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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Clin Cancer Res. Author manuscript; available in PMC Sep 1, 2012.
Published in final edited form as:
PMCID: PMC3167033

MK-1775, a Novel Wee1 Kinase Inhibitor, Radiosensitizes p53-defective Human Tumor Cells



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.


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 anti-tumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors.


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.

Keywords: Radiation, wee1, p53-dependent, MK-1775, DNA damage


Tumor cells typically have high rates of cell proliferation that render them especially sensitive to DNA-damaging agents, which include several classes of conventional cancer chemotherapy drugs and ionizing radiation, and these agents continue to have important roles in therapeutic treatments. Unfortunately, in some critical normal tissues, homeostasis is maintained by the proliferation of their stem cell compartments, and the cytotoxic effects of these DNA-damaging agents on such stem cells severely limits their efficacy for certain cancers. This has prompted the development of a new approach to cancer treatment, referred to as molecularly targeted therapy, and several molecularly targeted drugs have been approved for clinical use in the past few years. Molecularly targeted therapy takes advantage of our increasing understanding of the molecular abnormalities associated with tumor cells that are responsible for their malignant phenotype, and these new drugs are designed to specifically target such molecules thereby offering specificity that is lacking for the conventional agents.

Of all of the molecular abnormalities associated with tumor cells, one of the most common, estimated to be present in 50% of all cancers, is loss of function of the tumor suppressor protein p53 (1). p53 is a transcription factor that plays a pivotal role in the cellular response to DNA-damaging agents by facilitating either cell cycle arrest or cell death (2). Generally, cells arrest in the cell cycle following damage to their DNA at 3 well-established checkpoints which occur in G1, S, and in G2 phase (3). Arresting at these checkpoints allows the cells more time to repair the DNA damage before initiating cell division at mitosis; cells that attempt to undergo mitosis while continuing to harbor DNA lesions will likely die by a combination of cell death processes including apoptosis, mitotic death, and senescence (4, 5). p53 is primarily responsible for mediating the G1 checkpoint and, therefore, DNA-damaged cells that lack normal p53 function extensively rely on the G2 checkpoint for survival (68). The concept emerges then that abrogation of the G2 checkpoint might preferentially sensitize p53-defective tumor cells to DNA-damaging agents and spare normal cells with intact p53-function (9). Thus, developing novel molecularly targeted drugs that abrogate the G2 checkpoint has become an active area of research. Several such drugs have already been developed and tested either as single agents or in combination with conventional, DNA-damaging cancer chemotherapy agents (9, 10). Considering the importance of the G2 block to the survival of irradiated cells (11, 12), it is logical to extend this approach to radiotherapy.

Design of agents that abrogate the G2 block depends on understanding the molecular mechanisms involved. In G2 phase, subsequent entry into mitosis is mainly controlled by activation of the cdc2/cyclin B complex, i.e. dephosphorylation of tyrosine 15 (Tyr15) of cdc2 by cdc25 activates this complex thereby allowing entry into mitosis (13, 14). Following DNA damage, the G2 checkpoint is initiated when cdc2 is inactivated by its phosphorylation on Tyr15 by the wee1 tyrosine kinase (15). In this latter situation, wee1 is activated and cdc25 inactivated downstream of the ATM-Chk1 signaling pathway that recognizes lesions in the DNA and signals cell cycle checkpoints (16). Abrogation of the G2 block could, therefore, be achieved through inhibition of the tyrosine kinase activity of wee1. In fact, it has been reported previously that abrogation of wee1 activity, either by decreasing its expression using siRNA (17), or by inhibiting its tyrosine kinase activity with a small molecule inhibitor (18), sensitizes tumor cells to DNA-damaging agents.

In 2009, Hirai and colleagues described a novel, small molecule inhibitor of wee1, MK-1775, that is both potent and selective (19). In their initial report, they demonstrate that MK-1775 inhibits the phosphorylation of cdc2 on Tyr15 with an IC50 of 5.2 nmol/L in in vitro kinase assays. In tests of activity using a panel of 223 kinases, MK-1775 displayed substantial selectivity for wee1, e.g. MK-1775 was 100 times more selective for wee1 as for Myt1, another kinase that suppresses cdc2 activity via phosphorylation at threonine 14 (20). Furthermore, they showed that MK-1775 abrogated the G2 checkpoint and potentiated the cytotoxicity of the DNA-damaging agents gemcitabine, carboplatin and cisplatin, selectively in p53-deficient cells. In a subsequent report, this group showed that MK-1775 enhances the antitumor effects of additional DNA-damaging agents with different modes of action including 5-FU, capecitibine, camptothecin, mitomycin C, doxorubicin, and pemetrexed (21). In light of the findings from these pre-clinical studies, MK-1775 has entered phase I clinical trials as a chemosensitizer in combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors (22).

The purpose of the present study was to investigate the ability of MK-1775 to radiosensitize human tumor cells in vitro and in vivo. Our data indicate MK-1775 preferentially radiosensitizes human tumor cells with defective p53 by abrogating the radiation-induced G2 block thereby allowing irradiated cells harboring unrepaired DNA lesions to pass into mitosis and enter the next cell cycle. This radiosensitizing effect was consistent with mitotic death but not apoptosis. Tumor cells with wild-type p53 and cells of normal tissue origin were not similarly radiosensitized. Overall, these results suggest that MK-1775 may have clinical utility when combined with radiotherapy for the treatment of solid tumors with defective p53.

