AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo. Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

Cells are subject to ongoing DNA damage resulting from physiologic processes (e.g., reactive oxygen species generated through metabolic processes), and exposure to external agents (e.g. chemicals and radiation), which if left unrepaired may compromise genome integrity (1). Consequently, networks of proteins involved in the repair of DNA damage, chromatin remodeling, and cell-cycle maintenance ensure a coordinated response to limit the detrimental effects of DNA damage (2, 3). Together these pathways comprise the DNA damage response (DDR). During cancer development, activation of oncogenes (e.g., MYC and RAS), may induce DNA damage, which if coupled with tumor DDR defects is proposed to drive genomic instability, a core feature of cancer progression (4, 5). The high burden of DNA damage can be exploited in cancer therapy using agents that induce DNA double-strand breaks (DSB), thereby increasing DNA damage to cytotoxic levels. Developing drugs that target DDR proteins presents an opportunity to enhance the therapeutic value of such regimens in the clinic and to overcome DDR-associated resistance (6).

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

The value of targeting DDR factors in the clinic is exemplified by PARP inhibitors (e.g., olaparib), for which benefit has been demonstrated in patients with tumors that harbor BRCA mutations (e.g., ovarian and breast cancers; refs. 17, 18). The activity of PARP inhibitors in BRCAmut tumors exploits the principle of synthetic lethality (19), and preclinical studies indicate that PARP synthetic lethal interactions extend beyond BRCA to DDR proteins including ATM (20–22). Consequently, inhibition of ATM is anticipated to potentiate the effects of PARP inhibitors in the clinic. Indeed, selective inhibitors of ATM have been described, such as KU-55933 and KU-60019 (Fig. 1C), and have been shown to sensitize cancer cells to classic DSB-inducing agents including topoisomerase inhibitors and irradiation (23), in addition to olaparib, in vitro (24–26). These studies provide proof-of-concept for the use of selective ATM inhibitors to potentiate the activity of DNA-damaging agents, including PARP inhibitors. While compounds such as KU-55933 and KU-60019 have proved valuable for probing the effects of ATM inhibition in vitro, the relatively modest cellular potency of the compounds (Table 1), combined with the reported low aqueous solubility and low oral bioavailability, means that these compounds are not considered suitable to deliver meaningful levels of ATM inhibition in the clinic (27). AZD0156 was discovered after chemical optimization of a novel series of ATM inhibitors resulting in a highly significant increase in both potency and selectivity, while also delivering a compound with good physicochemical properties, good preclinical pharmacokinetic profiles, and an acceptable predicted clinically efficacious dose (ref. 21; Fig. 1C). As a result, AZD0156 was considered a suitable molecule to explore the effects of ATM inhibition in humans and was subsequently selected for development as a clinical agent. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

Figure 1.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation. A, FaDu WT and KO cells were pretreated with increasing doses of AZD0156 for 1 hour prior to receiving 5 Gy irradiation (IR). After 1 hour, whole-cell lysates were generated, and phosphorylation of ATM substrates and DNA-PKcs was measured by Western blotting. 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 with γH2AX antibody. Images were captured on the Cell Insight, and the percent of cells with greater than five nuclear foci, as measured by Hoechst staining, was recorded. Data represent the mean of two independent experiments conducted in triplicate ± SEM. Images depict γH2AX foci in green and nuclear staining by Hoechst in blue. C, Chemical structure of AZD0156, KU-55933, and KU-60019.

Figure 1.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation. A, FaDu WT and KO cells were pretreated with increasing doses of AZD0156 for 1 hour prior to receiving 5 Gy irradiation (IR). After 1 hour, whole-cell lysates were generated, and phosphorylation of ATM substrates and DNA-PKcs was measured by Western blotting. 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 with γH2AX antibody. Images were captured on the Cell Insight, and the percent of cells with greater than five nuclear foci, as measured by Hoechst staining, was recorded. Data represent the mean of two independent experiments conducted in triplicate ± SEM. Images depict γH2AX foci in green and nuclear staining by Hoechst in blue. C, Chemical structure of AZD0156, KU-55933, and KU-60019.

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Table 1.

Comparison of ATM inhibitor properties in cell assays.

TargetKU559933 IC50 (μmol/L)KU60019 IC50 (μmol/L)AZD0156 IC50 (μmol/L)
ATM (pATMS19811.13 (N = 222) 0.15 (N = 223) 0.00058 (N = 16) 
TargetKU559933 IC50 (μmol/L)KU60019 IC50 (μmol/L)AZD0156 IC50 (μmol/L)
ATM (pATMS19811.13 (N = 222) 0.15 (N = 223) 0.00058 (N = 16) 

Importantly, another potent ATM inhibitor under clinical evaluation, AZD1390 has been recently reported to radiosensitize preclinical brain tumor models inducing tumor regression and shows efficient blood–brain barrier penetration in vivo (28).

