The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo

Abstract

ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.

Keywords: ATM and Rad-3-related (ATR); DNA damage response; ataxia telangiectasia mutated (ATM); cisplatin; non-small cell lung cancer (NSCLC).

Figure 1. Inhibition of ATR by AZD6738 inhibits growth of NSCLC cells and induces a DNA damage response

A. Log dose response curves for NSCLC cell lines (H23, H460, A549, H358) treated with AZD6738 for 48 hours. Curves from representative experiments with 5 replicates per dose tested and depict the mean percentage of viable cells (± SD) relative to the mean of control cells. B. Western blots for ATR, phospho-Chk1 (S345), total Chk1, phospho-ATM (S1981), total ATM, phospho-H2A.X (S139), p53, p21, cleaved PARP, and p27 following 24 hour treatment of H23, H460, and A549 cells with 0.3 μM or 1.0 μM AZD6738. C. H23, H460, and A549 cells were treated for 48 hours with 0.3 μM or 1.0 μM AZD6738 and incubated in drug-free media for an additional 3 (H460, A549) or 4 (H23) days. Cells were then stained with crystal violet to visualize colony formation. D. H23, H460, and A549 cells were treated for 48 hours with 0.3 μM or 1.0 μM AZD6738, harvested, and re-seeded at equal density in 96-well plates. Cells were grown an additional 6 days in the absence of AZD6738 and viability was assessed on day 8. Bars represent the mean percentage of viable cells (± SD) relative to the mean of control cells, averaged from 2 independent experiments, each with 4 replicates per condition (n = 8 total). Statistical significance by ANOVA with Dunnett's multiple comparison test denoted as follows: ****P ≤ 0.0001, ns (not significant). E–F. H23, H460, and A549 cells were treated for 48 hours with 0.3 μM or 1.0 μM AZD6738 and incubated in drug-free media for an additional 2–3 days. Cells were then stained for senescence associated β-galactosidase activity. E. Quantitation of SA-β-gal positive A549 cells at day 5. Bars represent the mean percentage of positive cells/field (± SD), averaged from 2 independent experiments, each with 3 fields/replicate and 3 replicates per condition (n = 18 fields total). Statistical significance by ANOVA with Dunnett's multiple comparison test denoted as follows: ***P ≤ 0.001, ns (not significant). F. Representative images (20x objective) of SA-β-gal staining in H23 (day 5), H460 (day 4), and A549 (day 5) following treatment with 1.0 μM AZD6738.

Figure 2. AZD6738 sensitizes NSCLC cell lines to cisplatin and synergizes strongly with cisplatin in ATM-deficient H23 cells

A–B. Cells were treated with select doses of AZD6738 and cisplatin (as indicated) for 48 hours and viability was assessed at the end of treatment. A. Color coded matrix displays the inhibition in excess of Loewe additivity, with brighter colors and corresponding higher inhibition values indicative of greater synergy. B. Bars represent the mean percentage of viable cells (± SD) relative to the mean of control cells, averaged from 2 independent experiments, each with 3–4 replicates per condition (n = 7–8 total). C. Shift in cisplatin sensitivity in H23, H460, and A549 cell lines upon addition of 0.3 or 1.0 μM AZD6738. Curves represent the mean percentage of viable cells (± SD) relative to the mean of 0 μM cisplatin controls within each AZD6738 treatment condition. Data averaged from 2 independent experiments, each with 3 replicates per condition (n = 6 total).

