Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities

Abstract

ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.

Keywords: ATR; CHK1; DNA damage; cancer; replication stress; synthetic lethality.

Graphical abstract

Figure 1

ATR Target Activation by the CHK1 Inhibitor AZD7762 in U2OS Cancer Cells (A) Western blot showing activation of ATR targets. U2OS cells were treated with the indicated concentrations for 30 and 60 min, lysed, and probed with anti-phospho (Serine 345) CHK1 and β-actin antibodies. (B) Induction of pre-apoptotic pan-nuclear γ-H2AX by ATR and CHK1 inhibitor in combination in cancer cells. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody. Scale bar, 20 μm. (C) Quantitative data of γH2AX- (nine or more foci per cells) positive cells or pan-nuclear γH2AX signal after indicated treatments are shown (n = 3, mean ± SEM). (D) Western blot showing increased phosphorylation of H2AX after combination treatment. U2OS cells were treated with the indicated concentrations for 24 hr. At the end of incubation time, western blotting was performed using anti-phospho (Serine 139) H2AX, anti-phospho (Serine 345) CHK1, cleaved PARP, anti-phospho (Serine 10) H3, and β-actin antibodies. (E) Comet assay showing DNA damage induction by ATR and CHK1 inhibitor in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr. At the end of incubation, cells were harvested and alkaline comet assay was performed. (F) Quantitative data of the tail moment are shown (n = 3, mean ± SEM, in each experiment ≥100 comets were measured). (G) Cancer-specific ssDNA formation by VE-821 and AZD7762, either alone or in combination. U2OS cells were treated with the indicated drug concentrations for 24 hr and pre-extracted using CSK buffer before fixation. Cells were stained with anti-RPA32 antibody; images were taken using a confocal microscope and were analyzed using ImageJ software. A mean intensity of ≥70 a.u. per cell was considered as positive. Quantitative data are presented as mean ± SEM from three independent experiments. (H) ssDNA formation in normal fibroblast VH-10 cells is shown. (I) Pre-apoptotic pan-nuclear γH2AX induction by combination treatment of ATR and CHK1 inhibitors in U2OS is mediated through the JNK pathway. U2OS cells were treated with the indicated drug concentrations for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody, and high-throughput microscopy was used to determine the percentage of γH2AX-positive cells (nine or more γH2AX foci per cell) or an average intensity of ≥2,000 a.u. for pan-nuclear γH2AX-positive cells (n = 2 with multiple wells, mean ± SEM). Statistical significance was determined using one-way ANOVA (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001).

Figure 2

Combination of the ATR Inhibitor VE-821 and the CHK1 Inhibitor AZD7762 Synergistically Kills Cancer Cells (A) Clonogenic survival of U2OS, VH-10, and MCF-7 cells. The 500 (U2OS and MCF-7) or 1,000 (VH-10) cells were seeded in 10-cm² dishes, and, after 5-hr incubation, the inhibitors were added directly to the media. After 72-hr incubation, drug-containing media were replaced with fresh media and cells were kept for another 5–8 days before colonies were stained with methylene blue. Quantitative data: n = 3, mean ± SEM. (B) Parental and cMYC-transformed cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (C) BJ-hTERT, BJ-hTERT SV40, and BJ SV40 RAS cells were treated with the indicated doses for 72 hr. At the end of the incubation period, resazurin was added and cell viability was measured. Quantitative data: n = 3, mean ± SEM. (D) CHK1 functionally compromised cells are sensitive to ATR inhibitor. Clonogenic survival of DLD-1, DLD-1 CHK1^S317A/−, DLD-1 CHK1^+/−, and DLD-1 ATR^S/S after ATR inhibitor VE-821 treatment is shown. A similar protocol was used as for U2OS and VH-10 cells. Quantitative data: n = 3, mean ± SEM. (E) Therapeutic efficacy of combined inhibition of ATR and CHK1 in mouse tumor models. Therapeutic efficacy of VX-970 and AZD7762 in H460 lung cancer xenografted mice is shown. BALB/c nude mice bearing H460 xenograft were divided in four groups (five animals in each group) with a tumor volume of ∼130 mm³ in each group. The first control group of animals was treated with vehicle (orally and intraperitoneally). The second group of animals was treated with 25 mg/kg body weight of CHK1 inhibitor AZD7762 (intraperitoneal route). The third group of animals was treated with 60 mg/kg body weight of ATR inhibitor VE-822 (oral administration), and the fourth group received a combination of both CHK1 and ATR inhibitors. Vehicle and drugs were administered on days 0–3, 10–12, and 18–20 irrespective of no mice survival in each group. Tumor volume was measured with calipers and is shown here as mean ± SEM. Statistical significance was determined using two-way ANOVA with repeated measurement (^∗p < 0.05 and ^∗∗p < 0.01). (F) Kaplan-Meier survival curve of H460-xenografted mice. When tumor size reached 1,000 mm³, the animal was sacrificed.