Materials and Methods

Cell cultures and reagents

The human cell lines A549, H1299, Calu-6, H460, CCD-16, MCF-7, MDA-MB-231, MCF-10A, PC3, and LnCaP were all obtained from the American Type Culture Collection (ATCC) and routinely maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10,000 U/ML of penicillin-streptomycin, and 2 mmol/L-glutamine. The identities of these cell lines were validated during the course of this study by short tandem repeat (STR) profiling conducted by the institution’s Characterized Cell Line Core using the AmpFlSTR Identifiler PCR amplification kit according to the manufacturer’s instructions (Applied Biosystems). The STR profiles for these cell lines matched their known ATCC fingerprints. The H1299 cells with ponesterone A (Pon A)-inducible p53 expression have been described previously (23) and were the kind gift of Dr. Jack Roth, Department of Thoracic Surgery, M. D. Anderson Cancer Center. MK-1775 was provided by Merck Sharp & Dohme Corp., and it’s chemical structure has been described previously (18). Cells were trapped in mitosis using 0.2 µg/mL of nocodazole (Sigma-Aldrich).


Antibodies to cdc2 (#9112), p-cdc2(Tyr15) (#9111s), β-actin (#4967L), and phospho-Histone H3 (p-HH3, #9706) were purchased from Cell Signaling Technology. Antibodies to p53 (sc-263) were purchased from Santa Cruz Biotechnology, and γ-H2AX (Ser139) clone JBW301 (05-636) antibody was purchased from Millipore.

Western blot analysis

Total protein was extracted from the cell pellet using a lysis solution containing 50 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, and 1 mmol/L EDTA and fortified with 10 µL/mL phosphatase inhibitor cocktail 1, 10 µL/mL phosphatase inhibitor cocktail 2, 10 µL/mL protease inhibitor purchased from Sigma-Aldrich, and 1% NP-40. Protein concentration of the lysates was determined by the Bio-Rad protein assay. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to an Immobilon membrane (Millipore). Nonspecific binding sites on the membrane were blocked in 5% nonfat dry milk in Tris (20 mmol/L)-buffered saline (150 mmol/L, pH 7.4) with 0.1% Tween (TBS-T). Protein signals were detected by incubating the membrane in primary antibody in 5% nonfat dry milk overnight at 4°C, followed by a 45-min incubation in the appropriate peroxidase-conjugated secondary antibody. The membrane was then developed by enhanced chemiluminescence with ECL plus Western Blotting Detection Reagents (Amersham) on a Typhoon 9400 scanner (GE Healthcare).

Clonogenic assay

The effectiveness of the combination of MK-1775 and ionizing radiation was assessed by clonogenic assays. Briefly, cells growing in log phase were treated with 200 nmol/L MK-1775 1 h prior to irradiation. Following irradiation, the cells were subjected to an 18-h post-irradiation treatment with 200 nmol/L MK-1775. The cells were then trypsinized and counted, and known numbers were seeded in 60-mm culture dishes in two sets of three for each dose of radiation. Sufficient numbers were seeded to ensure that about 30–100 macroscopic colonies would appear in each plate after 10–14 days. Colonies were stained with 0.5% gentian violet in methanol and counted. The plating efficiency (PE) for each dose was calculated by dividing the number of colonies by the number of cells plated and expressing the result as a percentage. The surviving fraction was calculated by dividing the PE of the treatment by the PE of the appropriate un-irradiated control.

Cell cycle analysis

Cells were treated for 1 h with 200 nmol/L MK-1775, irradiated at 7.5 Gy, and then harvested at 0, 4, 8, 12, 16, and 24 h later. The cells were then washed with PBS and fixed in 70% ethanol in PBS overnight at 4°C. The fixed cells were washed in Buffer A (0.5% bovine serum albumin (BSA) and 2% FBS in PBS) and then incubated in lysis buffer (0.1% Triton X-100, 0.5% BSA, and 2% FBS in PBS) on ice for 5 min. The cells were pelleted by centrifugation and incubated in Buffer B (2% BSA and 10% FBS in PBS.) Again, cells were pelleted by centrifugation and then incubated with p-HH3 antibody at a dilution of 1:50 in Buffer A overnight at 4°C. The cells were then washed with Buffer A at room temperature and incubated for 1 h in anti-mouse FITC secondary antibody at a dilution of 1:100 in Buffer A. Cells were again washed with Buffer A, pelleted by centrifugation, and incubated in 2% BSA, 2% Tween-20, 5 µg/mL propidium iodide (PI, Sigma-Aldrich), and 2 µg/mL RNAse A (Sigma-Aldrich) for 1 h in the dark, and flow cytometric analysis was performed immediately thereafter. Flow cytometry was performed using a Beckman Coulter EPICS-ALTRA with Hypersort system equipped with a water-cooled Argon laser emitting at 488 nm. Analysis was performed using EXPO32 software. p-HH3 was measured using a 525-nm band pass filter. A minimum of 10000 events were collected for analysis. Gates were set to exclude cellular debris, and the fluorescence intensity of events within the gated region was measured.


