Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Abstract

Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

Keywords: Chk1; DNA-damage response; cancer; cell cycle; cell division cycle 7-related protein kinase (Cdc7); cyclin-dependent kinase (CDK); gemcitabine; replication catastrophe; single-strand binding protein RPA.

Figure 1.

Delayed CHK1i causes significantly more DNA damage than concurrent inhibition or gemcitabine alone. A, schedules of drug administration used in this study. B, MDA–MB-231 cells were incubated with gemcitabine and MK-8776 (CHK1i) as indicated. In the right lane, cells were incubated with 50 nm gemcitabine (Gem) for 0–6 h and 20 μm CHK2 inhibitor II (CHK2i) for 18–24 h. Cell lysates were analyzed by Western blotting. Densitometry values of γH2AX represent the mean of three replicates; see Fig. S2 for full densitometric analysis. C, similarly incubated cells were analyzed by alkaline single-cell gel electrophoresis. Inverse images are shown. Cells with a tail moment of >1 S.D. of the mean tail moment of control cells were counted as positive. Graphs represent the mean ± S.D. for percent positive cells. #, p value < 0.0001.

Figure 2.

Mitotic entry is not required for CHK1i-mediated DNA damage following gemcitabine treatment. MDA–MB-231 and HT29 cells were incubated with gemcitabine 0–6 h, MK-8776 (CHK1i) 18–24 h, and CVT-313 (CDK1/2i) 18–24 h. Cells were immunostained for γH2AX and pHH3, stained with propidium iodide, and analyzed by flow cytometry. Representative contour plots are shown for 2D analysis of γH2AX or pHH3 versus DNA content. Graphs represent the mean ± S.D. percent of cells positive for γH2AX. *, p value < 0.05; **, p value < 0.005; #, p value < 0.0001.

Figure 3.

Sensitization to gemcitabine by CHK1i is independent of homologous recombination. A, cells were incubated with or without gemcitabine (Gem) for 6 h and then harvested at 24 h. DNA-bound RAD51 was immunostained, and DNA was stained with DAPI and imaged by confocal microscopy. Cells with nuclear RAD51 intensity of >2 S.D. of the mean of control cells were counted as positive. Graphs represent the mean ± S.D. of positive cells (n = 3). B, MDA–MB-231, HCC1937, and HT29 cells were incubated with gemcitabine either alone for 0–6 h, concurrently with 2 μm MK-8776 (CHK1i), or with 2 μm MK-8776 at 18–24 h. Following treatment, cells were allowed to recover in fresh media for 6 days. DNA content was stained with Hoechst 33258 and analyzed with a fluorescent plate reader. The GI₅₀ graph represents mean ± S.D. of the concentration of gemcitabine required to inhibit growth. *, p value < 0.05; **, p value < 0.005; #, p value < 0.0001; N.S., not significant.

Figure 4.

MRE11 activity is not required for CHK1i-mediated sensitization to gemcitabine. MDA–MB-231, HCC1937, and HT29 cells were incubated with gemcitabine (Gem) 0–6 h, MK-8776 (CHK1i) 18–24 h, and mirin (MRE11i) 18–24 h. ASPC-1 cells (sensitive to CHK1i alone) were incubated with 2 μm MK-8776 alone or in combination with 100 μm mirin for 6 h as a positive control for mirin activity. Cell lysates were analyzed by Western blotting.

Figure 5.

Gemcitabine plus delayed CHK1i increases markers of DNA helicase activity and depletes soluble RPA. A, MDA–MB-231 cells were incubated with gemcitabine (Gem) and MK-8776 (CHK1i), and harvested at times shown. DNA-bound proteins were separated from soluble proteins by chromatin fractionation and analyzed by Western blotting. B, incubations as in A but harvested from 0 to 24 h. See Fig. S2B for densitometric analysis of DNA-bound CDC45 and MCM2–pSer-53.

Figure 6.

Majority of cell lines phosphorylate a majority of RPA32 in response to hydroxyurea plus CHK1i. 12 cell lines were incubated with 2 mm hydroxyurea (HU) for 0–24 h plus 2 μm MK-8776 (CHK1i) administered at 18–24 h. Lysates were analyzed by Western blotting. Representative of two replicates.

Figure 7.

Gemcitabine plus delayed CHK1i increases ssDNA that co-localizes with DNA-bound PCNA. A, MDA–MB-231 cells were incubated with 10 μm BrdU for 48 h then with gemcitabine (Gem) and MK-8776 (CHK1i). Cells were immunostained for DNA-bound PCNA and BrdU and imaged by confocal microscopy. Cells exhibiting nuclear signal >2 S.D. of the mean of the reference sample were counted as positive; BrdU reference = control, PCNA reference = Gem 24. Scatter plots represent mean ± S.D. percent of cells positive for each respective signal. *, p value < 0.05; **, p value < 0.005; #, p value < 0.0001. B, enlarged view of a cell from A incubated with gemcitabine plus delayed CHK1i. Co-localization of PCNA and BrdU signal was quantified using Cell Profiler analysis software, n = 3.

