Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735
Molecular
Cancer
Therapeutics
Chemical Therapeutics
WEE1 Kinase Inhibition Enhances the Radiation Response of
Diffuse Intrinsic Pontine Gliomas
Viola Caretti1,2,4, Lotte Hiddingh1,2,4, Tonny Lagerweij1,2,4, Pepijn Schellen1,2,4, Phil W. Koken3,
Esther Hulleman1,2,4, Dannis G. van Vuurden1,4, W. Peter Vandertop2, Gertjan J.L. Kaspers1, David P. Noske2,4,
and Thomas Wurdinger2,4,5
Abstract
Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric disease. Thus far, no therapeutic agent has proven
beneficial in the treatment of this malignancy. Therefore, conventional DNA-damaging radiotherapy remains
the standard treatment, providing transient neurologic improvement without improving the probability of
overall survival. During radiotherapy, WEE1 kinase controls the G2 cell-cycle checkpoint, allowing for repair of
irradiation (IR)-induced DNA damage. Here, we show that WEE1 kinase is one of the highest overexpressed
kinases in primary DIPG tissues compared with matching non-neoplastic brain tissues. Inhibition of WEE1 by
MK-1775 treatment of DIPG cells inhibited the IR-induced WEE1-mediated phosphorylation of CDC2,
resulting in reduced G2–M arrest and decreased cell viability. Finally, we show that MK-1775 enhances the
radiation response of E98-Fluc-mCherry DIPG mouse xenografts. Altogether, these results show that inhibition
of WEE1 kinase in conjunction with radiotherapy holds potential as a therapeutic approach for the treatment of
DIPG. Mol Cancer Ther; 12(2); 141–50. 2012 AACR.
Introduction
Diffuse intrinsic pontine glioma (DIPG) is an almost
invariably fatal brain neoplasm affecting mainly children,
with a 2-year survival rate less than 10% (1, 2). Its hallmarks are the specific anatomic location from which it
originates, the pons (3), its diffuse phenotype, often
spreading to the cerebellum and brain areas as far as the
cerebral hemispheres (4), and its bleak prognosis (3).
Despite various clinical trials, the standard treatment for
DIPG patients remains conventional radiotherapy, which
provides transient neurologic improvement, resulting in a
better quality of life, but does not improve probability of
overall survival (1, 2, 5). Therefore, novel treatment strategies to increase the efficacy of radiotherapy in DIPG are
urgently needed.
We have previously shown that inhibition of WEE1
kinase, one of the main gatekeepers of the G2 cell-cycle
checkpoint, is a potential therapeutic target for radiosensitization of adult gliomas (6) and of other type of
Authors' Affiliations: Departments of 1Pediatric Oncology, 2Neurosurgery, 3Radiation Oncology, 4Neuro-oncology Research Group, VU University Medical Center, Amsterdam, the Netherlands; and 5Molecular
Neurogenetics Unit, Department of Neurology, Massachusetts General
Hospital and Harvard Medical School, Boston, Massachusetts
Both V. Caretti and L. Hiddingh contributed equally to this work.
Corresponding Author: Thomas Wurdinger, VU University Medical Center, CCA Room 3.36, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Phone: 31-24447909; Fax: 31-204442126; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-12-0735
2012 American Association for Cancer Research.
cancers (7–9). Normal cells have functional cell-cycle
checkpoints as compared with cancer cells, which often
have a deficient G1 arrest due to aberrant p53 signaling
and, therefore, heavily rely on the G2 checkpoint to repair
DNA damage caused by irradiation (IR; ref. 10). Abrogation of the G2 checkpoint pushes glioma cells with
unrepaired DNA damage into mitotic catastrophe, resulting in subsequent cell death (11). Interestingly, in DIPG,
recent genomic studies have revealed aberrations in genes
regulating the G1 checkpoint, including TP53, MDM2,
CDKN2A, and ATM (12–19), suggesting a dysfunctional
G1 arrest in DIPG cells. Therefore, inhibition of WEE1
could be a potential strategy to enhance the response to IR
in DIPG cells.