A549 or H1299 cells were cultivated on coverslips placed in 35-mm dishes and treated with 0.2 µg/mL nocodazole, irradiated with 1 Gy, and treated with 200 nmol/L MK-1775 as indicated. The medium was then aspirated, and the cells were rinsed briefly in PBS and then fixed with 2% paraformaldehyde for 15 min. Permeabilization was achieved by a 10-min incubation with 100% methanol at −20°C. After three 5-min rinses in PBS, the cells were incubated in blocking buffer (1X PBS, 50 µL/mL normal goat serum, and 0.3% Triton X-100) for 1 h at room temperature. Next, the cells were incubated in γ-H2AX (Millipore) primary antibody in antibody dilution buffer (1X PBS, 10 mg/mL bovine serum albumin, 0.3% Triton X-100) overnight at 4°C with gentle shaking. After being washed with PBS, primary antibodies were visualized after a 2-h incubation with the appropriate Alexa Fluor-conjugated secondary antibody (goat anti-rabbit FITC or goat anti-mouse Alexa Fluor 594) at a 1:500 dilution. Nuclei were counterstained with 1:500 4’6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS, and the coverslips were mounted on slides with Vectashield (Vector Laboratories). Slides were examined using a Leica fluorescence microscope equipped with a CCD camera and images were imported into Advanced Spot Image analysis software. To quantify γ-H2AX foci, 50 nuclei were evaluated. Micronucleated cells were identified by DAPI staining and quantified (200 cells/coverslip).

Animals, tumor treatments, and tumor growth delay

Male Ncr Nu/Nu mice were used for tumor studies. Animals were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care approved facility in accordance with current regulations of the United States Department of Agriculture and Department of Health and Human Services. Experimental methods were approved by and in accordance with institutional guidelines established by the Institutional Animal Care and Use Committee.

Tumor xenografts were produced in the leg by im inoculation of 1 × 106 Calu-6 cells in 10 µL. Irradiation and MK-1775 treatment were started when tumors reached 8 mm diameter and continued for 5 days. Gamma-rays were delivered locally to the tumor-bearing legs of unanesthetized mice using a small-animal irradiator (Atomic Energy of Canada) consisting of two parallel-opposed 137Cs sources, at a dose rate of 5 Gy/min. Tumors were irradiated twice daily separated by 6 h. MK-1775 was given by gavage in 0.1 mL volumes 1 h before and 2 h after the first daily radiation dose.

Three mutually orthogonal tumor diameters were measured 2–3 times/week with a Vernier caliper and means calculated for plotting tumor growth delay. Mice were euthanized when tumors grew to 15 mm diameter. Tumor regrowth was expressed as the time in days for tumors in the treated groups to grow from 8 mm to 12 mm diameter minus the time for control tumors to reach the same size (absolute growth delay [AGD]). For treatment with both MK-1775 and radiation, normalized growth delay (NGD) was determined as time for tumors in the combined therapy group to grow to 12 mm minus time for tumors treated with drug alone to grow to 12 mm. Radiation enhancement factor (EF) was determined by dividing NGD for MK-1775 plus radiotherapy by the AGD for irradiation plus vehicle. p values for EFs were determined by Student’s t-test comparing NGD for MK-1775 plus irrradiation versus AGD for irradiation plus vehicle. Differences were considered significant if p≤ 0.05.

Statistical Analysis

Statistical significance was assessed by t test (two sample assuming unequal variances) and expressed as mean ± standard error. A difference was considered significant if p<0.05.


MK-1775 radiosensitizes human tumor cells in a p53-dependent manner

We assessed the ability of MK-1775 to radiosensitize human tumor cells using clonogenic survival curve assays. Various cell lines were tested including lines derived from non-small cell lung cancer (NSCLC), breast cancer, and prostate cancer. The p53 status of all of these lines had been previously established. In their original report on MK-1775, Hirai et al. showed that a concentration of 200 nmol/L of MK-1775 resulted in substantial suppression of p-cdc2 and that the optimal schedule was to first treat with the chemotherapy drug followed by a subsequent treatment with MK-1775 (19). The optimal concentration of MK-1775 and sequence of administration relative to irradiation were validated in preliminary, pilot studies using the NSCLC lines A549 and H1299. We determined that 24 h treatments with 200 nmol/L prior to irradiation were ineffective but 18 h treatments after irradiation modestly radiosensitized p53-null H1299 cells. Subsequent experiments showed that concentrations higher than 200 nmol/L were too toxic for these cells. Furthermore, in a detailed test of additional sequences using H1299 cells, we found that the optimum sequence consisted of a 1 h pre-treatment followed by an 18 h post-irradiation treatment (Fig. S1).