Here we present preclinical data characterizing the pharmacology of AZD0156 with properties considered suitable for clinical development. Our work presents a comprehensive data package providing mechanistic insights into how AZD0156 potentiates the effects of olaparib, thus providing supporting evidence for the evaluation of this combination in the clinic (NCT02588105).

Compounds/chemicals

Compounds (AZD0156 and olaparib) were synthesized internally at AstraZeneca laboratories (21) and dissolved in 100% DMSO at 1 mmol/L (AZD0156) or 10 mmol/L (olaparib) stocks, which were used for in vitro studies.

Cell culture

All cell lines were obtained from the ATCC and were authenticated by short tandem repeat profile and tested negative for Mycoplasma contamination. Cells were passaged at least twice following thawing and cultured in RPMI media supplemented with 10% FCS and 2 mmol/L l-glutamine and maintained under standard cell culture conditions at 37°C, 5% CO2. Cells were routinely passaged using 1× TrypleX to detach cells from the tissue culture vessel. Only exponentially growing cells below passage 10 and with a viability greater than 95% as measured by Trypan blue exclusion were used during experiments.

In vitro growth inhibition assays; sytox green assay

Cells were seeded into clear bottom, black 96-well plates at a preoptimized seeding density and left to adhere overnight. The following day, cells were dosed with 10× concentrated compound to achieve a 5-point dose response of olaparib and doses of AZD0156 as stated in figure legends. After 5–7 days, depending on cell doubling time, cells were permeabilized by the addition of saponin and stained with sytox green DNA stain. Cell number was quantified using the Acumen.

Colony formation assay

Exponentially growing cells were seeded into 6-well plates at 1,000 cells/well [FaDu wild-type (WT)] or 2,000 cells/well [FaDu ATM knockout (KO)] and dosed the following day with AZD0156 (30 nmol/L). After 1 hour, cells were dosed with olaparib (10–0.1 μmol/L) or irradiated at the stated dose using a bench top CellRad X-ray Irradiator (Faxitron). After 7–10 days, once colonies of >50 cells had formed in the DMSO control wells, cells were fixed and stained. Plates were scanned, and colonies quantified using the Gel Count System (Oxford Optronics Ltd).

Analysis of proliferation assay data

Data obtained from the sytox green and colony formation assays was normalized to DMSO control samples, and dose–response curves for olaparib or irradiation +/− AZD0156 were plotted in GraphPad Prism to generate GI50 values (the concentration of treatment required to inhibit cell growth by 50%).

Protein extraction and Western blot analysis

For protein extraction, cells were lysed in NP-40 lysis buffer (50 mmol/L Tris-HCl pH7, 150 mmol/L NaCl, 1 mmol/L EDTA, and 1% NP-40 supplemented with complete protease and phosphatase inhibitor cocktail, Roche). A total of 30 μg protein was separated on SDS-PAGE Bis-Tris 4%–12% or Tris-Acetate 3%–8% for high molecular weight proteins and transferred by iBLOT (Invitrogen) for 10 minutes at 20 V onto nitrocellulose membranes. Membranes were blocked in 3% BSA:0.1% Tween 20:TBS and incubated with primary antibody at 4°C overnight. Membranes were washed in 0.05% T-TBS, incubated with horseradish peroxide (HRP)-coupled secondary antibodies (1:4,000) then incubated with ECL Reagent (Pierce), and visualized using the ODYSSEY CLX system or film. For pharmacodynamic studies, the intensity of Western blot analysis signal was measured and normalized to vinculin expression.

The following antibodies were used at 1:1,000 dilution unless otherwise 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), β-Actin (Sigma, 1:10,000), PAR (Trevigen, 1:1,000), pDNA-PKcs (developed internally at AstraZeneca, 1:1,000), tDNA-PKcs (Cell Signaling Technology, 1:1,000), and γH2AX (Millipore, 1:500).

Cell-cycle studies

Cells were compound treated in 6-well plates for the specified time (24–72 hours) then harvested and fixed in ice-cold 70% EtOH for 1 hour. Cells were washed in PBS and incubated with 5 μg/mL DAPI for 30 minutes. Samples were acquired on the FACS Aria I, and data were analyzed in FlowJo to determine the percent of cells in each phase of the cell cycle based on DNA content.

Caspase-3/7 assay

FaDu cells were seeded in 96-well plates (2,000/well) and the following day compound and NucView caspase Glo reagent was added (following the manufacturer's instructions, Essen BioSciences). Images were captured in the green fluorescent and phase contrast channels every 4 hours, on the IncuCyte Zoom (Essen BioScience). Images were subsequently analyzed using the Incucyte Zoom software to report the area of apoptosis-positive cells in the green fluorescent channel relative to cell confluence, as determined in the phase contrast channel.

