, we performed the sytox greenproliferation assay across a panel of cancer cell lines in vitro. AZD0156potentiated the effects of olaparib across all cell lines tested, whichincluded TNBC, gastric cancer, and non–small cell lung cancer cells(Fig. 5A and B). A broader screen of gastric cell lines using short-termassays was also conducted, which confirmed the combination effectacross the majority of ATM-proficient cells tested (SupplementaryFig. S2). This suggests broad potential for combining AZD0156 with

Figure 5.

AZD0156 potentiates the effects of olaparib across a broad range of cancer cell lines in vitro.A, Example graphs of growth inhibition curves of cells dosedwith 0 or 33nmol/L AZD0156þ/� increasing doses of olaparib for 5 to 8 days depending on cell doubling rate. B, Cell number was determined using the sytox green assay. GI50values were derived from growth inhibition curves generated in GraphPad Prism. GC; gastric cancer.

Figure 4.AZD0156 impairs olaparib-induced DNA damage repair in FaDu WT cells, resulting in cell death through apoptosis. A, gH2AX foci in green, and nuclear staining byHoescht in blue (left). gH2AX foci formationwasmeasured at 48 hours following olaparib and in combinationwith AZD0156 (30 nmol/L) in the FaDuWT cell line. Thegraph represents the percent of cellswith gH2AX foci (green; right). Data are represented as themean of three independent experiments conducted in triplicateþ/�SEM. Cell lysates prepared from cells dosed with olaparibþ/�AZD0156were analyzed for pCHK1-S345 and gH2AX at 48 hours (right). B, FaDuWT cells were dosedwith DMSO, 30 nmol/L AZD0156, or 3 mmol/L olaparibþ/� 30 nmol/L AZD0156. After 48 hours, cells were processed, and the alkaline comet assay was conducted(left). Data are presented as the percent tail intensity of cells from a single experiment� SEM (scatter plot), and as the mean fold increase in tail intensity relative toDMSOcontrol cells across three independent experiments�SEM (right).C,FaDuWTcellswere treatedwith increasingdoses ofAZD0156andolaparib and incubatedwith caspase-Glo reagent. Images of cells were captured on the IncuCyte every 4 hours. Apoptosis is reported relative to cell confluence. Data represent themean oftriplicate samples from a single experiment � SEM.






PARP inhibitors across multiple disease segments. We next exploredthe effect of duration of AZD0156 treatment on olaparib sensitivityusing HCC1806 TNBC cells. Treating cells with two cycles ofAZD0156 (30 nmol/L)þ olaparib (0.3 mmol/L) for 24 hours followedby 5 days exposure to olaparib alone had a moderate combinationeffect, while a 3-day on 4-day off schedule achieved comparable growthinhibition to a continuous schedule of AZD0156 and olaparib com-bination (Supplementary Fig. S3). This indicates that inhibiting ATMfor 3 days is sufficient to sensitize cells to olaparib treatment.

AZD0156 improves the response of olaparib treatment inpatient-derived TNBC xenografts

To determine whether AZD0156 could potentiate olaparib in vivo,two patient-derived TNBC xenograft models with different sensitiv-ities to olaparib were selected, HBCx-10 and HBCx-9 (29). In eachmodel, two tolerated doses and schedules were used, schedule 1:AZD0156 was administered at 5 mg/kg for 3 consecutive days per

week and schedule 2: AZD0156 was administered at 2.5 mg/kgfor 5 consecutive days per week. In both schedules olaparib wasadministered continually at 50 mg/kg. Previously published datademonstrates that HBCx-10model is known to be sensitive to olaparibtreatment alone and demonstrates regressions in combination withAZD0156 using schedule 1 (28). Using schedule 2 in this HBCx-10model, olaparib alone induced regressions in one of 10 tumors,however in combination with AZD0156 tumor, regression wasachieved in 3 of the 8 treated mice and additionally tumor stasis wasseen in 4 of the remaining 5 mice (Fig. 6A). Although olaparibtreatment alone had little effect on tumor growth in the olaparib-insensitive HBCx-9 model, tumor growth inhibition was improvedwith the addition of AZD0156 using schedule 1 (Fig. 6B). Interestinglyschedule 2 in this model did not enhance tumor growth inhibitionbeyond the olaparib response alone (Supplementary Fig. S4).AZD0156 monotherapy treatment did not impact tumor growth ineither model.

Figure 6.