Using this treatment protocol, complete clonogenic survival curves for the 4 NSCLC lines examined consisting of two with wild-type p53, A549 and H460, and two that are null for p53, H1299 and Calu-6, were generated (Fig. 1A). Lines with defective p53, H1299 and Calu-6, were significantly radiosensitized but lines with wild-type p53, A549 and H460, were not and this pattern extended to two breast cancer lines and two prostate cancer lines (Fig. S2). The degree of radiosensitization was quantified from the survival curves in two different ways; by comparing the surviving fractions at the radiation dose of 2 Gy (SF2) and by calculating the dose enhancement factor (DEF), i.e. the ratio of radiation doses to achieve 10% survival. The DEF values for all of the cell lines examined are provided in Table 1. SF2 is a particularly relevant parameter in the context of clinical radiotherapy since 2 Gy is the typical dose given on a daily basis. Some of the p53-defective cell lines had substantial and significant changes in SF2 values. For example, for H1299 cells, SF2 was reduced from 0.65 ± 0.0496% in the control to 0.42 ± 0.0777% (p<0.01) by MK-1775. Such a radiosensitizing effect would be projected to result in 6 additional logs of cell kill if repeated for 30 fractions.

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 ...
Table 1
The DEF values calculated from the survival curves shown in Figures 1A and S2 for the p53-defective and p53 wild-type cell lines.

MK-1775 at the concentration of 200 nmol/L was slightly toxic to these cell lines, typically lowering PE by about 20%. For example, for the two cell lines used in subsequent experiments, the PE for A549 cells was reduced from 89.5 ± 1.1% in controls to 72.1 ± 1.0% in MK-1775 treated cells and in H1299 cells PE was reduced from 78.1 ± 2.9% to 58.5 ± 4.4% by MK-1775. These effects were typical for the other lines tested, independently of their p53 status, with the exception of the two cell lines derived from normal tissues, CCD-16 (normal lung fibroblasts) and MCF-10A (normal breast epithelial cells); PE was not affected by MK-1775 in these cell lines. Moreover, neither of these two lines was radiosensitized by MK-1775 (Fig. S2 and Table 1).

Although the correlation shown in Table 1 between p53 status of a cell line and its radiosensitization by MK-1775 was evident for the panel of eight tumor and two normal cell lines, we tested this relationship further using a cell line in which p53 expression is under exogenous control. Thus, we tested a cell line that we have reported on previously (24); H1299 cells that had been transfected with a Pon A-inducible p53 construct (23). Immunoblot analysis (Fig. 1C) showed that this cell line did not express p53 when cultured in medium without Pon A but robustly expressed it when cultured for 24 h with Pon A. Clonogenic survival analysis of this cell line confirmed the p53 dependency of radiosensitization by MK-1775; radiosensitization was suppressed in these H1299 cells when p53 expression was induced by Pon A treatment compared to the radiosensitization seen when Pon A treatment was withheld (Fig. 1B).

MK-1775 abrogates the radiation-induced G2 block in a p53-dependent manner by accelerating p53-defective cells into mitosis prematurely

We analyzed the effect of MK-1775 on cell cycle progression following irradiation in H1299 cells to determine if abrogation of the G2 block explained the radiosensitization effect of MK-1775 in this cell line. First, we performed mitotic trap experiments. H1299 cells were treated with 200 nmol/L MK-1775 for 1 h, irradiated with 4 Gy, and then incubated for 4 h in medium containing nocodazole and MK-1775. These samples were compared to control samples consisting of nocodazole alone (4 h), MK-1775 and nocodazole (4 h), 4 Gy and nocodazole (4 h), and 4 Gy followed by MK-1775 and nocodazole (4 h). At the end of the nocodazole treatment, the mitotic cells (which detach and float in the medium) were gently collected for each sample and counted. That these cells were mitotic was validated by cytospins and Giemsa staining; the mitotic index (MI) was typically > 95%. The results, depicted in Fig. 2A, show that MK-1775 alone accelerated unirradiated cells into mitosis compared to the nocodazole alone control. Cells irradiated with 4 Gy displayed a reduced level of mitotic cells compared to the control consistent with a radiation-induced G2 block but the block was reversed when MK-1775 was present during the post-irradiation nocodazole treatment and reversed to an even greater extent, i.e above the nocodazole only control, when the cells were pre-treated with MK-1775 for 1 h prior to irradiation and post-irradiation incubation in nocodazole plus MK-1775.

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 ...

To validate that the results in Fig. 2A actually represented changes in MI, a similar set of samples was generated except that all of the cells in the dishes were harvested at the end of the nocodazole treatment and the proportion of cells in mitosis was ascertained on the basis of p-HH3 stained cells as detected by flow cytometry. In this case, the response of H1299 cells was compared to A549 cells. The results (Fig. S3) show that MK-1775 accelerates unirradiated A549 and H1299 cells into mitosis when compared to the nocodazole control and, in both A549 and H1299 cell lines, 4 Gy followed by a 4 h incubation with nocodazole resulted in an MI lower than the level observed in the nocodazole only control indicative of the G2 block induced by radiation in these cells. However, a 1-hour pretreatment with MK-1775 followed by 4 Gy and then a 4 h incubation in MK-1775 + nocodazole accelerated H1299 cells into mitosis compared to the radiation alone sample illustrating abrogation of the G2 block as was observed in the prior experiment. A similar result was not seen in the p53 wild-type A549 cells where treatment with MK-1775 resulted in only a small increase in MI following irradiation but to a level below the MK-1775 and radiation alone controls indicating that the G2 block in these cells is substantially maintained (Fig. S3).