Immunofluorescence (γH2AX)

FaDu cells cultured in clear bottom, black 96-well plates (2,000 cells/well) were compound treated with AZD0156 for 1 hour prior to Olaparib or radiation treatment. At the stated time, cells were fixed in 3.7% paraformaldehyde for 20 minutes, washed in PBS, and permeabilized with 0.5% Triton-X100 for 5 minutes. Cells were blocked in 3% BSA, incubated with γH2AX antibody (Millipore clone JBW301; 1:500) at 4°C overnight, then washed with tween, and incubated with Alexa-488–conjugated secondary antibody (1:500; Invitrogen Molecule Probes) for 45 minutes. Cells were stained with Hoechst prior to imaging on the Cell Insight at 20 × magnification. The number of nuclei foci was measured using the Cell Insight spot detector application. Only foci within the nuclear region as defined by Hoechst staining were classified. A minimum of 100 cells were analyzed per well.

Comet assay

Cells were treated with compound in 6-well plates and samples processed at 48 hours. Single-cell suspensions were mixed with low melting agarose and transferred onto 20-well slides (Trevigen) then lysed in Trevigen lysis solution overnight. Samples were incubated in alkaline solution (pH 13) for 20 minutes and electrophoresed in the 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 microscopy at 10 × magnification and a minimum of 100 cells were scored across duplicate samples using the Comet IV Software (Perspective). Data are represented as percent of DNA in the tail (% tail intensity–TI).

Xenograft-targeted irradiation study

NCI-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 with 50% Matrigel at 5 × 106 per mouse. Male nude mice (Harlan UK) at greater than 18 g had tumor size monitored twice weekly by bilateral caliper measurements. Treatments were started when tumor volumes reached an average of approximately 0.26 cm3. Targeted irradiation of 2 Gy was delivered over 2 minutes daily over the first 5 days. AZD0156 (10 mg/kg) was given orally for the duration of the study. This study was run in the United Kingdom in accordance with UK Home Office legislation, the Animal Scientific Procedures Act 1986, and with AstraZeneca Global Bioethics policy. Experimental details are outlined in Home Office project license 40/3451.

Patient-derived xenograft studies

Triple-negative breast cancer (TNBC) HBCx-10 and HBCx-9 (29) patient-derived xenograft studies were carried out at XenTech, France in accordance with French regulatory legislation concerning the protection of laboratory animals. Female athymic nude mice (Harlan France) greater than 18 g were implanted with HBCx-10 or HBCx-9 tumor derived from a primary ductal adenocarcinoma. Donor mice were sacrificed to provide tumor fragments, which were surgically implanted subcutaneously. Tumor size was monitored twice weekly by bilateral caliper measurements. Mice body weights were recorded at the same time. For efficacy studies, treatment started when tumor volume averaged approximately 0.15 cm3 and for pharmacodynamic studies treatment started at approximately 0.5 cm3. In efficacy and pharmacodynamic studies, control mice were dosed orally with vehicle, AZD0156, or olaparib alone or in combination using different dosing schedules.

Pharmacodynamic analysis

For 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 cycles at 6.5 m/s. Samples were sonicated for 15 seconds at 50% amplitude and incubated on ice for 30 minutes. The supernatant was collected by centrifugation and lysates analyzed by Western blotting for pATM and γH2AX expression.

PARylation was analyzed using the HT PARP in vivo Pharmacodynamic Assay II ELISA 2nd generation ELISA Kit (Trevigen catalog no. 4520-096-K; Trevigen Inc.). Protein (1 mg/mL in Lysis buffer) was incubated at 100°C for 5 minutes, diluted to 40 ng/μL in kit diluent buffer and incubated for 16 hours at 4°C on precoated ELISA plates. After washing in PBS Tween (0.1%), secondary antibody was added for 1 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 estimated from comparisons with standard curves.

Plasma analysis

Each plasma sample (25 μL) was prepared using an appropriate dilution factor and compared against an 11-point standard calibration curve (1–10,000 nmol/L) prepared in DMSO and spiked into blank plasma. Acetonitrile (100 μL) was added with the internal standard, followed by centrifugation at 3,000 rpm for 10 minutes. Supernatant (50 μL) was then diluted in 300 μL water and analyzed via UPLC-MS/MS (Supplementary Tables S1 and S2).

AZD0156 is a potent inhibitor of ATM, with an IC50 of 0.58 nmol/L in cell assays, as measured by inhibition of ATM auto-phosphorylation at serine 1981, at 1 hour following radiation treatment in HT29 cells (Table 1; Fig. 1C). Selectivity of AZD0156 was 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 in these assays (21).

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation

ATM undergoes rapid auto-phosphorylation in response to irradiation (9) and so we employed irradiation to validate AZD0156 activity preclinically, using isogenic FaDu ATM proficient (FaDu WT) and ATM triple knockout (FaDu KO) cell lines. The FaDu-KO cell line was generated at AstraZeneca using Zinc finger nuclease technology. Cells were pretreated with increasing doses of AZD0156 (1–30 nmol/L) for 1 hour prior to radiation (5 Gy). In FaDu WT cells, AZD0156 inhibited irradiation-induced ATM signaling in a dose-dependent manner as measured by auto-phosphorylation on serine 1981 of ATM, and phosphorylation of ATM substrates including KAP1, RAD50, and CHK2 (Fig. 1A). AZD0156 did not modulate DNA-PKcs phosphorylation following irradiation, indicating that AZD0156 selectively abrogates ATM signaling (Fig. 1A). As expected, no phosphorylation of ATM or its substrates were observed in the FaDu KO cells. To further confirm inhibition of ATM activity by AZD0156, γH2AX foci formation, a universal biomarker of DSB and direct target of ATM (30), was measured by immunofluorescence. Irradiation (5 Gy) alone resulted in a 20% increase in the number of FaDu WT cells with >5 nuclear γH2AX foci cells at 4 hours, which was reduced to less than 2% when pretreated with 30 nmol/L AZD0156 (Fig. 1B). This indicates that H2AX phosphorylation was strongly inhibited in the absence of ATM function following irradiation.

