MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells

Abstract

Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells.

Experimental design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G₂ block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice.

Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G₂ block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors.

Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.

Fig. 1

MK-1775 radiosensitizes NSCLC cells in a p53-dependent manner. A, clonogenic survival curves for A549 and H460 (both p53 wild-type) and H1299 and Calu-6 (both p53-defective) cells treated or not with 200 nmol/L of MK-1775 for 1 h prior to irradiation followed by an additional 18 h post-irradiation incubation in MK-1775 containing medium. The results shown represent the average of 3 or more independent determinations. Error bars are shown when larger than the symbol plotted and represent the standard error. * indicates p<0.05. B, clonogenic survival curves for H1299 cells harboring a Pon A inducible p53 expression vector treated or not with MK-1775 as in panel A above. Cells were treated with Pon A (5 µmol/L) for 24 h prior to irradiation (left panel) or not (right panel). The results shown represent the average of 3 or more independent determinations. Error bars are shown when larger than the symbol plotted and represent the standard error. * indicates p<0.05. C, immunoblot showing p53 expression in H1299 cells harboring a Pon A inducible p53 expression vector. Cells were treated or not with Pon A for 24 h prior to harvest.

Fig.2

MK-1775 abrogates the radiation-induced G2 block and prematurely accelerates cells into mitosis. A, H1299 cells were irradiated with 4 Gy and the cells were then incubated in medium containing nocodazole for 4 h. MK-1775 (200 nmol/L) was added either 1 h before irradiation or immediately thereafter. The mitotic cells were selectively harvested at the end of the 4 h nocodazole treatment by gentle shaking and counted. 1. nocodazole alone, 2. 4 Gy+nocodazole, 3. MK-1775+nocodazole, 4. 4 Gy+MK-1775+nocodazole, 5. MK-1775 for 1 h followed by 4 Gy+MK-1775+nocodazole. The values shown represent the average of 5 independent experiments. Error bars represent the standard error. The counts for samples 2–5 were normalized to the nocodazole alone sample in each experiment. B,C, asynchronously growing H1299 and A549 cells were treated with MK-1775 for 1 h or not and then irradiated with 7.5 Gy. Samples were taken as a function of time thereafter and analyzed by flow cytometry for mitosis (B) on the basis of p-HH3 and for percent G2/M (C) on the basis of DNA content. Drug treatment was continued after irradiation in the MK-1775 treated cells.

Fig. 3

Cells prematurely accelerated into mitosis by MK-1775 harbor unrepaired DNA double strand breaks and undergo mitotic death. A, H1299 and A549 cells growing on cover slips were irradiated with 1 Gy and then incublated in medium containing nocodazole for 4 h. MK-1775 (200 nmol/L) was added either 1 h before irradiation or immediately thereafter. At the end of the 4 h nocodazole treatment, the mitotic cells (identified by their distinctive morphology) were analyzed for DSBs on the basis of γ-H2AX foci as detected by immunofluorescence. 1. nocodazole alone, 2. 1 Gy+nocodazole, 3. MK-1775+nocodazole, 4. 1 Gy+MK-1775+nocodazole, 5. MK-1775 for 1 h followed by 1 Gy+MK-1775+nocodazole. Error bars represent the standard error. *, ** indicates p<0.05. B, Cells growing in 100 mm dishes were treated as in A above except that the dose of radiation was 4 Gy. At the end of the 4 h nocodazole treatment, mitotic cells were harvested by gentle shaking and replated on cover slips in medium without nocodazole for 18 h. Cells were then harvested, stained with DAPI and analyzed for the presence of micronuclei.. Error bars represent the standard error. *, **, *** indicates p<0.05. C, western blot for A549 and H1299 cells treated for 1 h with 200 nmol/L of MK-1775 and analyzed for p-cdc2 expression. D, representative photomicrographs of H1299 cells treated as in panel B above illustrating the presence of micronuclei; a) nocodazole alone, b) 4 Gy + nocodazole, c) MK-1775 + nocodazole, d) 4 Gy + MK-1775 + nocodazole, e) MK-1775 (1 h) + 4 Gy + MK-1775 + nocodazole.

Fig. 4

MK-1775 radiosensitized Calu-6 xenograft tumors growing in nude mice. Xenograft tumors made using p53 null Calu-6 cells were treated with MK-1775, 60 mg/kg twice daily (BID), or radiation, 1 Gy twice daily, or the combination for 5 consecutive days starting when the tumors reached 8 mm in diameter. Tumor growth was followed until the tumors reached 12 mm in diameter and the time for the tumors to grow from 8 mm to 12 mm was calculated for the treated groups and compared to the growth in the untreated controls. The error bars are the standard error of the mean tumor diameter for the group on that day and there were 8 mice in each group.