We also determined whether the radiosensitizing effects of MK-1775 correlated with abrogation of the radiation-induced G2/M block in asynchronously growing cells. A549 and H1299 cells were treated or not with 200 nmol/L MK-1775 for 1 h, irradiated with 7.5 Gy, returned to MK-1775-containing medium or not, and then harvested at 4 h intervals for up to 24 h. Cell cycle arrest as a function of time was determined on the basis of MI by p-HH3 staining and G2/M-associated DNA content, both assessed by flow cytometry. For both cell lines, irradiation alone caused a prompt decline in MI that reached a nadir by 4 h (Fig. 2B). Over time, MI recovered and peaked about 16 h after irradiation. The pattern was quite different for the two cell lines for the combination of MK-1775 and radiation. In H1299 cells, treatment with MK-1775 completely abrogated the decline in MI seen for irradiation alone and, instead, accelerated irradiated cells into mitosis, peaking about 8 h after irradiation. This effect was not seen in the A549 cells; the initial decline in MI after irradiation was identical whether the cells were treated with MK-1775 or not; and cells responding to both treatments reached a nadir of 0% MI at 4 h.

The results for the assessment of G2/M (Fig. 2C) were consistent with those seen for the MI above. H1299 cells treated with radiation alone accumulated in G2/M over time peaking at 12 h after irradiation, consistent with a radiation-induced G2 block. In the H1299 cells that were treated with MK-1775 + radiation, the cells continued to progress through G2/M with no substantial accumulation. MK-1775 did not substantially alter the accumulation of irradiated A549 cells in G2/M compared to that seen for radiation alone. Overall, these results are consistent with a p53-dependent abrogation of the radiation-induced G2 block by MK-1775 in p53-defective cells. Flow cytometry profiles for some of the key time points in Figure 2C are provided in Figure S4.

Abrogation of the G2 block with MK-1775 causes p53-defective cells to enter mitosis and into the next cell cycle harboring radiation-induced DNA lesions

The radiosensitizing effect of MK-1775 could be explained if p53-defective cells enter mitosis prematurely and progressed into the next cell cycle before they completed repair of the radiation-induced DNA damage. Unrepaired DNA lesions, especially DSBs, present at the time of mitosis would in that case be expected to have lethal consequences. To test this hypothesis, H1299 and A549 cells growing on cover slips were treated with MK-1775 for 1 h or not, irradiated with 1 Gy, and trapped in mitosis with nocodazole for 4 h. The dose of 1 Gy was used in this experiment due to the sensitivity of γ-H2AX foci detection. The mitotic cells in the samples were identified on the basis of their distinct morphology and γ-H2AX foci were scored in these mitotic cells by immunofluorescent staining as indicators of radiation-induced DNA damage, specifically DSBs. To underscore the relative influence of the 1 h pre-irradiation treatment with MK-1775, cells treated with this protocol were compared to cells that only received drug during the post-irradiation incubation. The results, shown in Figure 3A, indicate that for both cell lines, cells that enter mitosis within 4 h after irradiation harbor unrepaired DSBs. When MK-1775 was added to the cultures immediately after irradiation this effect was not increased. However, mitotic H1299 cells that received a 1 h pre-irradiation treatment followed by continued incubation with MK-1775 harbored significantly more DSBs compared to radiation alone (p<0.01) indicating that MK-1775, due to its abrogation of the G2 block, allows irradiated cells to prematurely enter mitosis harboring un-repaired DSBs. MK-1775 treatments did not similarly affect the levels of γ-H2AX foci in the A549 cells. For both cell lines, there was a slight increase in γ-H2AX foci in unirradiated cells that were treated with MK-1775, consistent with the toxicity of the drug on these cells and suggesting that the premature entry of cells into mitosis seen with this drug, i.e. Figures 2A and S3, may lead to lethal events due to the incomplete repair of DNA replication errors. Representative photomicrographs illustrating the presence of γ-H2AX foci in H1299 cells following these different treatments are presented in Figure S5.

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 ...

In a follow-up experiment examining the induction of micronuclei, the same protocol was followed except that the cells were grown in 100 mm culture dishes irradiated with 4 Gy, and, after the 4 h incubation in nocodazole, the mitotic cells were preferentially harvested by gentle shaking and replated onto cover slips in fresh medium without nocodazole or MK-1775. After 18 h of incubation, the cover slips were collected, stained with DAPI, and scored for micronuclei; the presence of cells with chromosome bridges was also noted. In both H1299 and A549 cells, the incidence of micronuclei increased with radiation alone compared to unirradiated control. Treatment of H1299 cells with MK-1775 led to substantially increased numbers of micronuclei compared to radiation alone, with this effect being more robust in the cells that were treated with MK-1775 1 h before and after irradiation compared to cells that received the drug only after irradiation (Fig. 3B). H1299 cells that were treated with MK-1775 both before and after irradiation also had substantial numbers of chromosome bridges compared to the other samples (data not shown). In A549 cells, MK-1775 given after irradiation led to increased numbers of micronuclei over the radiation alone control but to a lesser degree compared to H1299 cells. This effect was not increased by adding the drug before irradiation. Representative photomicrographs illustrating the presence of micronuclei in H1299 cells following these different treatments are presented in Figure 3D.