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

Figure 2.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation. A, FaDu WT and KO cells were pretreated +/− 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 a single 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 receiving 2 Gy irradiation. After 14 days, colonies were scored. Data are represented as the mean of two independent experiments conducted in duplicate ± SD. C, NCI-H441 non–small cell lung cancer xenograft grown subcutaneously was treated with 5 days of targeted irradiation (2 Gy over 2 minutes daily) combined with 38 days of once daily oral dosing AZD0156 10 mg/kg, AZD0156 administered 1 hour prior to irradiation (IR; initial group sizes n = 9–12). PO, orally; QD, every day.

Figure 2.

AZD0156 inhibits ATM signaling and potentiates the effects of irradiation. A, FaDu WT and KO cells were pretreated +/− 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 a single 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 receiving 2 Gy irradiation. After 14 days, colonies were scored. Data are represented as the mean of two independent experiments conducted in duplicate ± SD. C, NCI-H441 non–small cell lung cancer xenograft grown subcutaneously was treated with 5 days of targeted irradiation (2 Gy over 2 minutes daily) combined with 38 days of once daily oral dosing AZD0156 10 mg/kg, AZD0156 administered 1 hour prior to irradiation (IR; initial group sizes n = 9–12). PO, orally; QD, every day.

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To investigate the potential for AZD0156 to combine with irradiation in a disease relevant setting, the non–small cell lung adenocarcinoma cell line NCI-H441 was treated with 3, 10, and 30 nmol/L AZD0156 prior to 2 Gy radiation. Consistent with data generated in the FaDu WT cell line, pretreatment of cells with AZD0156 reduced colony formation 24-fold compared with irradiation treatment alone (Fig. 2B). We next determined whether this radiosensitization would translate to the corresponding in vivo xenograft model. Irradiation treatment alone, administered during the first 5 days of the study to induce DNA damage, inhibited tumor growth in the NCI-H4441 xenograft model, which was enhanced by the addition of AZD0156 (Fig. 2C).

AZD0156 impairs olaparib-induced activation of ATM and potentiates 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 that ATM deficiency drives PARP inhibitor sensitivity (20, 25, 31), and therefore we investigated the ability of AZD0156 to potentiate olaparib treatment in vitro. Activation of ATM was observed following 2 and 6 hours olaparib treatment (1–3 μmol/L), as measured by auto-phosphorylation of ATM and induction of pCHK2-T68, which was reduced by pretreatment with AZD0156 (30 nmol/L; Fig. 3A). To establish 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 FaDu WT cells, however no effect was observed in the FaDu KO cell line (Fig. 3B). This result confirmed that the combination effect was mediated through inhibition of ATM (Fig. 3B). We noted that in this assay, the FaDu KO cells were intrinsically more sensitive to olaparib treatment alone, reinforcing the notion that ATM deficiency potentiates the effects of olaparib treatment (Fig. 3B). Further mechanistic studies were conducted in the FaDu ATM-proficient cell line.

Figure 3.

AZD0156 impairs olaparib-induced activation of ATM and potentiates the activity of olaparib in vitro. A, Cells were treated with olaparib +/− 30 nmol/L AZD0156 for 2 to 6 hours. pATM-S1981 and pCHK2-T68 were measured as markers of ATM signaling by Western blotting. B, Cells were treated with 30 nmol/L AZD0156 (red) or DMSO (black) and increasing doses of olaparib. After 7 to 10 days, colonies were scored and the surviving fraction plotted relative to DMSO control cells. Data represent the mean of two independent repeats run in triplicate ± SD. C, FaDu WT and KO cells treated with olaparib +/− AZD0156 were processed for flow cytometry 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 +/− AZD0156 at 24, 48, or 72 hours.

Figure 3.

AZD0156 impairs olaparib-induced activation of ATM and potentiates the activity of olaparib in vitro. A, Cells were treated with olaparib +/− 30 nmol/L AZD0156 for 2 to 6 hours. pATM-S1981 and pCHK2-T68 were measured as markers of ATM signaling by Western blotting. B, Cells were treated with 30 nmol/L AZD0156 (red) or DMSO (black) and increasing doses of olaparib. After 7 to 10 days, colonies were scored and the surviving fraction plotted relative to DMSO control cells. Data represent the mean of two independent repeats run in triplicate ± SD. C, FaDu WT and KO cells treated with olaparib +/− AZD0156 were processed for flow cytometry 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 +/− AZD0156 at 24, 48, or 72 hours.