Figure 3. The combination of AZD6738 and cisplatin causes accumulation of cells in early S-phase and at the G1/S border

A–B. Cells were treated with 1.0 μM AZD6738, 5.0 μM (H23) or 1.67 μM (H460) cisplatin, combination, or mock control for the durations indicated, and cell cycle profiles were determined using propidium iodide staining of DNA content. A. Representative cell cycle profiles for H23 (left) and H460 (right) cells following 8, 16, and 24 hour treatment with AZD6738, cisplatin, combination, or mock. B. Quantitation of the percentage of cells in G1, S, and G2/M phases of the cell cycle at the specified time points. Bars represent the mean percentage of gated cells (± SD). Data averaged from 2 independent experiments, each with 1–2 replicates per condition. Statistical significance by ANOVA with Tukey's multiple comparison test denoted for AZD + Cis compared to other treatments as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. C. Cells were pulsed with 10 μM EdU for 15 min (H23) or 10 min (H460) and then treated with AZD6738, cisplatin, combination, or mock for 4 or 8 hours. Representative dot plots show EdU incorporation versus DNA content (stained with propidium iodide), with red boxes denoting cells that have incorporated EdU, and blue boxes denoting early S-phase cells that have incorporated EdU but have not progressed in S-phase (eg. have not increased DNA content).

Figure 4. The combination of AZD6738 and cisplatin causes dramatic cell death of ATM-deficient cells independent of the ATM-p53 signaling pathway

H23 and H460 cells were treated with 1.0 μM AZD6738, 5 μM (H23) or 1.67 μM (H460) cisplatin, combination, or mock control. A. Cell death assessed by analysis of the sub-G1 population following 16, 24, and 48 hour treatment. Bars represent the mean percentage of gated cells (± SD). Data averaged from 2 independent experiments, each with 1–2 replicates per condition. Statistical significance by ANOVA with Tukey's multiple comparison test denoted for AZD + Cis compared to other treatments as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. B–C. Western blots following 24 hour treatment with AZD6738, cisplatin, combination, or mock. B. Blots for phosphorylation of Chk1 (S345), ATM (S1981), and H2A.X (S139), and induction of p53 and p21. C. Blots depicting cleavage of caspase-3 and PARP. D–E. Cell death assessed by DNA dye (SYTOX AADvanced) exclusion following 16, 24 and 48 hour treatment. D. Representative histograms depicting the population of cells that stained positive for the DNA dye following 16, 24, or 48 hour treatment with AZD6738, cisplatin, combination, or mock. E. Quantification of cell death. Bars represent the mean percentage of gated cells (± SD) that stained positive. Data averaged from 2 independent experiments, each with 2 replicates per condition (n = 4 total). Statistical significance by ANOVA with Tukey's multiple comparison test denoted for AZD + Cis compared to other treatments as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 5. AZD6738 sensitizes ATM knockdown cells to cisplatin

A. Western blot depicting successful knockdown of ATM protein in shATM H460 and A549 cells compared to scrambled control. B. Relative sensitivity of shATM and scrambled control cells to AZD6738. Bars represent the mean percentage of viable cells (± SD) relative to the mean of untreated control cells. Data averaged from 2 independent experiments, each with 3 replicates per condition (n = 6 total). C. Shift in cisplatin sensitivity in H460 and A549 ATM knockdown cell lines compared to scrambled control lines. Curves represent the mean percentage of viable cells (± SD) relative to the mean of 0 μM cisplatin controls within each AZD6738 treatment condition. Data averaged from 2 independent experiments, each with 3 replicates per condition (n = 6 total).

Figure 6. AZD6738 potentiates cisplatin efficacy in NSCLC xenografts, and the combination causes rapid regression of ATM-deficient H23 tumors

Mice bearing H460 or H23 xenografts were treated with 3 mg/kg cisplatin (IP, q7d x 2), 50 mg/kg (H460) or 25 mg/kg (H23) AZD6738 (PO, qd x 14), the combination of cisplatin and AZD6738, or vehicle. A–B. Curves depicting mean% change in body weight (± SEM) for H460 (A) and H23 (B) bearing mice. C–D. Tumor growth curves depicting response of H460 (C) and ATM-deficient H23 (D) tumors to treatment. Curves represent mean tumor volume (± SEM). Mice per treatment arm: H460, n = 6 (Vehicle) or n = 7 (remaining arms); H23, n = 6. Statistical significance (compared to Vehicle) determined by ANOVA with Tukey's multiple comparison test.