Article Figures & Data
Figures
- 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 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 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 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 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 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 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.
Tables
- Table 1.
Comparison of ATM inhibitor properties in cell assays.
Target KU559933 IC50 (μmol/L) KU60019 IC50 (μmol/L) AZD0156 IC50 (μmol/L) ATM (pATMS1981) 1.13 (N = 222) 0.15 (N = 223) 0.00058 (N = 16)
Supplementary Data
- Supplementary Data - Table S1. Mass Specrometer and UPLC system parameters; Table S2. Optimization parameters for mass spectrometry analysis; Figure S1.Spontaneous micronuclei formation with AZD0156; Figure S2. In vitro potentiation of AZD0156 and Olaparib across a gastric cell panel; Figure S3. In vitro washout experiments; Figure S4. In vivo anti-tumor efficacy of AZD0156 in patient-derived explant HBCx-9 in combination with olaparib; Figure S5. Individual animal bodyweights (g) of mice with HBCx-10 patient-derived breast tumour explant (BRCA-2 mut).
Volume 19, Issue 1