In vivo antitumor efficacy of AZD0156 in patient-derived explants in combination with olaparib. In all studies when delivered in combination, olaparib is dosed first,followed 1 hour later by AZD0156. Adjacent to each efficacy figure (AþB), group plots demonstrate the growth of individual tumors over the study time frame. A,HBCx-10 patient-derived TNBC tumor explant (BRCA2 mut) grown subcutaneously and treated with olaparib (50 mg/kg oral days 1–5 each week for 7 weeks) orAZD0156 either aloneor in combination (monotherapy, 20mg/kgoral alternate day schedule; combination, 2.5mg/kgorally ondays 1–5 eachweek for 7weeks; initialgroup sizes n¼ 8–10). B, HBCx-9 patient-derived TNBC tumor explant (BRCA2WT) grown subcutaneously and treated with olaparib (50 mg/kg oral once daily) orAZD0156 either alone or in combination (monotherapy, 2.5 mg/kg oral on days 1–5 each week; combination, 5 mg/kg oral on days 1–3 each week; initial group sizesn ¼ 10). PO, orally.

Riches et al.





Olaparib sensitivity in the HBCx-10 model may be attributed to amutation in the BRCA2 gene, a known genetic driver of olaparibsensitivity, while no mutations in DDR genes have been described inthe HBCx-9 model. The observation that AZD0156 improved theresponse to olaparib in both models indicates benefit of this combi-nation, irrespective of drivers of PARP inhibitor sensitivity. The dataalso highlights that higher dose of AZD0156 for a shorter period oftime is more efficacious than lower doses over longer periods.

The treatment regimens tested in the HBCx-10 and HBCx-9experiments were well tolerated over the study time frame withindividual animal body weight profiles shown in SupplementaryFig. S5.

Assessment of pharmacodynamic biomarkers of ATM activityTo confirm modulation of ATM following treatment with

AZD0156, tumor samples were collected from the HBCx10 model at

1 and 14 days following daily dosing (olaparib 50 mg/kg once daily,2 mg/kg AZD0156 once daily, combination treatment, and vehiclecontrol) for pharmacokinetic and pharmacodynamic analysis.

Expression of pATM-S1981was quantified byWestern blotting, as ameasure of ATM activity. After a single dose of olaparib (1 hour posttreatment), pATM-S1981 was elevated in olaparib treatment groups at1 hour, indicating that olaparib treatment activates ATM signaling intheHBCx10 xenograft model. Olaparib-induced expression of pATM-S1981 was reduced in AZD0156 combination treatment group, dem-onstrating that AZD0156 effectively inhibits ATM signaling (Fig. 7A).PARylation was also quantified by ELISA, to confirm inhibition ofPAR following olaparib treatment after 1 day of dosing at 50 mg/kgolaparib (Fig. 7B), demonstrating that PARP activity was effectivelyinhibited. Plasma pharmacokinetic exposure was not altered by theaddition of olaparib, and there was no alteration in exposure between 1or 14 days dosing (Fig. 7D).

Figure 7.

Assessment of pharmacodynamic biomarkers of ATM activity and DNA damage HBCx10 patient-derived TNBC xenograft models were dosedwith vehicle, AZD0156(2mg/kg) oncedaily, olaparib (50mg/kg) oncedaily, or olaparibþAZD0156 (oncedaily). Protein isolated from tumors derived fromanimals after 1 dayof dosingwasanalyzed for pATM-S1981 expression by Western blotting (A) or PARylation by ELISA (B). For Western blotting analysis, protein expression was normalized tovinculin and the geometric mean of each animal group is presented relative to the geometric mean of the vehicle group � SEM. C,Western blot analysis of gH2AX(geometric mean relative to vehicle groups � SEM). D, Plasma pharmacokinetics was measured 1 hour after compound administration on day 1 and on day 14.






Expression of gH2AX was quantified by Western blotting, as abiomarker of DNA DSB. A modest increase in the expression ofgH2AX was at 1 hour following a single dose of olaparib, which wasmoderately reduced in combination with AZD0156 (Fig. 7C). On day14, the magnitude of gH2AX expression was greater in the olaparibmonotherapy group compared with day 1, and in combination withAZD0156 treatment, gH2AX was further elevated (Fig. 7C). Thisresult indicates an accumulation of DNA DSB with continuousolaparib treatment, which is exacerbated by ATM inhibition. In thisscenario, H2AX is presumably phosphorylated by ATR or DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is inagreement with in vitro data generated in the FADU cell line, at48 hours.