A number of small-molecule compounds that inhibit
WEE1 have been developed. These include PD0166285
(20, 21), PD0407824 (22, 23), WEE1 inhibitor II, and
PHCD (23–25). The most promising WEE1 inhibitor
may be MK-1775, a pyrazolopyrimidine derivative,
because of its selectivity and potency to inhibit WEE1
kinase (26, 27). In vivo, WEE1 inhibition has resulted in
tumor growth reduction, increased survival, and
absence of significant toxicity in several studies using
xenograft animal models (6, 9, 26–30). Moreover, preliminary results of a phase 1 study of oral MK-1775 as
monotherapy and in combination with gemcitabine,
cisplatin, or carboplatin reported good tolerance and
strong target engagement (31).
We have previously shown that inhibition of WEE1
could function as a potential radiosensitizer of adult
gliomas, both in vitro and in vivo. As an extension to these
previous results, in this study, we investigated the
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Caretti et al.
potential radiation-enhancing effects of a more potent and
clinically relevant WEE1 inhibitor, MK-1775, in DIPG cells
in culture and in vivo using the E98-Fluc-mCherry (E98FM) DIPG mouse model, closely resembling the DIPG
phenotype in humans (32).
Materials and Methods
Ethics statement
All animal experiments were conducted according to
the guidelines established by the European community
and following a protocol (NCH10-05) approved by the
institutional ethical committee on animal experiments
of the VU University (Amsterdam, the Netherlands). All
patient samples, including the de novo cell line VUMCDIPG-A, were used after appropriate written informed
consent and under approval of the institutional medical
ethical committee of the VU University Medical Center
(VUmc). The research described here has been conducted according to the principles expressed in the
Declaration of Helsinki.
In silico analysis of DIPG kinase expression
R2, a microarray analysis and visualization platform,
provided by the Department of Oncogenomics of the
Academic Medical Centre, Amsterdam, The Netherlands
(http://r2.amc.nl), was used to obtain an overview of
kinase mRNA expression in DIPG. A MAS5.0 normalized
dataset of post-mortem DIPG tissues (ref. 17; n ¼ 27; GSE
GSE26576) was compared with post-mortem normal brain
regions (ref. 33; n ¼ 172; GSE11882), consisting of the
hippocampus, entorhinal cortex, superior frontal gyrus,
and postcentral gyrus, and 2 samples of post-mortem
normal brainstem from the DIPG dataset (ref. 17; ref.
GSE26576). To evaluate statistical significance, a false
discovery rate (FDR)-corrected, moderated t test (Linear
Models for Microarray Data - Limma) was used to compare mean kinase mRNA expression levels between datasets. An FDR P value less than 0.000005 was considered
significant. To compare WEE1 mRNA expression
between groups within DIPG dataset and normal brain
tissue datasets, 1-sided ANOVA was used.
Cells and tissue samples
The primary low-passage VUMC-DIPG-A cells were
derived from tumor tissue surgically removed from a
patient diagnosed with DIPG at the VUmc. E98 cells were
obtained from the Radboud University Nijmegen Medical
Centre (Nijmegen, the Netherlands; ref. 34) and transduced with a lentiviral vector containing Fluc and
mCherry at our institution (32). Cells used in this study
were not authenticated. The primary VUMC-DIPG-A and
E98-FM cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; PAA) containing 10% FBS and
antibiotics. DIPG tissue and control non-neoplastic brain
tissue were obtained post-mortem from 5 DIPG patients
(VUMC-DIPG-1, 2, 3, 4, and 5; ref. 35), while VUMCDIPG-A cells were isolated from surgical specimen after
written informed consent.
142
Mol Cancer Ther; 12(2) February 2013
WEE1 inhibitor and IR
The WEE1 inhibitor MK-1775 (Axon Medchem) was
resuspended in dimethyl sulfoxide (DMSO) to a concentration of 100 mmol/L and diluted in PBS for the in vivo
experiments and in medium for the in vitro experiments.
Cells were irradiated in a Gammacell 220 Research Irradiator (MDS Nordion).
Western blotting
Expression levels of WEE1 were assessed by Western
blot analysis in both tissue samples and cell lines as
described previously (6). In brief, after cell lysis (for
CDC2-pY15 a phospholysis buffer was used), 30 mg protein
was transferred to a polyvinylidene difluoride (PVDF)
membrane and incubated with the primary antibodies:
mouse anti-WEE1 (1:1,000; Santa Cruz Biotechnology),
mouse anti-b-actin (1:10,000; Santa Cruz Biotechnology),
and rabbit anti-CDC2-pY15 (1:2,000; Abcam), and subsequently incubated with horseradish peroxidase (HRP)labeled goat-anti-mouse or HRP-labeled goat-anti-rabbit
immunoglobulins (DAKO). Protein detection and visualization was carried out using ECLþ Western Blotting
Detection Reagents (Pierce).
