Enhancement of radiation response in p53 deficient cancer cells by the Aurora-B kinase inhibitor AZD1152

A256 Background: Overexpression of aurora kinases has been correlated with cancer susceptibility and poor prognosis in many human cancers. The aim of this study was to evaluate the effect of inhibition of aurora kinases on radiosensitivity and cell cycle progression. AZD1152 is a specific aurora kinase inhibitor with selectivity for Aurora B kinase (Ki 0.4 nM). AZD1152 is a phosphate pro-drug that is converted in vivo to the active moiety AZD1152-HQPA.
 Methods AZD1152-HQPA was used in in vitro studies, whilst AZD1152 was used in in vivo studies. Clonogenic survival assays and cell cycle analyses were performed in HCT 116 (p53wt and p53-/-) and HT-29 (p53 and ras mutant) colorectal cancer cell lines and an A549 (p53wt) lung cancer cell line.
 Results: Incubation of HCT 116 cells with AZD1152-HQPA for 24 hours resulted in the complete inhibition of histone H3 phosphorylation, and clonogenic survival assays further revealed that AZD1152-HQPA effectively inhibited colony formation in various cancer cell lines.
 Studies of sequential treatment showed that treatment with AZD1152-HQPA 24 hours before irradiation (IR) resulted in pronounced cell killing compared with IR alone in p53wt HCT 116 and A549 cells. This enhancement of the radiation effect was more pronounced in p53-/- HCT 116 and p53 mutant HT-29 cells, as well as in A549 cells treated with the p53 inhibitor cyclic pifithrin-α.
 Cell cycle analyses demonstrated that AZD1152-HQPA-treated cells progressively accumulated a DNA content >4N, indicating polyploidization due to mitotic failure, and was more pronounced in p53-/- than in p53wt HCT 116 cells. Notably, when cells were exposed to both IR and AZD1152-HQPA, the accumulation of >4N DNA cells was reduced compared with AZD1152-HQPA treatment alone in both cell lines. HT-29 cells in either the G2/M or G1/S phase of the cell cycle were selected by FACS. Populations enriched with cells in the G2/M phase were highly radiosensitized by AZD1152-HQPA. This radiosensitizing effect was less pronounced in the population enriched with cells in the G1/S phase. This suggests that AZD1152-mediated radiosensitization is cell cycle dependent.
 In vivo experiments in subcutaneous xenograft models showed an enhancement of the radiation effect by AZD1152 (10-40 mg/kg/day ip, 4 days) combined with IR (4-6 Gy/day, 2 days; 24 hours after the first injection of AZD1152) compared with IR alone in p53wt HCT 116 cell xenografts. Interestingly, this effect was more pronounced in p53-/- HCT 116 and HT-29 cell xenografts.
 Conclusions: This study reveals that sequential treatment with AZD1152 and IR enhances cell death in HCT 116, HT-29, and A549 cancer cells. This effect was more pronounced in the absence of functional p53 and in G2/M enriched cells. Preclinical data from mice bearing human cancer xenografts strongly suggest that the radiosensitizing effect of AZD1152 warrants further clinical exploration.