To confirm that MK-1775 affects its wee1 target in both A549 and H1299 cells, we treated cells for 1 h with 200 nmol/L and assessed the levels of p-cdc2 by western blot. The results (Fig. 3C) indicated that MK-1775 leads to a de-phosphorylation of cdc2 downstream of wee1 in both A549 and H1299 cells. This observed decrease in p-cdc2 below control levels is presumably due to the increased de-phosphorylation of Tyr15 on cdc2 by cdc25 when the balance of wee1 and cdc25 competing activities is upset by inhibition of wee1 by MK-1775. This effect was recapitulated in H460 and Calu-6 cells (Fig. S6A). Additionally, we tested whether p-cdc2 levels were suppressed in cells irradiated with 7.5 Gy and incubated with MK-1775 after irradiation. Although is was difficult to observe a substantial activation of wee1 activity by radiation due to the fact that almost all of the cdc2 is normally already phosphorylated in asynchronously growing cells (cdc2 is normally de-phosphorylated by cdc25 during the cell cycle only in the small subpopulation of G2-phase cells about to enter mitosis), the western blot analyses shown in Figure S6B and C indicate that MK-1775 leads to a de-phosphorylation of p-cdc2 in irradiated H1299, A549, H460 and Calu-6 cells independently of their p53 status.

We also tested whether radiosensitization by MK-1775 involved enhanced apoptosis. H1299 cells were treated for 1 h with 200 nmol/L MK-1775, irradiated with 7.5 Gy, incubated for an additional 18 h in MK-1775, and harvested for assessment of apoptosis at 24, 48, and 72 h post-irradiation. Apoptosis levels were determined on the basis of Annexin 5 staining and sub-G1 DNA content, both assessed by flow cytometry. The results indicated that the dose of 7.5 Gy induced levels of apoptosis of only about 5% above control at any time point and these levels of apoptosis were not significantly enhanced by MK-1775 (data not shown).

MK-1775 enhances H1299 xenograft tumor response to fractionated radiotherapy

Based on the substantial radiosensitization by MK-1775 in the p53-defective NSCLC cell lines (Table 1), we determined whether this effect extended to the in vivo situation. We performed a series of experiments to examine this question using xenograft tumors growing in nude mice made from one of the p53-defective NSCLC lines and treated with the combination of MK-1775 and external beam radiation where tumor growth delay was used as the endpoint for analysis. The Calu-6 cell line was chosen for this study based on its substantial radiosensitization by MK-1775 in the in vitro survival curve analysis (Fig. 1A and Table 1). Various treatment protocols were investigated including testing different sequences of drug and radiation, different doses of drug and different radiation fractionation schemes. Many of these protocols indicated that tumor growth delay was significantly enhanced by the drug/radiation combination compared to radiation alone. The greatest response was observed when tumors were irradiated twice a day with 1 Gy for 5 days and 60 mg/kg given twice a day on the same days as irradiation. The results of this experiment are presented in Figure 4. The enhancement factor for this treatment protocol was 3.2 (p<0.01). These results underscore the importance of sequencing the drug and radiation treatment close in time and demonstrate that the radiosensitizing effect of MK-1775 extends to the in vivo setting.

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 ...


In this study, we investigated the radiosensitizing abilities of a novel, potent and highly selective inhibitor of the wee1 kinase, MK-1775. Although previous reports have demonstrated that MK-1775 sensitizes p53-defective tumor cells to other DNA-damaging agents such as gemcitibine and cisplatin (19), its radiosensitizing properties have not been previously demonstrated. We focused our tests of MK-1775 on cell lines derived from three types of human tumors, i.e. NSCLC, breast and prostate, where radiotherapy typically plays a key role in the management of patients with these tumors and where improvements in radioresponse in these disease sites would be expected to provide clinical benefit. As shown in Figures 1A and S2 and summarized in Table 1, four p53-defective human tumor cell lines were radiosensitized by nanomolar concentrations of MK-1775 whereas four tumor cell lines with wild-type p53 and two cell lines of normal tissue origin were not. This comparison of p53-defective and p53 wild-type cell lines makes a convincing argument that the radiosensitizing effect of MK-1775 is p53-dependent. However, to bolster that argument, we also tested H1299 cells in which p53 expression had been restored using a Pon A-inducible vector. These results (Fig. 1B) confirmed the p53 dependence of MK-1775’s radiosensitizing effect.

Only one other small molecule wee1 kinase inhibitor has been previously reported. In 2001, Wang et al. described the development of the wee1 inhibitor PD166285 and showed that it abrogated the G2 checkpoint and radiosensitized p53-defective human colon carcinoma cells in vitro (18). In a more recent study, PD166285 was shown to radiosensitize wee1-overexpressing glioblastoma cells that were not p53-defective by abrogating their radiation-induced G2 checkpoint upon which they had become dependent (25). Thus, the radiosensitizing effects of PD166285 and the results with MK-1775 reported here are consistent and once again illustrate the profound importance of the G2 checkpoint in mediating the response of cells to radiation (11). Although, this has been well-understood for many years, the identification of wee1 as a viable drug target offers a unique opportunity for enhancing the therapeutic effects of DNA damaging agents such as radiation in p53-defective and wee1-overexpressing tumor cells (26, 27).