Immunohistochemistry
Paraffin-embedded DIPG tissue samples and
matched non-neoplastic brain samples were deparaffinized and rehydrated. Endogenous peroxidase was
inhibited by 30-minute incubation in 0.3% H2O2, diluted
in methanol. Antigens were retrieved by boiling in Tris/
EDTA buffer (pH 9.0) in a microwave for 10 minutes,
followed by washing 3 times in PBS. Slides were incubated with mouse anti-WEE1 (1:50; Cell Signaling Technology) overnight at 4 C. Slides were washed 3 times in
PBS and incubated with the secondary antibody,
Envisionþ Poly-HRP immunohistochemistry (IHC) Kit
(Immunologic) for 30 minutes at room temperature.
Positive reactions were visualized by incubation with
DAB chromogen solution. Slides were counterstained
with hematoxylin, dehydrated, mounted, and analyzed
by microscopy.
Flow cytometry
At 16 hours after treatment, cells were washed twice
with PBS containing 1% FBS and subsequently fixed in
70% ethanol for 24 hours. Next, the cells were washed
once in PBS containing 1% FBS followed by RNAse A
treatment (0.15 mg/mL) for 20 minutes and subsequent
DNA staining with 50 mg/mL propidium iodide (PI) for
30 minutes. Cell-cycle distribution assessment was
conducted using a FacsCalibur Flow Cytometer and
CellQuest Pro software (Becton-Dickinson). Subsequently, data were analyzed using ModfitLT software
(Verity Software House).
Immunofluorescence staining
Cells were fixed in 3% paraformaldehyde at different
time points post irradiation (15 minutes, 30 minutes, and
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WEE1 Inhibition Enhances the Radiation Response in DIPG
1, 24, 48, and 72 hours). Before staining, the cells were
rinsed in PBS and permeabilized in PBS containing 0.1%
Trition X-100 for 30 minutes at room temperature and
blocked in PBS containing 5% FCS. Coverslips were incubated with both mouse-anti-g-histone-H2AX (1:100; Millipore) and rabbit-anti-53BP1 (1:100; Novus Biologicals) in
PBS containing 5% FCS overnight at 4 C. This was followed by secondary antibody incubation with goat-antirabbit/Alexa568 immunoglobulins (1:100; Invitrogen)
and, subsequently, with rabbit-anti-mouse/fluorescein
isothiocyanate (FITC) immunoglobulins (1:100; DAKO)
both in PBS containing 5% FCS for 30 minutes at room
temperature in the dark. Slips were rinsed in PBS 3 times
and nuclei were stained with 40 , 6-diamidino-2-phenylindole (DAPI; 1:10,000) in PBS at room temperature in the
dark. This was followed by successive rinses in PBS and
sterile water. The slips were then mounted on glass slides
using Vectashield (Vector Laboratories) and visualized
with a Carl Zeiss Axioskop 20 microscope at 100
objective.
Cell counts
At 4 days after treatment, cells were fixed with 3.7%
formaldehyde and DNA was stained with DAPI
(0.3 mg/mL). Cell numbers were assessed by counting
the number of DAPI-stained cells using the Acumen Ex3
laser scanning cytometer (TTP LabTech).
In vivo analysis using the orthotopic E98-FM DIPG
mouse model
Female athymic nude mice (age 6–8 weeks; Harlan,
Zeist, the Netherlands) were kept under specific pathogen-free conditions in air-filtered cages and received
food and water ad libitum. E98-FM cells were injected as
described previously (32). In short, tumors were generated via stereotactic injection of 0.5 106 E98-FM cells
in a volume of 5 mL into the murine pons (coordinates
from lambda: 1.0 mm X, 0.8 mm Y, 5.0 mm Z). Tumor
growth was monitored twice-weekly by bioluminescence imaging (BLI), as previously reported (32). At
day 7 after intracranial injection, mice were stratified
on the basis of BLI signal intensities into 4 treatment
groups (n ¼ 12) with comparable mean Fluc activity
and, then, received MK-1775 (90 mg/kg) or vehicle (PBS
containing 18% DMSO) in a total volume of 200 mL via
intraperitoneal injection every other day for a total of 6
injections per mouse. At day 8, mice received head-only
IR with a single dose of 2 Gy using Clinac D/E (Varian
Medical Systems), as described previously (32). Mice
were sacrificed after humane endpoints were reached
via sedation (1.5 L O2/min and 2.5% isoflurane), followed by cervical dislocation. To assess significant differences between treatment groups mice with BLI
values within each treatment group outside the mean
2 SD range were excluded from further analysis.
Next, BLI signals were log10-transformed and unpaired
Student t test was carried out. A difference was considered significant when P < 0.05.