DiscussionGiven the prominent role of ATM in DNA DSB signaling, ATM

is a promising therapeutic target in cancer biology. AlthoughAZD0156 is not the first reported ATM inhibitor, AZD0156 hasa strong selectivity profile and vastly improved pharmacokineticproperties enabling its use in vivo. Our studies validate AZD0156preclinically, as a potent inhibitor of ATM, which effectively radio-sensitized cancer cell lines in vitro and enhanced the antitumoractivity of radiation in the non–small cell lung cancer xenograftNCI-H441 model in vivo. We anticipate that AZD0156 will alsopotentiate the effects of other clinically relevant DSB-inducingagents such as topoisomerase inhibitors (e.g., irinotecan and topo-tecan; refs. 21, 38).

In addition to enhancing the activity of standard-of-care treatments,our studies support combining AZD0156 with the PARP inhibitorolaparib. Using the early ATM inhibitor KU-55933 and gene silencingtechniques, several groups have reported that abrogation of ATMpotentiates the effects of olaparib in vitro (25, 37). By utilizingAZD0156 and olaparib, our studies build on these observations,demonstrating that AZD0156 is a potent sensitizer of olaparibacross a large panel of cancer cell lines, including TNBC and gastriccancer cells, which is representative of the clinical positioning ofolaparib. Our data indicates that ATM plays an important role inthe response to olaparib, which is supported by the observation ofATM signaling at early time points following olaparib treatment.We demonstrate that AZD0156 impacts the repair of olaparib-induced DNA damage, resulting in a modest increase in DNAstrand breaks and a significant increase in cell death, in vitro. Theobservation that olaparib-induced H2AX phosphorylation waselevated following AZD0156 treatment in vitro and in the HBCx10xenograft model suggests that multiple DDR kinases phosphorylateH2AX in response to olaparib treatment (e.g., ATR or DNA-Pkcs).Despite a degree of redundancy between kinases, the sensitization ofolaparib by AZD0156 reported here, confirms that ATM is animportant factor in determining cell fate following PARP inhibition.In these studies, the elevated gH2AX foci observed followingcombination treatment presumably represents unrepaired DNADSB that when sustained contributes to cell death.

Clinical efficacy has been seen with olaparib in patients with tumorscontaining BRCA mutations (17, 18), and our studies using TNBCpatient-derived xenograft models demonstrate that AZD0156enhances the antitumor activity of olaparib irrespective of intrinsicolaparib sensitivity and DDR deficiencies. Here we present datademonstrating that intermittent dosing of AZD0156 enhances ola-parib activity in vivo, which should prove valuable in facilitating the

development of well-tolerated combination regimes in the clinic. Ourdata provides proof-of-concept for the assessment of AZD0156 andolaparib combinations in the clinic. Furthermore, AZD0156 provides avaluable tool for preclinical target validation and research into the rolesof ATM in the DDR and noncanonical pathways.

Disclosure of Potential Conflicts of InterestA.G. Trinidad has ownership interest (including patents) in AstraZeneca

shares. G. Hughes is a senior scientist (paid consultant) at and has ownershipinterest (including patents) in AstraZeneca shares. G.N. Jones is an associateprinciple scientist (paid consultant) at AstraZeneca and has ownership interest(including patents) in AstraZeneca (owns shares). A.M. Hughes is a seniorscientist (paid consultant) at and has ownership interest (including patents) inAstraZeneca. S. Ling is an associate principal scientist (paid consultant) atAstraZeneca. J. Stott is a senior research scientist (paid consultant) at andhas ownership interest (including patents) in AstraZeneca Pharmaceuticals Ltd.S. Peel is a principal scientist (paid consultant) at AstraZeneca. A. Smith is ascientist (paid consultant) at and has ownership interest (including patents) inAstraZeneca. K.G. Pike is an associate director (paid consultant) at AstraZenecaand has ownership interest (including patents) in AstraZeneca (ordinary shares).B. Barlaam is an associate director (paid consultant) at AstraZeneca. M. Pass isvice president projects (paid consultant) and global project manager at and hasownership interest (including patents) in AstraZeneca. M.J. O’Connor is a full-time employee (paid consultant) at and has ownership interest (including patents)in AstraZeneca (shareholder). G. Smith is an employee (paid consultant) atAstraZeneca and Artios Pharma and has ownership interest (including patents)in AstraZeneca. Elaine B. Cadogan is an associate director (paid consultant) at andhas ownership interest (including patents) in AstraZeneca shares. No potentialconflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: L.C. Riches, A.G. Trinidad, A.G. Thomason, K.G. Pike,M. Pass, M.J. O'Connor, G. Smith, E.B. CadoganDevelopment of methodology: L.C. Riches, A.G. Trinidad, A. Cui, S. Ling, R. Clark,A. SmithAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): L.C. Riches, A.G. Trinidad, A.M. Hughes, P. Gavine, A. Cui, S. Ling,S. Peel, P. Gill, L.M. Goodwin, A. Smith, B. BarlaamAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.C. Riches, A.G. Trinidad, G. Hughes, A.M. Hughes,A.G. Thomason, P. Gavine, A. Cui, S. Ling, J. Stott, R. Clark, P. Gill, A. Smith,E.B. CadoganWriting, review, and/or revision of the manuscript: L.C. Riches, A.G. Trinidad,G. Hughes, G.N. Jones, A.G. Thomason, S. Ling, J. Stott, A. Smith, K.G. Pike,B. Barlaam, M. Pass, M.J. O'Connor, G. Smith, E.B. CadoganAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): L.C. Riches, P. Gavine, J. Stott, P. GillStudy supervision: G. Hughes, P. GavineOther (responsible for designing the molecule AZD0156 used in these studies aswell as ensuring that budget/resource was made available to supply suitablematerial for the study, heavily involved in the discussions around the studiesto be performed, and involved in the analysis/conclusions around the dataobtained and wrote part of/was heavily involved in the review of the article):K.G. Pike

