Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the cell checkpoint and Chk1-Chk2 inhibitor AZD7762

Abstract

Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (CPT). Here, we report molecular investigations into LMP-400, an indenoisoquinoline Top1 inhibitor in phase 1 clinical trial, by itself and in combination with the cell-cycle checkpoint inhibitor AZD7762. We examined drug effects on DNA replication and killing of cancer cells and found that LMP-400 showed synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation were similarly induced by LMP-400 and CPT and were abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. AZD7762 inhibited both the activation/autophosphosphorylation of Chk1 and Chk2 at nanomolar concentrations in LMP-400-treated cells. This potent dual inhibition of Chk1 and Chk2 by AZD7762 was below the drug concentrations required to abrogate cell-cycle inhibition and produce synergism with LMP-400. Also, the synergism was independent of Chk2 both in Chk2-complemented cells and Chk2 knockout cells, suggesting additional mechanisms for cell-cycle abrogation by AZD7762. Together, our findings show a rationale for combining cell-cycle checkpoint inhibitors with the novel non-CPT indenoisoquinoline Top1 inhibitors.

Figure 1. Synergistic inhibition of cellular proliferation in human colon carcinoma HT29 cells treated with LMP-400 and AZD7762

A) LMP-400. B) AZD7762. C) Viability of HT29 cells treated with increasing concentrations of LMP-400 alone or in the presence of fixed non-cytotoxic concentrations of AZD7762 (16, 31, 62 and 125 nM) for 48 h. Data represent mean ± S.E. of four independent experiments. D) Graph of CI (combination Index) values vs F_a (Fraction affected) for data points shown in panel C on the combination (AZD7762 + LMP-400).

Figure 2. AZD7762 abrogates the S-phase arrest induced by LMP-400 or CPT

A) Treatment schedule for the time course experiments shown in panel B. HT29 cells were treated with LMP-400 for 1 h. AZD7762 was added immediately after removal of LMP-400 and kept for up to 6 h. B) Representative time-course experiments showing loss of S-phase progression in response to LMP-400 and recapitulation of cell cycle by AZD7762. DNA synthesis was monitored by adding 50 μM BrdU 30 min prior to cell harvesting. C) Treatment schedule for the AZD7762 concentration-response experiments shown in panel D. D) Representative concentration-response FACS experiments showing LMP-400-and CPT-mediated S-phase arrest and S-phase arrest abrogation by AZD7762. DNA synthesis was monitored by adding 50 μM BrdU 30 min prior to cell harvesting. Data are representative of at least three independent experiments.

Figure 3. S-phase arrest induced by LMP-400 is abrogated by AZD7762

A) Treatment schedule. B) DNA synthesis was monitored by adding 50 μM BrdU 30 min prior to harvesting HT29 cells treated as indicated. C) Cell cycle effects were determined simultaneously by propidium iodide staining. Data are representative of at least three independent experiments.

Figure 4. Effects of LMP-400 and AZD7762 on replication fork velocity and origin firing

A) Treatment protocol. B) Distribution of Idu:CldU ratios (red:green signals) determined in single DNA molecules after molecular combing; N = number of signals measured. C) Fork velocity responses to LMP-400 and AZD7762 alone or in combination. Each dot represents a data point for an individual DNA fiber. Mean, standard deviation (SD) and number of signals measured (N) are indicated under each scatter plot. D) Inter-origin distances measured for each treatment. A minimum of 30 inter-origin distances were taken to measure their average distance. The bars represent SD. All data are representative from at least three independent experiments.

Figure 5. AZD7762 abrogates both the Chk1 and Chk2 activations induced by LMP-400 and enhances the histone H2AX response

A) Time-dependent phosphorylations/activations of Chk1 and Chk2 by LMP-400 and their inhibition by AZD7762. The treatment schedule is shown at the top. HT-29 cells were harvested post LMP-400 treatment as indicated (upper panel). Autophosphorylation of Chk1 at Ser²⁹⁶ and Chk2 at Ser⁵¹⁶ were analyzed by Western blotting. Chk1 and Chk2 total proteins were both used as loading controls. B) AZD7762-dependent concentration-response for abrogation of Chk1 or Chk2 autophosphorylations. Chk2 activation/phosphorylation (Chk2 T68) and histone H2AX phosphorylation (γH2AX) by PI3 kinases were also determined. Actin, Chk1 total and Chk2 total were used as loading controls.

Figure 6. The synergistic effects of AZD7762 and LMP-400 in a panel of colon carcinoma cell lines is independent of Chk2

A) HCT15 cells complemented with wild-type Chk2 (HCT15 Chk2 WT), kinase-dead Chk2 (HCT15 KD) were treated with LMP-400 alone or in combination with various fixed concentrations of AZD7762 for 48 h. B) Graph of CI (combination Index) values vs F_a (Fraction affected) for data points shown in panel A on the combination (AZD7762 + LMP-400) for HCT15 cells. C) wild-type HCT116 (HCT116 WT) and Chk2 knockout HCT116 cells (HCT116 Chk2 −/−) were treated with LMP-400 alone or in combination with various fixed concentrations of AZD7762 for 48 h. D) Graph of CI (combination Index) values vs F_a (Fraction affected) for data points shown in panel C on the combination (AZD7762 + LMP-400) for HCT116 cells. Cell survival was assessed by MTS assays. Data represent mean ± S.E. of three independent experiments.