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Results
In silico analysis of kinase mRNA expression
identifies WEE1 as a potential drug target in DIPG
Using the publicly available microarray analysis and
visualization platform R2 (http://r2.amc.nl), we analyzed a DIPG gene expression data set (17) and compared
it with nonmalignant brain tissue (33, 36), including 2
tissue samples of normal brainstem (17), to identify the top
upregulated kinases for this malignancy. To compare the
differential kinase gene expression of cancer versus normal samples, the fold-change to nonmalignant brain tissue was determined for all kinases within the dataset. A
FDR-corrected moderated t test was used to compare
means of kinase gene expression levels between the datasets and a P value less than 0.000005 was considered
significant. Analysis of the kinase expression data showed
that WEE1 ranked seventh among the overexpressed
kinases (Table 1). Given our previous experience on the
radiosensitizing effect of WEE1 inhibition in adult glioma
and the existence of a selective inhibitor, MK-1775, currently being tested in clinical trials, we decided to further
investigate WEE1 as a potential target to enhance the IR
response in DIPG cells. Using the R2 platform, we specifically investigated WEE1 mRNA expression in DIPG,
low-grade glioma (LGG) in the brainstem, normal brainstem, nonmalignant brain regions, and normal cerebellum
(Fig. 1A). Using 1-way ANOVA, WEE1 mRNA levels
were determined to be significantly higher in DIPG than
in different regions of normal control brain. Interestingly,
WEE1 mRNA levels were significantly higher in DIPG
compared with LGG of the brainstem, as we previously
observed in adult low- and high-grade gliomas (6).
WEE1 protein is overexpressed in DIPG patient
material and cell lines
Next, we analyzed the protein expression of WEE1
kinase by Western blot analysis in 5 different post-mortem
DIPG tissues (35) isolated from the pons and matched these
to non-neoplastic brain tissues of the same patient (Fig. 1B).
We observed significant overexpression of WEE1 protein in
4 out of 5 tumors compared with the non-neoplastic brain
tissues. For patient VUMC-DIPG-1, we were not able to
detect WEE1 protein overexpression in the tumor in the
pons region, which may be due to the presence of tumor
necrosis in the sample used. However, we detected significant WEE1 protein expression in VUMC-DIPG-1 tissue
isolated from the frontal lobe of the same patient (Fig. 1B). In
addition, we analyzed WEE1 protein expression by Western blot analysis in a primary DIPG cell line and in E98-FM
glioma cells (32), again showing significant WEE1 protein
levels (Fig. 1C). Furthermore, by IHC we found WEE1
protein overexpression, as shown by a clear nuclear staining, in DIPG tumors located in the pons (Fig. 1D–G and I–K,
left) as compared with matched nonneoplastic brain tissue
(Fig. 1L). In particular, high levels of WEE1 were detected in
the tumor bulk (Fig. 1D, asterisk), as well as in the infiltrative DIPG component (Fig. 1E), and in tumor cells surrounding the blood vessels (Fig. 1F). Nuclei in the
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Table 1. Top 20 upregulated kinases in DIPG
Rank
Gene
Name
Probeset
Expression
normal
brain (log2)
Expression
DIPG (log2)
Fold
increase
(log2)
P value
(FDR)
1
2
TOP2A
MELK
201292_at
204825_at
1,05
1,61
7,85
7,49
7,45
4,66
3,30E-68
6,64E-62
3
BUB1
209642_at
1,98
6,69
3,38
3,70E-44
4
5
6
7
8
TTK
PBK
OSR1
WEE1
NEK2
204822_at
219148_at
228399_at
212533_at
204641_at
2,12
2,46
1,87
3,60
3,44
6,83
7,61
5,66
8,81
6,74
3,23
3,10
3,03
2,45
1,96
2,78E-51