AcknowledgmentsThe authors would like to thankAlan Lau and StephenDurant for contributions to

the ATM project, Xentech SAS for conducting the in vivo studies, Elisabetta Leo forreviewing the article, and AstraZeneca Animal Sciences and Technology and Oncol-ogy in vivo teams for their expert technical assistance.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 20, 2018; revised June 13, 2019; accepted September 11, 2019;published first September 18, 2019.

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References1. Jackson SP, Bartek J. The DNA-damage response in human biology and disease.

Nature 2009;461:1071–8.2. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with

knives. Mol Cell 2010;40:179–204.3. Jeggo PA, Pearl LH, Carr AM. DNA repair, genome stability and cancer: a

historical perspective. Nat Rev Cancer 2016;16:35–42.4. Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature

2012;481:287–94.5. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage

model for cancer development. Science 2008;319:1352–5.6. Curtin NJ. DNA repair dysregulation from cancer driver to therapeutic target.

Nat Rev Cancer 2012;12:801–17.7. Kastan MB, Lim DS. The many substrates and functions of ATM. Nat Rev Mol

Cell Biol 2000;1:179–86.8. 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.

9. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecularautophosphorylation and dimer dissociation. Nature 2003;421:499–506.

10. Gately DP, Hittle JC, Chan GK, Yen TJ. Characterization of ATM expression,localization, and associated DNA-dependent protein kinase activity. Mol BiolCell 1998;9:2361–74.

11. Kastan MB, Lim DS, Kim ST, Xu B, Canman C. Multiple signaling pathwaysinvolving ATM. Cold Spring Harb Symp Quant Biol 2000;65:521–6.

12. Goldberg M, Stucki M, Falck J, D'Amours D, Rahman D, Pappin D, et al.MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature2003;421:952–6.

13. Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cellsignalling and cancer. Nat Rev Mol Cell Biol 2008;9:759–69.

14. Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ. Ataxiatelangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc NatlAcad Sci U S A 2000;97:10389–94.

15. GateiM, Jakob B, Chen P, Kijas AW, BecherelOJ, GuevenN, et al. ATMprotein-dependent phosphorylation of Rad50 protein regulatesDNA repair and cell cyclecontrol. J Biol Chem 2011;286:31542–56.

16. MoralesM, Theunissen JWF, KimCFB, Kitagawa R, KastanMB, Petrini JHJ. TheRad50S allele promotes ATM-dependent DNA damage responses and sup-presses ATM deficiency: implications for the Mre11 complex as a DNA damagesensor. Genes Dev 2005;19:3043–54.

17. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, Bell-McGuinnKM, et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients withBRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concepttrial. Lancet 2010;376:245–51.

18. Tutt A, RobsonM, Garber JE, Domchek SM, AudehMW,Weitzel JN, et al. Oralpoly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 orBRCA2 mutations and advanced breast cancer: a proof-of-concept trial. Lancet2010;376:235–44.

19. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al.Targeting the DNA repair defect in BRCAmutant cells as a therapeutic strategy.Nature 2005;434:917–21.

20. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, et al.Deficiency in the repair of DNA damage by homologous recombination andsensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res 2006;66:8109–15.

21. Pike KG, Barlaam B, Cadogan E, Campbell A, Chen Y, Colclough N, et al. Theidentification of potent, selective, and orally available inhibitors of ataxia

telangiectasia mutated (ATM) kinase: the discovery of AZD0156 (8-{6-[3-(dimethylamino)propoxy]pyridin-3-yl}-3-methyl-1-(tetrahydro-2 H-pyran-4-yl)-1,3-dihydro-2 H-imidazo[4,5- c]quinolin-2-one). J Med Chem 2018;61:3823–41.

