AZD1152 rapidly and negatively affects the growth and survival of human acute myeloid leukemia cells in vitro and in vivo

Abstract

Aurora kinases play a critical role in regulating mitosis and cell division, and their overexpression has been implicated in the survival and proliferation of human cancer. In this study, we report the in vitro and in vivo activities of AZD1152, a compound that has selectivity for aurora B kinase, in acute myeloid leukemia (AML) cell lines, primary AML samples, and cord blood cells. AZD1152 exerted antiproliferative or cytotoxic effects in all cell lines studied, inhibited the phosphorylation of histone H3 (pHis H3) on Ser10 in a dose-dependent manner, and resulted in cells with >4N DNA content. THP-1 cells treated with AZD1152 accumulated in a state of polyploidy and showed a senescent response to the drug, in contrast to the apoptotic response seen in other cell lines. Accordingly, AZD1152 profoundly affected the growth of AML cell lines and primary AML in an in vivo xenotransplantation model. However, concentration-dependent effects on cell growth, apoptosis, and cell cycle progression were also observed when human cord blood and primary lineage-negative stem and progenitor cells were analyzed in vitro and in vivo. These data suggest that the inhibition of aurora B kinase may be a useful therapeutic strategy in the treatment of AML and that further exploration of dosing and treatment schedules is warranted in clinical trials.

Figure 1

AZD1152-HQPA inhibited cell proliferation, induced cytotoxicity and inhibited phosphorylation of histone H3 (ser10) in AML cell lines. Effects of AZD1152-HQPA on cell number (Fig 1A) and cell viability (Fig 1B) at 48hr and 96 hrs; changes in histone H3 phosphorylation in HL-60 and THP-1 cells by flow cytometry at 18 hours (Fig 1C); IgG staining and colcemid treated cells were used as negative and positive controls respectively; the decrease in H3 phosphorylation was concentration-dependent in all cell lines and was fully inhibited at 100 nM AZD1152-HQPA (Fig 1D).

Figure 2

The induction of polyploidy by AZD1152-HQPA in HL-60 and THP-1 cells. Changes in cell-cycle distribution were investigated after exposure of HL-60 and THP-1 cells to the indicated concentrations of AZD1152-HQPA for 48hr (Fig 2A). HL-60 cells showed a marked increase in the apoptotic fraction after AZD1152-HQPA for 96 hr, whereas little change was seen in THP-1 cells (Fig 2B). AZD1152-HQPA treated THP1 showed the enlarged flattened morphology of senescent cells (Fig 2C), stained positive for senescence associated β-galactosidase activity (Fig 2C), and showed an increase in the anti-apoptotic TRAIL decoy receptor DcR2 with 100 nM AZD1152 (Fig 2D).

Figure 3

In vivo effect of AZD 1152 treatment on HL-60 and primary AML. One million HL-60 cells were injected i.v. into sub-lethally irradiated mice. 4 weeks later, one group of mice were treated with AZD1152. One week after treatment the level of HL-60 xenograft were compared to untreated controls (Fig 3A). Example of FACS analysis that displays human, myeloid cell content in the murine marrows. Upper panel is from AZD1152-treated mice and lower panel is from untreated mice (Fig 3B). Three bar charts that summarize the frequency of human cells in murine marrows as a percentage of total nucleated cells. The number of mice in each group is represented by N. (Fig 3C-D). Summary of the effect of one-week (Fig 3C) and two-weeks (Fig 3D) of 25mg/kg/day AZD1152 treatment on the human cell content from various AML cases.

Figure 4

Summary of the number of progenitor colonies formed/ml of methylcellulose medium supplemented with differing concentrations of AZD1152 (Fig 4A). Summary of the accumulative cell count/well (ml) achieved for each AZD1152 condition. AZD1152 was added to the culture for the first week only (Fig 4B). Summary of the number of colonies produced each week displayed as the number of colonies produced/well per week (Fig 4C). Histogram analysis of PI fluorescence revealing the cell cycle status of CB Lin- cells after 1 week of culture in the presence of differing concentrations of AZD1152 (Fig 4D upper panel). Density-plot analysis of fluorescence due to Annexin-V and DAPI fluorescence. Displayed below each plot is the percentage of apoptotic cells (Annexin-V⁺/Dapi⁻ cells) (Fig 4D lower panel).

Figure 5

In vivo effect of AZD 1152 treatment on Umbilical Cord Blood Cells. One round of AZD1152 treatment at 25mg/kg/day severely reduced the level of human CB-derived xenografts. Although when left post treatment for 5 more weeks before analysis the level of CB-derived graft was slightly higher. Further treatment with another round of 1-week AZD1152 treatment did not completely eradicate the human grafts. Statistical significance (p<0.05) is indicated by *.