5,90E-61
7,03E-22
2,60E-46
8,43E-24
9
10
11
12
13
STK33
TEX14
CHEK1
AURKB
BUB1B
228035_at
221035_s_at
205394_at
209464_at
203755_at
3,18
2,92
2,90
3,05
3,98
6,14
5,50
5,37
5,32
6,84
1,93
1,88
1,85
1,74
1,72
2,03E-19
5,24E-14
2,57E-30
2,45E-20
4,08E-32
14
15
16
17
HK2
CHEK2
AURKA
ROR2
202934_at
210416_s_at
204092_s_at
205578_at
5,54
3,51
4,26
2,41
8,95
5,43
6,57
3,71
1,62
1,55
1,54
1,54
2,81E-32
3,41E-15
4,37E-33
3,62E-06
18
19
PLAU
DYRK3
205479_s_at
210151_s_at
3,52
4,28
5,41
6,32
1,54
1,48
2,29E-14
6,32E-15
20
MASTL
Topoisomerase (DNA) II alpha
Maternal embryonic leucine
zipper kinase
Budding uninhibited by
benzimidazoles 1 homolog
TTK protein kinase
PDZ binding kinase
Odd-skipped related 1 (Drosophila)
WEE1 homolog
NIMA (never in mitosis
gene a)-related kinase 2
Serine/threonine kinase 33
Testis expressed 14
CHK1 checkpoint homolog (S. pombe)
Aurora kinase B
Budding uninhibited by benzimidazoles
1 homolog beta (yeast)
Hexokinase 2
CHK2 checkpoint homolog (S. pombe)
Aurora kinase A
Receptor tyrosine kinase-like
orphan receptor 2
Plasminogen activator, urokinase
Dual-specificity tyrosine-(Y)phosphorylation
regulated kinase 3
Microtubule-associated
serine/threonine kinase-like
228468_at
4,21
6,13
1,46
3,76E-47
NOTE: Top 20 upregulated kinases in DIPG tumor samples (n ¼ 27; ref. 17) sorted on log2 fold increase as compared with nonmalignant
brain tissues (n ¼ 174; ref. 33) including 2 samples of normal brain stem tissue from the DIPG dataset (17). Wee1 homolog (WEE1) is
identified as a highly differentially overexpressed kinase in DIPG.
leptomeningeal tumor component also revealed WEE1
nuclear staining (Fig. 1G). Notably, WEE1 was found to
be overexpressed in tumor cells invading brain areas
beyond the brainstem (Fig. 1H and I–K, right). Finally,
clear WEE1 nuclear staining was also detected in tumor
tissue derived from the orthotopic E98-FM DIPG mouse
model (Fig. 1M, arrow).
MK-1775 inhibits WEE1-regulated CDC2, IRinduced G2 arrest, and repair of IR-induced DNA
damage in DIPG cells
E98-FM and VUMC-DIPG-A cells were used to study
the effect of the WEE1 kinase inhibitor MK-1775 in vitro.
Because WEE1 phosphorylates CDC2 on being subjected
to IR (37–39), we conducted Western blot analysis for
phosphorylated CDC2 (Y15) 16 hours after treatment with
0.1 mmol/L MK-1775 and IR (Fig. 2B). IR at 6 Gy resulted in
increased CDC2-pY15 levels, which were reduced on treatment with MK-1775, indicating functional inhibition of
144
Mol Cancer Ther; 12(2) February 2013
WEE1 kinase activity in these cells. The effect of MK-1775mediated WEE1 inhibition on the IR-induced G2 arrest
was determined by cell-cycle analysis using flow cytometry (Fig. 2C). In both cell lines, IR at 6 Gy induced a G2–M
arrest after 16 hours, indicated by an accumulation of cells
with doubled DNA content and indicative for a dysfunctional G1 arrest, which was reduced on treatment with 0.1
mmol/L MK-1775. Subsequently, we examined the effect
of WEE1 inhibition on the repair of IR-induced DNA
damage within these cells (Fig. 2D and E). The IR-induced
DNA damage was visualized using the DNA doublestrand break (DSB) markers gH2AX and 53BP1 at 15
minutes, 30 minutes, and 1, 24, 48, and 72 hours after
treatment with IR at 4 Gy in the presence or absence of 0.1
mmol/L MK-1775. As shown in Fig. 2D gH2AX and 53BP1
colocalize. These results indicate DNA damage in both
cell lines after IR treatment as compared with untreated
cells. Cells treated with IR showed fast onset of DSBs (as
early as 15 minutes after IR); however, a decreased
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WEE1 Inhibition Enhances the Radiation Response in DIPG