22. Murai J, Huang S-yN,Das BB, RenaudA, ZhangY,Doroshow JH, et al. Trappingof PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res 2012;72:5588–99.

23. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI, et al.Identification and characterization of a novel and specific inhibitor of theataxia-telangiectasia mutated kinase ATM. Cancer Res 2004;64:9152–9.

24. Hong R, Ma F, ZhangW, Yu X, Li Q, Luo Y, et al. 53BP1 depletion causes PARPinhibitor resistance in ATM-deficient breast cancer cells. BMC Cancer 2016;16:725.

25. Wang C, Jette N, Moussienko D, Bebb DG, Lees-Miller SP. ATM-deficientcolorectal cancer cells are sensitive to the PARP inhibitor olaparib. Transl Oncol2017;10:190–6.

26. Kubota E, Williamson CT, Ye R, Elegbede A, Peterson L, Lees-Miller SP, et al.Low ATM protein expression and depletion of p53 correlates with olaparibsensitivity in gastric cancer cell lines. Cell Cycle 2014;13:2129–37.

27. Hickson I, Pike KG,Durant ST. Targeting ATM for cancer therapy: prospects fordrugging ATM. In: Pollard J, Curtin N, editors. Targeting the DNA damageresponse for anti-cancer therapy. Cham, Switzerland: Humana Press; 2018.p.185–208.

28. Durant ST, Zheng L, Wang Y, Chen K, Zhang L, Zhang T, et al. The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves sur-vival of preclinical brain tumor models. Sci Adv 2018;4:eaat1719.

29. Marangoni E, Vincent-Salomon A, Auger N, Degeorges A, Assayag F, deCremoux P, et al. A new model of patient tumor-derived breast cancerxenografts for preclinical assays. Clin Cancer Res 2007;13:3989–98.

30. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J BiolChem 1998;273:5858–68.

31. Schmitt A, Knittel G, Welcker D, Yang TP, George J, Nowak M, et al. ATMdeficiency is associated with sensitivity to PARP1 and ATR inhibitors in lungadenocarcinoma. Cancer Res 2017;77:3040–56.

32. Pommier Y, O'Connor MJ, de Bono J. Laying a trap to kill cancer cells: PARPinhibitors and their mechanisms of action. Sci Transl Med 2016;8:362ps17.

33. Yazdi PT,WangY, Zhao S, Patel N, Lee EY,Qin J. SMC1 is a downstream effectorin theATM/NBS1 branch of the human S-phase checkpoint. Genes Dev 2002;16:571–82.

34. Dale Rein I, Solberg Landsverk K, Micci F, Patzke S, Stokke T. Replication-induced DNA damage after PARP inhibition causes G2 delay, and cellline-dependent apoptosis, necrosis and multinucleation. Cell Cycle 2015;14:3248–60.

35. Podhorecka M, Skladanowski A, Bozko P. H2AX phosphorylation: its role inDNA damage response and cancer therapy. J Nucleic Acids 2010;2010. doi:10.4061/2010/920161.

36. Weston VJ, Oldreive CE, Skowronska A, Oscier DG, Pratt G, Dyer MJ, et al. ThePARP inhibitor olaparib induces significant killing of ATM-deficient lymphoidtumor cells in vitro and in vivo. Blood 2010;116:4578–87.

37. Williamson CT, Kubota E, Hamill JD, Klimowicz A, Ye R, Muzik H, et al.Enhanced cytotoxicity of PARP inhibition in mantle cell lymphoma harbouringmutations in both ATM and p53. EMBO Mol Med 2012;4:515–27.

38. Greene J, Nguyen A, Bagby SM, Jones GN, TaiWW,Quackenbush KS, et al. Thenovel ATM inhibitor (AZ31) enhances antitumor activity in patient derivedxenografts that are resistant to irinotecan monotherapy. Oncotarget 2017;8:110904–13.






2020;19:13-25. Published OnlineFirst September 18, 2019.Mol Cancer Ther Lucy C. Riches, Antonio G. Trinidad, Gareth Hughes, et al. Irradiation and Olaparib Responses PreclinicallyPharmacology of the ATM Inhibitor AZD0156: Potentiation of

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Pharmacology of the ATM Inhibitor AZD0156: Potentiation of … · the clinic and to overcome DDR-associated resistance (6). Ataxia-telangiectasia mutated (ATM) kinase belongs to a