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Mechanistically, olaparib inhibits single-strand break repair and traps PARP onto DNA, creating complexes that interfere with DNA replication and manifest as DSBs (32). Therefore, olaparib-induced DNA damage was anticipated to occur predominantly in S-phase. We hypothesized that the synergy observed between olaparib and AZD0156 may be in part due to abrogation of checkpoint signaling by AZD0156, because ATM contributes to S-phase checkpoint signaling (33). To investigate this, we measured the impact of AZD0156–olaparib combinations on the cell cycle by flow cytometry. At 24 hours following treatment, olaparib treatment alone had a modest effect on cell-cycle parameters compared with control samples, as measured by DNA 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 effect observed using the colony formation assay (Fig. 3B). Cell-cycle parameters were also measured at 48 and 72 hours to investigate longer term effects of olaparib (1 μmol/L)–AZD0156 combinations on the cell cycle. A time-dependent decrease in the G2–M-phase population was accompanied by an increase in the sub-G1 peak, indicative of cell death and at 72 hours we noted the appearance of cells with >2N DNA content (Fig. 3C and D). The latter finding is similar to data reported in malignant lymphocyte cells, in which concomitant PARP and ATM inhibition, by olaparib and KU55933, respectively, resulted in a delayed G2 transition and an increase in DNA content in some cell lines tested (34).

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

Our cell-cycle observations support the hypothesis that when ATM is inhibited, cells containing olaparib-induced DNA damage in S-phase continue through the cell cycle, and the presence of unrepaired DNA damage activates the G2–M checkpoint. Because combination of KU55933 and olaparib was previously reported to exacerbate γH2AX in CAL51 cells (24), we measured the formation of γH2AX foci, a universal biomarker of DSB, by immunofluorescence. In FaDu WT cells, olaparib treatment (1 and 3 μmol/L) resulted in an elevated proportion of cells with >5 γH2AX foci at 48 hours, (Fig. 4A), indicative of DSB, presumably ensuing from unrepaired single-strand breaks or collapsed replication forks. Combining olaparib (3 μmol/L) with 30 nmol/L AZD0156 resulted in a significant increase (2.67-fold) in the percent of γH2AX-positive cells at 48 hours compared with olaparib treatment alone (Fig. 4A). Although H2AX is a substrate of ATM, in this scenario, it is feasible that prolonged inhibition of ATM (48 hours) may result in activation of other kinases, for example ATR or DNA-PKcs, which can also phosphorylate H2AX (35). To explore whether checkpoint signaling was affected, we measured CHK1 phosphorylation at serine 345. After 48 hours of treatment, pCHK1 was elevated in the combination treated cells above monotherapy treatment (Fig. 4A). CHK1 is a substrate of ATR, and contributes to the G2 checkpoint signaling, which may indicate increased activation of ATR when ATM is inhibited. Despite activation of γH2AX in the absence of ATM, the profound growth inhibitory effect observed between olaparib and AZD0156 (Fig. 3B) suggests that other DDR kinases cannot fully compensate in FaDu cells. Rather, the presence of γH2AX foci following combination treatment likely represents persistent DSB due to inefficient repair of olaparib-induced damage when ATM is inhibited. This is similar to findings in ATM-deficient lymphoid cells following prolonged olaparib treatment (36).

Figure 4.

AZD0156 impairs olaparib-induced DNA damage repair in FaDu WT cells, resulting in cell death through apoptosis. A, γH2AX foci in green, and nuclear staining by Hoescht in blue (left). γH2AX foci formation was measured at 48 hours following olaparib and in combination with AZD0156 (30 nmol/L) in the FaDu WT cell line. The graph represents the percent of cells with γH2AX foci (green; right). Data are represented as the mean of three independent experiments conducted in triplicate +/− SEM. Cell lysates prepared from cells dosed with olaparib +/− AZD0156 were analyzed for pCHK1-S345 and γH2AX at 48 hours (right). B, FaDu WT cells were dosed with DMSO, 30 nmol/L AZD0156, or 3 μmol/L olaparib +/− 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 to DMSO control cells across three independent experiments ± SEM (right). C, FaDu WT cells were treated with increasing doses of AZD0156 and olaparib and incubated with caspase-Glo reagent. Images of cells were captured on the IncuCyte every 4 hours. Apoptosis is reported relative to cell confluence. Data represent the mean of triplicate samples from a single experiment ± SEM.

Figure 4.