Following our appreciation of the fact that the 1 h pre-irradiation treatment added significant additional radiosensitization to the 18 h post-irradiation protocol, we conducted additional experiments to understand its impact. Apparently, this effect is due to a requirement for a finite amount of time for MK-1775 to affect its target and we showed that MK-1775 leads to the de-phosphorylation of cdc2, wee1’s substrate, by cdc25 within 1 h (Fig. 3C). Then, due to this de-phosphorylation of cdc2, the drug accelerates both unirradiated and irradiated cells into mitosis prematurely as demonstrated in the mitotic trap experiments (Figs. 2A and S3). In asynchronously growing cells, irradiation promptly blocked cells in G2 causing a sharp decline in mitotic cells (Fig. 2B). However, this block was abrogated by MK-1775 in H1299 cells leading to a significantly higher level of γ-H2AX foci in cells trapped in mitosis and a higher level of micronuclei in cells allowed to progress into the next cell cycle when treatment with MK-1775 commenced 1 h prior to irradiation compared to initiating drug treatment immediately after irradiation (Figs. 3A and B). It is these increased levels of DSBs and their subsequent conversion to micronuclei in the next cell cycle observed when irradiated cells are prematurely accelerated into mitosis that explain MK-1775’s radiosensitizing effect. Thus, radiosensitization my MK-1775 is not due to an inhibition of DNA repair but an abrogation of the extra repair time that would normally be allowed during a G2 block. When the drug treatment is only given after irradiation, a proportion of cells may already be blocked in G2 and cannot be radiosensitized by MK-1775. It is also conceivable that MK-1775, when given 1 h prior to irradiation accelerates a proportion of unirradiated cells into a more radiosensitive phase of the cell cycle, i.e. mitosis. Although, PD166285 was shown to accelerate irradiated cells into mitosis prematurely, the premature acceleration of unirradiated cells into mitosis by a wee1 inhibitor, as shown here for MK-1775, has not been reported previously to our knowledge. This may be an important feature of this drug as it may partially explain its activity as a single agent.

The question remains, why is MK-1775 ineffective as a radiosensitizer in tumor cells and normal cells that have wild-type p53 status? The answer appears to involve a critical role for p53 in governing the G2/M transition in DNA-damaged cells in addition to its well-known role in blocking such cells in G1 phase. What is known of this role for p53 in the G2/M transition has recently been reviewed and may involve a number of possible mechanisms (28). Following DNA damage, activation of p53 leads to induced expression of p21/waf1, GADD45 and 14-3-3σ. It has been proposed that each of these proteins may bind to the cdc2/cyclinB complex and modulate its function in such a manner as to inactivate it thereby causing an arrest of cells in G2 independently of the action of the wee1 kinase (29). However, in this case, a finite amount of time would be required for this p53-induced expression and, therefore, a small proportion of irradiated cells may escape a G2 block and progress into mitosis. Such an effect may explain the small increase in mitotic cells (Fig. S3) and micronuclei (Fig. 3B) observed in p53 wild-type A549 cells following MK-1775 treatment.

In addition to its ability to sensitize human tumor cells to DNA-damaging agents in vitro, MK-1775 has similar activity against human xenograft tumors growing in vivo. Hirai et al. reported that MK-1775 enhanced anti-tumor efficacy of gemcitabine, carboplatin, cisplatin and 5-FU in a model consisting of nude rats bearing WiDr human colon carcinoma xenografts (19, 21). Here, we demonstrate that this anti-tumor efficacy extends to NSCLC xenografts growing in nude mice treated with the combination of MK-1775 and external beam radiation. The wee1 inhibitor, PD166285, has also been examined in combination with radiation for the treatment of glioblastoma in an orthotopic mouse model (25). The combination significantly extended the survival of the mice compared to mice treated with either agent when used alone or to untreated controls. Due to a combination of factors related to the compound (27), PD166285 has not been investigated clinically. However, MK-1775 is currently being evaluated in phase I clinical trials in combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors. A preliminary report indicated that MK-1775 is well tolerated in monotherapy and in combination with chemotherapy (22).

In conclusion, we have shown that the wee1 kinase inhibitor, MK-1775, at nanomolar concentrations, potently radiosensitizes human tumor cells derived from lung, breast and prostate cancers in a p53-dependent manner. Lung cancer cells growing as xenograft tumors were also radiosensitized by this combination. The mechanism to explain this sensitization appears to involve a drug-induced, premature acceleration of G2 phase cells into mitosis. Such cells harbor unrepaired DNA lesions that lead to abnormal cell divisions and cell death. These findings support the continued clinical assessment of MK-1775 in combination with DNA damaging agents including radiation.

Translational Relevance

There is increasing interest in combining molecularly targeted drugs with radiotherapy as a strategy for enhancing the therapeutic ratio and, thus, patient response. Considering that many if not most human tumors have defective p53 function, an attractive approach to this strategy is to use agents that abrogate the radiation-induced G2 block as radiosensitizers. One such agent, MK-1775, a wee1 kinase inhibitor, is currently in phase 1 clinical trials as a chemosensitizer in combination with the DNA damaging agents gemcitibine, carboplatin, and cisplatin. The present study demonstrates that MK-1775 also potently radiosensitizes p53-defective human tumor cells in vitro and in vivo. If the degree of radiosensitization observed in these preclinical experiments could be recapitulated in patients undergoing fractionated radiotherapy, this strategy would be predicted to have significant benefit. Thus, these results strongly support the clinical development of MK-1775 in combination with radiation.

