In vitro and in vivo radiation sensitization of human tumor cells by a novel checkpoint kinase inhibitor, AZD7762

Abstract

Purpose: Inhibition of checkpoint kinase 1 has been shown to enhance the cytotoxicity of DNA-damaging targeted chemotherapy through cell cycle checkpoint abrogation and impaired DNA damage repair. A novel checkpoint kinase 1/2 inhibitor, AZD7762, was evaluated for potential enhancement of radiosensitivity for human tumor cells in vitro and in vivo xenografts.

Experimental design: Survival of both p53 wild-type and mutant human cell lines was evaluated by clonogenic assay. Dose modification factors (DMF) were determined from survival curves (ratio of radiation doses for control versus drug treated at 10% survival). Flow cytometry, Western blot, and radiation-induced tumor regrowth delay assays were conducted.

Results: AZD7762 treatment enhanced the radiosensitivity of p53-mutated tumor cell lines (DMFs ranging from 1.6-1.7) to a greater extent than for p53 wild-type tumor lines (DMFs ranging from 1.1-1.2). AZD7762 treatment alone exhibited little cytotoxicity to any of the cell lines and did not enhance the radiosensitivity of normal human fibroblasts (1522). AZD7762 treatment abrogated radiation-induced G(2) delay, inhibited radiation damage repair (assessed by gamma-H2AX), and suppressed radiation-induced cyclin B expression. HT29 xenografts exposed to five daily radiation fractions and to two daily AZD7762 doses exhibited significant radiation enhancement compared with radiation alone.

Conclusions: AZD7762 effectively enhanced the radiosensitivity of mutated p53 tumor cell lines and HT29 xenografts and was without untoward toxicity when administered alone or in combination with radiation. The results of this study support combining AZD7762 with radiation in clinical trials.

Fig. 1

Radiation survival curves for cell lines treated with AZD7762 (100 nM) 1 hr prior to and 24 hr post-radiation (closed circles) or without AZD7762 treatment (open circles). A) HT29, B) 1522, C) H460, and D) H460 DN p53. DMFs and AZD7762 survival alone are shown in Table 1. Survival curve data from 2–3 independent experiments were fitted using a linear-quadratic model (11).

Fig. 2

Percentage of cells in G2/M assessed by flow cytometry for cells treated with radiation (open circles) or radiation plus AZD7762 (closed circles) (100 nM, added 1 hr prior to and 24 hr post-radiation) as a function of time. A) HT29, B) 1522, C) H460, and D) H460 DN p53. Dashed lines represent untreated control G2/M percentages. Data were derived from flow cytometry profiles (Supplementary Fig. S4–S5) and were in qualitative agreement with replicate experiments.

Fig. 3

The effects of radiation with and without AZD7762 treatment (100 nM, added 1 hr prior to and 24 hr post-radiation) on radiation-induced damage repair and mitotic catastrophe. Western analysis of phosphorylated γH2AX from HT29 (A) and 1522 (B) cells as a function of time following treatment with AZD7762 and radiation (8 Gy, HT29; 7 Gy, 1522). Normalized density levels of protein bands from westerns using H2A as the loading control are indicated by values in parenthesis. Additional data are provided for DU145 and A549 cells in Supplementary Fig. S7. Mitotic catastrophe as a function of time following treatment with radiation (2 Gy) or radiation with AZD7762 (100 nM, 1 hr prior to and 24 hr post-radiation) in H460 DN p53 cells (C) or H460 WT p53 cells (D); p values shown according to Student’s t test.

Fig. 4

A) Fractionated radiation (daily 2 Gy fractions × 5) with and without AZD7762 treatment (25 mg/kg, one i.p. injection immediately after each radiation fraction) on radiation-induced tumor regrowth for HT29 xenografts. B) Western analysis of pChk1 from HT29 tumors as a function of time following treatment with radiation (5 Gy). Numbers in parenthesis indicate normalized densities of pChk1 bands. C) Fractionated radiation (daily 2 Gy fractions × 5) with and without AZD7762 treatment (25 mg/kg, two i.p. injections immediately and 8 hr later for each radiation fraction) on radiation-induced tumor regrowth for HT29 xenografts. *p < 0.0038, relative to fractionated radiation alone.

Fig. 5

Western analysis of γ-H2AX, pChk1, and cyclin B proteins taken from HT29 tumors as a function of time following treatment with radiation (5 Gy) with or without AZD7762 (25 mg/kg administered immediately after radiation). Numbers in parenthesis beneath each row indicate fold increase/decrease of proteins relative to respective loading controls.