AZD0156 impairs olaparib-induced DNA damage repair in FaDu WT cells, resulting in cell death through apoptosis. A, γH2AX foci in green, and nuclear staining by Hoescht in blue (left). γH2AX foci formation was measured at 48 hours following olaparib and in combination with AZD0156 (30 nmol/L) in the FaDu WT cell line. The graph represents the percent of cells with γH2AX foci (green; right). Data are represented as the mean of three independent experiments conducted in triplicate +/− SEM. Cell lysates prepared from cells dosed with olaparib +/− AZD0156 were analyzed for pCHK1-S345 and γH2AX at 48 hours (right). B, FaDu WT cells were dosed with DMSO, 30 nmol/L AZD0156, or 3 μmol/L olaparib +/− 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 to DMSO control cells across three independent experiments ± SEM (right). C, FaDu WT cells were treated with increasing doses of AZD0156 and olaparib and incubated with caspase-Glo reagent. Images of cells were captured on the IncuCyte every 4 hours. Apoptosis is reported relative to cell confluence. Data represent the mean of triplicate samples from a single experiment ± SEM.

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From images captured to measure γH2AX foci formation, we noted an increase in the proportion of cells containing micronuclei (Supplementary Fig. S1), a characteristic of DNA damage. To confirm the presence of DNA damage, we performed the alkaline comet assay in FaDu WT cells. Following 30 nmol/L AZD0156 treatment alone, we observed a modest but consistent increase in tail intensity, indicative of DNA damage (Fig. 4B). In combination with 3 μmol/L olaparib, the mean tail intensity of comets was elevated 1.48-fold above 3 μmol/L olaparib treatment alone, and we noted a large heterogeneity in tail intensity, with a small proportion of cells with a tail intensity greater than 50% (Fig. 4B). This likely reflects the mode of action of olaparib, in which replicating cells are expected to be more susceptible to the effects of PARP inhibition.

Having observed an increase in the sub-G1 population of cells treated with olaparib and AZD0156 combination by flow cytometry analysis (Fig. 3D), which is indicative of cell death, we investigated whether this proceeded through apoptosis. Using the caspase3/7 NucView assay we measured the kinetics of apoptosis using the IncuCyte. A relatively small increase in cleaved caspase3/7 was observed with olaparib or AZD0156 treatment alone, indicating that few cells underwent apoptosis in these treatment groups (Fig. 4C). In combination, however, the cleaved caspase3/7 signal was elevated 4-fold with 30 nmol/L AZD0156 + 1 μmol/L olaparib compared with corresponding single-agent control samples at 96 hours (Fig. 4C). This demonstrates that concomitant inhibition of ATM and PARP drives FaDu WT cells into apoptosis.

Our data suggests that AZD0156 creates a DDR-deficient phenotype that exacerbates the effects of olaparib in FaDu ATM proficient cells and impedes the repair of olaparib-induced DNA damage in vitro. As such, AZD0156 presents an opportunity to create a contextual DDR-deficient phenotype to sensitize cancer cells to PARP inhibitors.

Potentiation of olaparib by AZD0156 across a panel of cancer cell lines

Previously published data has indicated that cancer cell lines including colorectal, mantle cell lymphoma, and gastric cancer cells can be sensitized to the effects of olaparib in vitro using the early ATM inhibitor KU59933 (25, 37). To determine whether the combination effect between AZD0156 and olaparib extended beyond FaDu cells and to build upon published findings, we performed the sytox green proliferation assay across a panel of cancer cell lines in vitro. AZD0156 potentiated the effects of olaparib across all cell lines tested, which included TNBC, gastric cancer, and non–small cell lung cancer cells (Fig. 5A and B). A broader screen of gastric cell lines using short-term assays was also conducted, which confirmed the combination effect across the majority of ATM-proficient cells tested (Supplementary Fig. S2). This suggests broad potential for combining AZD0156 with PARP inhibitors across multiple disease segments. We next explored the effect of duration of AZD0156 treatment on olaparib sensitivity using HCC1806 TNBC cells. Treating cells with two cycles of AZD0156 (30 nmol/L) + olaparib (0.3 μmol/L) for 24 hours followed by 5 days exposure to olaparib alone had a moderate combination effect, while a 3-day on 4-day off schedule achieved comparable growth inhibition to a continuous schedule of AZD0156 and olaparib combination (Supplementary Fig. S3). This indicates that inhibiting ATM for 3 days is sufficient to sensitize cells to olaparib treatment.

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 dosed with 0 or 33 nmol/L AZD0156 +/− increasing doses of olaparib for 5 to 8 days depending on cell doubling rate. B, Cell number was determined using the sytox green assay. GI50 values were derived from growth inhibition curves generated in GraphPad Prism. GC; gastric cancer.

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 dosed with 0 or 33 nmol/L AZD0156 +/− increasing doses of olaparib for 5 to 8 days depending on cell doubling rate. B, Cell number was determined using the sytox green assay. GI50 values were derived from growth inhibition curves generated in GraphPad Prism. GC; gastric cancer.