Figure 8.

DNA replicative helicase is regulated by both CDC7 and CDK2 following gemcitabine plus delayed CHK1i. A, MDA–MB-231 cells were incubated with gemcitabine (Gem) 0–6 h, MK-8776 (CHK1i) 18–24 h, and CVT-313 (CDK1/2i) 18–24 h. Lysates were analyzed by Western blotting. See Fig. S4 for densitometric quantification of pTPXK bands; values represent mean ± S.D. of the concentration of CVT-313, which reduced the band intensity by 50% compared with gemcitabine plus CHK1i, n = 3. B, MDA–MB-231 cells were incubated with gemcitabine 0–6 h, MK-8776 (CHK1i) 18–24 h, CVT-313 (CDK1/2i) 18–24 h, or XL413 (CDC7i) 18–24 h. DNA-bound proteins were separated from soluble proteins by chromatin fractionation and analyzed by Western blotting. C, MDA–MB-231 and HT29 cells were incubated with gemcitabine 0–6 h and then MK-8776 (CHK1i) and harvested from 18 to 24 h. Western blottings were probed for pTyr-15–CDK1/2 with fluorescent-tagged secondary antibodies. Values indicate the mean band intensity of each phosphorylated band relative to gemcitabine alone at 18 h (n = 4). See Fig. S5 for full densitometric analysis of the band intensities.

Figure 9.

CDK1/2i and CDC7i reduce ssDNA and DNA-bound RPA32. A, MDA–MB-231 cells were incubated with gemcitabine (Gem) and MK-8776 (CHK1i) as indicated, with the addition of 40 μm CVT-313 (CDKi) or 20 μm XL413 (CDC7i) administered at 18–24 h. Cells were immunostained for DNA-bound RPA32, pMCM2, and BrdU. Cells exhibiting nuclear signal of pMCM2 and BrdU >2 S.D. of the mean of the control sample were counted as positive; a threshold of >1 S.D. of the mean of the control sample was used for RPA32 positivity. *, p value < 0.05; **, p value < 0.005; #, p value < 0.0001, n = 3. B, zoomed in view of a cell from A, treated with gemcitabine plus CHK1i. Pearson's correlation Coefficient represents three replicates, mean ± S.D.

Figure 10.

CDC7i, CDK1/2i, and CDC25i prevent CHK1i-mediated sensitization to gemcitabine. A, MDA–MB-231 cells were incubated with gemcitabine (Gem) and MK-8776 (CHK1i) as indicated, with the addition of 40 μm CVT-313 (CDKi) or 20 μm XL413 (CDC7i) administered at 18–24 h. Cells were analyzed by alkaline single-cell gel electrophoresis. Inverse images are shown. Cells with a tail moment >1 S.D. of the mean tail moment of control cells were counted as positive. Graphs represent the mean ± S.D. for percent positive cells. #, p value < 0.0001. B, MDA–MB-231 cells were incubated with gemcitabine 0–6 h, 2 μm MK-8776 (CHK1i) 18–24 h, 20 μm CVT-313 (CDK1/2i) 18–24 h, and 20 μm XL413 (CDC7i) 18–24 h. Following treatment, cells were allowed to recover in fresh media for 6 days. Plates were analyzed for DNA content with Hoechst 33258 dye. The GI₅₀ graph represents the mean ± S.D. of the concentration of gemcitabine that resulted in 50% growth inhibition. #, p value < 0.0001. C, MDA–MB-231 cells were incubated with gemcitabine 0–6 h, MK-8776 (CHK1i) 18–24 h, and NSC663284 (pan-CDC25i) 18–24 h. Lysates were analyzed by Western blotting.

Figure 11.

Proposed mechanism of replication catastrophe from gemcitabine plus delayed CHK1i. The normal firing of early replication origins involves CDC7-dependent phosphorylation of the MCM2–7 DNA helicase core and CDK2-dependent loading of the helicase co-factor, CDC45. Gemcitabine arrests replication by depleting dNTPs. Accumulation of RPA at stalled replication forks activates the DNA-damage response through ATR and CHK1. Over time, replication machinery dissociates from stalled replication forks, preventing their restart. CHK1 prevents firing of dormant origins. Inhibition of CHK1 activates CDK2 which, in concert with CDC7, leads to CDC45 loading and dormant origin firing. Active helicases unwind DNA, but in the continued absence of dNTPs, extensive single-strand DNA is produced that exceeds the ability of replication protein A to protect it from nuclease activity and replication catastrophe.