Supplementary Material


We gratefully acknowledge the technical assistance of David Molkentine and Mayra Vasquez. We also thank Dr. Jack Roth for the kind gift of the H1299 cells with the ponesterone A-inducible p53 construct. STR DNA fingerprinting was done by the institution’s Cancer Center Support Grant (NCI CA16672) funded Characterized Cell Line Core.

Grant Support

Merck Sharp & Dohme Corporation.


Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Disclosure of Potential Conflicts of Interest

CAB is an employee of Merck Sharp & Dohme. HH is an employee of Taiho Pharmaceutical Co. Ltd. KAB, CB, TAB, JMM, KAM, and REM received research support for this project from Merck Sharp & Dohme.


1. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323–331. [PubMed]
2. Vousden KH, Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer. 2002;2:594–604. [PubMed]
3. Su TT. Cellular responses to DNA damage: one signal, multiple choices. Annu Rev Genet. 2006;40:187–208. [PubMed]
4. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science (New York, NY. 1996;274:1664–1672. [PubMed]
5. Eriksson D, Stigbrand T. Radiation-induced cell death mechanisms. Tumour Biol. 2010;31:363–372. [PubMed]
6. Yao SL, Akhtar AJ, McKenna KA, et al. Selective radiosensitization of p53-deficient cells by caffeine-mediated activation of p34cdc2 kinase. Nat Med. 1996;2:1140–1143. [PubMed]
7. Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor PM. UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst. 1996;88:956–965. [PubMed]
8. Powell SN, DeFrank JS, Connell P, et al. Differential sensitivity of p53(−) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay. Cancer Res. 1995;55:1643–1648. [PubMed]
9. Leijen S, Beijnen JH, Schellens JH. Abrogation of the G2 checkpoint by inhibition of Wee-1 kinase results in sensitization of p53-deficient tumor cells to DNA-damaging agents. Curr Clin Pharmacol. 2010;5:186–191. [PubMed]
10. Kawabe T. G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther. 2004;3:513–519. [PubMed]
11. Iliakis G, Wang Y, Guan J, Wang H. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene. 2003;22:5834–5847. [PubMed]
12. Huang H, Fletcher L, Beeharry N, et al. Abnormal cytokinesis after X-irradiation in tumor cells that override the G2 DNA damage checkpoint. Cancer Res. 2008;68:3724–3732. [PubMed]
13. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990;344:503–508. [PubMed]
14. Nurse P. Checkpoint pathways come of age. Cell. 1997;91:865–867. [PubMed]
15. O'Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. Embo J. 1997;16:545–554. [PMC free article] [PubMed]
16. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature. 2000;408:433–439. [PubMed]
17. Wang Y, Decker SJ, Sebolt-Leopold J. Knockdown of Chk1, Wee1 and Myt1 by RNA interference abrogates G2 checkpoint and induces apoptosis. Cancer Biol Ther. 2004;3:305–313. [PubMed]
18. Wang Y, Li J, Booher RN, et al. Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator. Cancer Res. 2001;61:8211–8217. [PubMed]
19. Hirai H, Iwasawa Y, Okada M, et al. Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther. 2009;8:2992–3000. [PubMed]
20. Mueller PR, Coleman TR, Kumagai A, Dunphy WG. Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15. Science (New York, NY. 1995;270:86–90. [PubMed]
21. Hirai H, Arai T, Okada M, et al. MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil. Cancer Biol Ther. 2010;9:514–522. [PubMed]
22. Leijen S, Schellens JH, Shapiro G, et al. A phase I pharmacological and pharmacodynamic study of MK-1775, a Wee1 tyrosine kinase inhibitor, in monotherapy and combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors. J Clin Oncol; 46th Ann Meet Am Soc of Clin Oncol (ASCO); Chicago. 2010. p. 28:15s. 2010.
23. Nakamura S, Roth JA, Mukhopadhyay T. Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Molecular and cellular biology. 2000;20:9391–9398. [PMC free article] [PubMed]
24. Sah NK, Munshi A, Nishikawa T, Mukhopadhyay T, Roth JA, Meyn RE. Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity. Mol Cancer Ther. 2003;2:1223–1231. [PubMed]
25. Mir SE, De Witt Hamer PC, Krawczyk PM, et al. In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell. 2010;18:244–257. [PMC free article] [PubMed]
26. Indovina P, Giordano A. Targeting the checkpoint kinase WEE1: selective sensitization of cancer cells to DNA-damaging drugs. Cancer Biol Ther. 2010;9:523–525. [PubMed]
27. Stathis A, Oza A. Targeting Wee1-like protein kinase to treat cancer. Drug News Perspect. 2010;23:425–429. [PubMed]
28. Stark GR, Taylor WR. Control of the G2/M transition. Molecular biotechnology. 2006;32:227–248. [PubMed]
29. Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene. 2001;20:1803–1815. [PubMed]
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