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AZD0156 improves the response of olaparib treatment in patient-derived TNBC xenografts

To determine whether AZD0156 could potentiate olaparib in vivo, two patient-derived TNBC xenograft models with different sensitivities to olaparib were selected, HBCx-10 and HBCx-9 (29). In each model, 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/kg for 5 consecutive days per week. In both schedules olaparib was administered continually at 50 mg/kg. Previously published data demonstrates that HBCx-10 model is known to be sensitive to olaparib treatment alone and demonstrates regressions in combination with AZD0156 using schedule 1 (28). Using schedule 2 in this HBCx-10 model, olaparib alone induced regressions in one of 10 tumors, however in combination with AZD0156 tumor, regression was achieved in 3 of the 8 treated mice and additionally tumor stasis was seen in 4 of the remaining 5 mice (Fig. 6A). Although olaparib treatment alone had little effect on tumor growth in the olaparib-insensitive HBCx-9 model, tumor growth inhibition was improved with the addition of AZD0156 using schedule 1 (Fig. 6B). Interestingly schedule 2 in this model did not enhance tumor growth inhibition beyond the olaparib response alone (Supplementary Fig. S4). AZD0156 monotherapy treatment did not impact tumor growth in either 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 (A+B), 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) or AZD0156 either alone or in combination (monotherapy, 20 mg/kg oral alternate day schedule; combination, 2.5 mg/kg orally on days 1–5 each week for 7 weeks; initial group sizes n = 8–10). B, HBCx-9 patient-derived TNBC tumor explant (BRCA2 WT) grown subcutaneously and treated with olaparib (50 mg/kg oral once daily) or AZD0156 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 sizes n = 10). PO, orally.

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 (A+B), 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) or AZD0156 either alone or in combination (monotherapy, 20 mg/kg oral alternate day schedule; combination, 2.5 mg/kg orally on days 1–5 each week for 7 weeks; initial group sizes n = 8–10). B, HBCx-9 patient-derived TNBC tumor explant (BRCA2 WT) grown subcutaneously and treated with olaparib (50 mg/kg oral once daily) or AZD0156 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 sizes n = 10). PO, orally.

Close modal

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

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

Assessment of pharmacodynamic biomarkers of ATM activity

To 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 vehicle control) for pharmacokinetic and pharmacodynamic analysis.

Expression of pATM-S1981 was quantified by Western blotting, as a measure of ATM activity. After a single dose of olaparib (1 hour post treatment), pATM-S1981 was elevated in olaparib treatment groups at 1 hour, indicating that olaparib treatment activates ATM signaling in the HBCx10 xenograft model. Olaparib-induced expression of pATM-S1981 was reduced in AZD0156 combination treatment group, demonstrating that AZD0156 effectively inhibits ATM signaling (Fig. 7A). PARylation was also quantified by ELISA, to confirm inhibition of PAR following olaparib treatment after 1 day of dosing at 50 mg/kg olaparib (Fig. 7B), demonstrating that PARP activity was effectively inhibited. Plasma pharmacokinetic exposure was not altered by the addition of olaparib, and there was no alteration in exposure between 1 or 14 days dosing (Fig. 7D).

Figure 7.

Assessment of pharmacodynamic biomarkers of ATM activity and DNA damage HBCx10 patient-derived TNBC xenograft models were dosed with vehicle, AZD0156 (2 mg/kg) once daily, olaparib (50 mg/kg) once daily, or olaparib + AZD0156 (once daily). Protein isolated from tumors derived from animals after 1 day of dosing was analyzed for pATM-S1981 expression by Western blotting (A) or PARylation by ELISA (B). For Western blotting analysis, protein expression was normalized to vinculin 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 γH2AX (geometric mean relative to vehicle groups ± SEM). D, Plasma pharmacokinetics was measured 1 hour after compound administration on day 1 and on day 14.

Figure 7.

Assessment of pharmacodynamic biomarkers of ATM activity and DNA damage HBCx10 patient-derived TNBC xenograft models were dosed with vehicle, AZD0156 (2 mg/kg) once daily, olaparib (50 mg/kg) once daily, or olaparib + AZD0156 (once daily). Protein isolated from tumors derived from animals after 1 day of dosing was analyzed for pATM-S1981 expression by Western blotting (A) or PARylation by ELISA (B). For Western blotting analysis, protein expression was normalized to vinculin 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 γH2AX (geometric mean relative to vehicle groups ± SEM). D, Plasma pharmacokinetics was measured 1 hour after compound administration on day 1 and on day 14.

Close modal

Expression of γH2AX was quantified by Western blotting, as a biomarker of DNA DSB. A modest increase in the expression of γH2AX was at 1 hour following a single dose of olaparib, which was moderately reduced in combination with AZD0156 (Fig. 7C). On day 14, the magnitude of γH2AX expression was greater in the olaparib monotherapy group compared with day 1, and in combination with AZD0156 treatment, γH2AX was further elevated (Fig. 7C). This result indicates an accumulation of DNA DSB with continuous olaparib treatment, which is exacerbated by ATM inhibition. In this scenario, H2AX is presumably phosphorylated by ATR or DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is in agreement with in vitro data generated in the FADU cell line, at 48 hours.