Figure 3

ATR and CHK1 Inhibitors, Alone or in Combination, Decrease Replication Fork Speed Only in Cancer Cells (A) Treatment regimen is shown. U2OS and VH-10 cells were treated for 60 min with the indicated drug concentrations and sequentially labeled with 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) for 30/20 min each in the presence of the inhibitors. DNA fibers were stained and replication speed was measured by IdU labeling. (B and C) Representative images show stained replication fork tracts for each treatment group. (D) Quantitative data of replication fork speed (kb/min), mean ± SEM, and p values were analyzed with one-way ANOVA for each condition and cell line. (E and F) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats.

Figure 4

Combination Treatment of VE-821 and AZD7762 Results in S Phase Arrest in U2OS Cells (A) U2OS cells were treated with the indicated drug concentrations for 24 hr and propidium iodide (PI) staining was carried out to measure cell-cycle profile using flow cytometry. (B) Quantitative data were obtained using Modfit software. (C) ATR and CHK1 inhibitors in combination decrease EdU incorporation in U2OS cells. U2OS cells were treated for 24 hr with the indicated doses. Images were taken with a confocal microscope and analyzed using ImageJ software. A mean intensity of ≥80 a.u. per cell was considered as EdU-positive cells. Quantitative data: n = 3, mean ± SEM. (D) No significant decrease in EdU incorporation in normal fibroblast VH-10 cells treated with the ATR and CHK1 inhibitors either alone or in combination. VH-10 cells were treated for 24 hr with the indicated doses. Quantitative data: n = 3, mean ± SEM. Statistical significance was determined using one-way ANOVA (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 5

Synergistic Cytotoxic Effect in U2OS Cancer Cells by Combination Treatment of AZD7762/VE-821 Is Mainly Due to CDK-Mediated Excess Origin Firing (A) U2OS cells were pretreated with the indicated concentrations of the CDK inhibitor Roscovitine for 1 hr prior to the addition of VE-821 and AZD7762 for 24 hr. Cells were probed with anti-phospho (Serine 139) H2AX antibody and anti-53BP1, and DNA was counterstained with ToPro. (B) Quantitative data of pan-nuclear γH2AX are shown (mean ± SEM from two independent experiments). (C) Treatment regimen for DNA fiber assay in U2OS cells is shown. (D) CDK inhibitors Roscovitine and PHA-767491 enhance the replication fork speed of U2OS cells treated with VE-821 and AZD7762 in combination. U2OS cells were pre-treated with Roscovitine or PHA-767491 for 1 hr prior to the addition of VE-821 and AZD7762. Representative images show stained replication fork tracts for each treatment group. (E) Average distribution of replication fork rates. A minimum of 450 forks per condition were analyzed from at least three independent repeats. (F) Roscovitine abolishes the synergistic cytotoxic effect of combination treatment of AZD7762 and VE-821 in U2OS cancer cells. U2OS cells were individually treated with VE-821, AZD7762, or the combination with our without Roscovitine for 24 hr, followed by recovery for another 48 hr. Cell viability was measured by using resazurin at 72 hr. (G) Model for ATR/CHK1 synthetic lethality. CHK1 is activated by replication stress both by ATR-dependent and -independent pathways (Yang et al., 2008) to suppress replication stress in cancer, promoting restart and survival. CHK1 inhibitors increase oncogene-activated CDK activity and origin firing, leading to replication stress and accumulation of stalled replication forks, requiring ATR activity to prevent replication collapse. Red arrows indicate primary route in the presence of both ATR and CHK1 inhibitors.

Figure 6

HU-Induced Replication Stress in Combination with VE-821 and AZD7762 Causes Fragmented Nuclei and the Early Onset of Apoptosis Only in U2OS Cells (A) U2OS cells were treated for 24 hr with the indicated drug concentrations and stained with anti-cleaved caspase 3 and β-actin antibodies. Representative confocal images are shown. (B) Quantitative data of fragmented nuclei and cleaved caspase-3 positive cells presented as mean ± SEM from three independent experiments. (C) Western blot showing apoptosis in U2OS cells treated with ATR and CHK1 inhibitors alone or in combination. U2OS cells were treated with the indicated concentrations for 24 hr; lysed; protein extracted; and western blotting was performed with anti-Cleaved PARP, anti-phospho (Serine 10) Histone H3, and anti-β-actin antibodies. (D) HU-induced replication stress does not cause fragmentation of nuclei or apoptosis in combination with VE-821 and AZD7762 in VH-10 normal fibroblast cells in 24 hr. Etoposide treatment (4 μM) was used to induce apoptosis as a positive control. Scale bar represents 20 μM.