Preclinical development of the novel Chk1 inhibitor SCH900776 in combination with DNA-damaging agents and antimetabolites

Abstract

Many anticancer agents damage DNA and arrest cell-cycle progression primarily in S or G(2) phase of the cell cycle. Previous studies with the topoisomerase I inhibitor SN38 have shown the efficacy of the Chk1 inhibitor UCN-01 to overcome this arrest and induce mitotic catastrophe. UCN-01 was limited in clinical trials by unfavorable pharmacokinetics. SCH900776 is a novel and more selective Chk1 inhibitor that potently inhibits Chk1 and abrogates cell-cycle arrest induced by SN38. Like UCN-01, abrogation of SN38-induced arrest enhances the rate of cell death but does not increase overall cell death. In contrast, SCH900776 reduced the growth-inhibitory concentration of hydroxyurea by 20- to 70-fold. A similar magnitude of sensitization was observed with cytarabine. A 5- to 10-fold sensitization occurred with gemcitabine, but no sensitization occurred with cisplatin, 5-fluorouracil, or 6-thioguanine. Sensitization occurred at hydroxyurea concentrations that marginally slowed DNA replication without apparent activation of Chk1, but this led to dependence on Chk1 that increased with time. For example, when added 18 hours after hydroxyurea, SCH900776 induced DNA double-strand breaks consistent with rapid collapse of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells required lower concentrations of SCH900776 to sensitize them to hydroxyurea. We conclude that some tumors may be very sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than concurrent treatment. SCH900776 is currently in phase I clinical trials, and these results provide the rationale and schedule for future clinical trials.

Fig. 1

Comparative efficacy of UCN-01 and SCH900776 at inhibiting Chk1 and abrogating SN38-induced cell cycle arrest. A. MDA-MB-231 cells were incubated with 10 ng/mL SN38 for 24 h. The drug was then replaced with either 0-50 nmol/L UCN-01 (left) or 0-1000 nmol/L SCH900776 (right) for an additional 6 or 24 h. Cells were harvested at the indicated times and analyzed for DNA content by flow cytometry. The experiment with UCN-01 is a replicate of previous publications (4,21), while results with SCH900776 are representative of at least three experiments. The bar reflects the mean and range (n=2) for the percentage of cells in the G2/M phase. B. MDA-MB-231 cells were incubated with 10 ng/mL SN38 for 24 h. The drug was then replaced with either UCN-01 (left) or SCH900776 (right) and the cells harvested after an additional 3 h. Cell lysates were analyzed for Chk1 phosphorylation. C. MCF10AΔp53 cells were incubated as in B, and additionally with the Chk2 inhibitor “2h.” Cell lysates were analyzed by western blotting for the indicated proteins. D. MDA-MB-231 cells were incubated with UCN-01 or SCH900776 for 3 h, then harvested and analyzed for Chk1 phosphorylation and Cdc25A .

Fig. 2

Comparative efficacy of UCN-01 and SCH900776 at abrogating SN38-induced cell cycle arrest in cells suppressed for Chk1. A. MDA-MB-231ΔChk1 cells were incubated with 10 ng/mL SN38 for 24 h. The drug was then replaced with either 0-150 nmol/L UCN-01 (left) or 0-1000 nmol/L SCH900776 (right) for an additional 6 or 24 h. Cells were harvested at the indicated times and analyzed for DNA content by flow cytometry. The experiment with UCN-01 is a replicate from a previous publication (17), while results with SCH900776 are representative of at least three experiments. The bar reflects the mean and range (n=2) for the percentage of cells in the G2/M phase. B. The level of Chk1 in the ΔChk1 cells was assessed by loading 15 μg of protein in the first lane (100%) followed by serial 2-fold dilutions. Comparison was made to the levels of Chk1 in the parent cell line.

Fig. 3

The impact of checkpoint inhibitors on sensitivity of cells to SN38, hydroxyurea and cisplatin. A. MDA-MB-231 (top), MDA-MB-231ΔChk1 (middle) and MCF10A cells (bottom) were incubated with SN38 (left), hydroxyurea (middle) or cisplatin (right) for 24 h either alone or in combination with 50 nmol/L UCN-01, 1 μmol/L SCH900776 or 10 μmol/L KU55933. The drugs were replaced with fresh media and analyzed after an additional 5-7 days for cell growth. B. MDA-MB-231 and MDA-MB-231ΔChk1 cells were incubated with hydroxyurea for 24 h in combination with a range of concentrations of UCN-01 (left) or SCH900776 (right). Cell growth was assessed after 6 days and are recorded as the 50% inhibitory concentration (IC₅₀) of hydroxyurea at each concentration of the Chk1 inhibitors. Values are highlighted for the concentration of each Chk1 inhibitor that reduces the IC₅₀ for hydroxyurea to 50 μmol/L (the dotted line). C. Lysates were made from the indicated cell lines and analyzed for expression of Chk1.

Fig. 4

Analysis of cell cycle perturbation induced by hydroxyurea. A. MCF10A and MDA-MB-231 cells were incubated with 0 – 4 mmol/L hydroxyurea for 6 h (left), 24 h (center), or were allowed to recover for 6 h after the initial 6-h incubation with hydroxyurea (right). Cells were analyzed by flow cytometry and the results analyzed by ModFit for the proportion in G1, S or G2/M phase. B. Representative examples of MCF10A cells incubated with 62-500 μmol/L hydroxyurea for 0-24 h. C. MCF10A cells were incubated for 24 h with 0 – 500 μmol/L hydroxyurea; 10 μmol/L EdU was added for the final 30 min. Cells were harvested and analyzed by 2-dimensional flow cytometry for DNA content and incorporation of EdU. The data were gated to calculate the mean incoporation of EdU into S phase cells.

Fig. 5

Impact of concentration and schedule of hydroxyurea and SCH900776 on DNA damage and cytotoxicity A. MCF10A (left) and MDA-MB-231 cells (right) were incubated with hydroxyurea for 24 h and lysates analyzed for the indicated proteins. MDA-MB-231 cells were also coincubated with 1 μmol/L SCH900776. B. MDA-MB-231 and MCF10A cells were incubated with 0.1 or 1 mmol/L hydroxyurea in combination with 1 μmol/L SCH900776 for 0-24 h. C. MDA-MB-231 and MCF10A cells were incubated with 0.1 or 1 mmol/L hydroxyurea for 0 – 24 h. 1 μmol/L SCH900776 was added as indicated at time 0 or after 18 h. In the latter case, the cells were harvested after an additional 2 h (20 h) or 6 h (24 h). D. MDA-MB-231 and U2OS cells were incubated with hydroxyurea and SCH900776 at the indicated conditions and schedules. Drugs were removed and growth inhibition (IC₅₀) assessed over the following 6 days.