Given the prominent role of ATM in DNA DSB signaling, ATM is a promising therapeutic target in cancer biology. Although AZD0156 is not the first reported ATM inhibitor, AZD0156 has a strong selectivity profile and vastly improved pharmacokinetic properties enabling its use in vivo. Our studies validate AZD0156 preclinically, as a potent inhibitor of ATM, which effectively radio-sensitized cancer cell lines in vitro and enhanced the antitumor activity of radiation in the non–small cell lung cancer xenograft NCI-H441 model in vivo. We anticipate that AZD0156 will also potentiate the effects of other clinically relevant DSB-inducing agents such as topoisomerase inhibitors (e.g., irinotecan and topotecan; refs. 21, 38).

In addition to enhancing the activity of standard-of-care treatments, our studies support combining AZD0156 with the PARP inhibitor olaparib. Using the early ATM inhibitor KU-55933 and gene silencing techniques, several groups have reported that abrogation of ATM potentiates the effects of olaparib in vitro (25, 37). By utilizing AZD0156 and olaparib, our studies build on these observations, demonstrating that AZD0156 is a potent sensitizer of olaparib across a large panel of cancer cell lines, including TNBC and gastric cancer cells, which is representative of the clinical positioning of olaparib. Our data indicates that ATM plays an important role in the response to olaparib, which is supported by the observation of ATM 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 DNA strand breaks and a significant increase in cell death, in vitro. The observation that olaparib-induced H2AX phosphorylation was elevated following AZD0156 treatment in vitro and in the HBCx10 xenograft model suggests that multiple DDR kinases phosphorylate H2AX in response to olaparib treatment (e.g., ATR or DNA-Pkcs). Despite a degree of redundancy between kinases, the sensitization of olaparib by AZD0156 reported here, confirms that ATM is an important factor in determining cell fate following PARP inhibition. In these studies, the elevated γH2AX foci observed following combination treatment presumably represents unrepaired DNA DSB that when sustained contributes to cell death.

Clinical efficacy has been seen with olaparib in patients with tumors containing BRCA mutations (17, 18), and our studies using TNBC patient-derived xenograft models demonstrate that AZD0156 enhances the antitumor activity of olaparib irrespective of intrinsic olaparib sensitivity and DDR deficiencies. Here we present data demonstrating that intermittent dosing of AZD0156 enhances olaparib activity in vivo, which should prove valuable in facilitating the development of well-tolerated combination regimes in the clinic. Our data provides proof-of-concept for the assessment of AZD0156 and olaparib combinations in the clinic. Furthermore, AZD0156 provides a valuable tool for preclinical target validation and research into the roles of ATM in the DDR and noncanonical pathways.

A.G. Trinidad has ownership interest (including patents) in AstraZeneca shares. G. Hughes is a senior scientist (paid consultant) at and has ownership interest (including patents) in AstraZeneca shares. G.N. Jones is an associate principle scientist (paid consultant) at AstraZeneca and has ownership interest (including patents) in AstraZeneca (owns shares). A.M. Hughes is a senior scientist (paid consultant) at and has ownership interest (including patents) in AstraZeneca. S. Ling is an associate principal scientist (paid consultant) at AstraZeneca. J. Stott is a senior research scientist (paid consultant) at and has ownership interest (including patents) in AstraZeneca Pharmaceuticals Ltd. S. Peel is a principal scientist (paid consultant) at AstraZeneca. A. Smith is a scientist (paid consultant) at and has ownership interest (including patents) in AstraZeneca. K.G. Pike is an associate director (paid consultant) at AstraZeneca and has ownership interest (including patents) in AstraZeneca (ordinary shares). B. Barlaam is an associate director (paid consultant) at AstraZeneca. M. Pass is vice president projects (paid consultant) and global project manager at and has ownership 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) at AstraZeneca and Artios Pharma and has ownership interest (including patents) in AstraZeneca. Elaine B. Cadogan is an associate director (paid consultant) at and has ownership interest (including patents) in AstraZeneca shares. No potential conflicts of interest were disclosed by the other authors.

Conception and design: L.C. Riches, A.G. Trinidad, A.G. Thomason, K.G. Pike, M. Pass, M.J. O'Connor, G. Smith, E.B. Cadogan

Development of methodology: L.C. Riches, A.G. Trinidad, A. Cui, S. Ling, R. Clark, A. Smith

Acquisition of data (provided animals, acquired and managed patients, provided facilities, 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. Barlaam

Analysis 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. Cadogan

Writing, 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. Cadogan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.C. Riches, P. Gavine, J. Stott, P. Gill

Study supervision: G. Hughes, P. Gavine

Other (responsible for designing the molecule AZD0156 used in these studies as well as ensuring that budget/resource was made available to supply suitable material for the study, heavily involved in the discussions around the studies to be performed, and involved in the analysis/conclusions around the data obtained and wrote part of/was heavily involved in the review of the article): K.G. Pike

The authors would like to thank Alan Lau and Stephen Durant for contributions to the ATM project, Xentech SAS for conducting the in vivo studies, Elisabetta Leo for reviewing the article, and AstraZeneca Animal Sciences and Technology and Oncology in vivo teams for their expert technical assistance.

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

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