B
C
200
D
C.bellum
Brain
Brainstem
VUMC-DIPG-A
E98-FM
Healthy brain
Healthy brain
Matched CTRL-5
VUMC-DIPG-5
Matched CTRL-4
VUMC-DIPG-4
WEE1
LGG
0
Matched CTRL-3
400
VUMC-DIPG-3
600
Matched CTRL-2
800
VUMC-DIPG-2
1,000
Matched CTRL-1
VUMC-DIPG-1
Frontal Lobe
1,200
VUMC-DIPG-1
1,400
DIPG
WEE1 mRNA Expression
A
β-Actin
E
F
G
H
J
K
L
M
*
I
Figure 1. WEE1 is overexpressed in DIPG tissues and cells. A, WEE1 mRNA level in 27 DIPG samples, 6 brainstem low-grade glioma (LGG) samples, 2 normal
brainstem samples, 172 nonmalignant brain region, and 9 normal cerebellum samples. B, Western blot analysis of WEE1 expression in DIPG tissues
and matching controls. C, Western blot analysis of WEE1 expression in E98-FM cells and primary DIPG cells. Nonmalignant brain tissue collected from a
pediatric patient that underwent surgery due to refractory epilepsy was used as a negative control. D–M, IHC of WEE1 expression in 5 DIPG patients,
VUMC-DIPG-1 (D–H), VUMC-DIPG-2 (I), VUMC-DIPG-3 (J), VUMC-DIPG-4 and -5 (K, left and right, respectively). D–G and I–K, left; H, I, and J, right;
and K, right, show DIPG at the level of the pons, cerebellum, and insula, respectively. H, arrows indicate DIPG cells subpial accumulation. L, matched control
tissue collected from the cerebellum and the pons for VUMC-DIPG-2 and VUMC-DIPG-1 patients, respectively. M, IHC of WEE1 expression in the E98-FM
tumor tissue. The triangle indicates absence of WEE1 nuclear staining in murine pons tissue microscopically free of disease. Size bar, 20 mm (E–G, L, K), 40 mm
(D, H–J, and M, right), and 80 mm (M, left).
number of gH2AX and 53BP1 foci was observed over time
in cells treated only with IR, indicating the occurrence of
DNA damage repair within these cells (Fig. 2D and E).
Notably, although no differences in the number of IRinduced DSB foci were observed at the early time points
after IR, the cells treated with both IR and MK-1775
showed persistence of DNA damage over time, as indicated by a higher number of gH2AX and 53BP1 foci in
these cells compared with cells treated with IR at 4 Gy
alone (Fig. 2D and E; E98-FM and VUMC-DIPG-A, P <
0.001). These results show that MK-1775 is capable of
functional WEE1 inhibition and, thereby, of attenuating
the IR-induced G2 arrest in G2-dependent E98-FM and
VUMC-DIPG-A cells and, subsequently, of inhibiting the
repair of IR-induced DNA damage in these cells.
MK-1775 enhances the radiation response in
cultured DIPG cells
Next, we determined whether the attenuation of IRinduced G2 arrest by MK-1775 reduces viability of these
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cells in vitro. A clonogenic survival assay is the gold
standard for this purpose (40), but because these cells do
not form appreciable colonies, we monitored cell viability
by counting DAPI-stained cells using a laser scanning
cytometer. We treated E98-FM cells with IR at 4 Gy in
the presence or absence of 0.1 mmol/L MK-1775. After 4
days, cells were stained with DAPI and cell numbers were
counted. Treatment of the E98-FM cells with 0.1 mmol/L
MK-1775 or IR monotherapy resulted in a significant but
moderate decrease in cell viability compared with
untreated cells. Treatment with MK-1775 in combination
with IR resulted in radiosensitization of E98-FM cells (P <
0.001; Fig. 3A and B). Next, we analyzed the effect of 0.1
mmol/L MK-1775, in combination with IR at 4 Gy, on the
cell viability of primary VUMC-DIPG-A cells. Both 0.1
mmol/L MK-1775 and IR monotherapy show only a limited effect on cell viability of VUMC-DIPG-A. However,
combined treatment with MK-1775 and IR resulted in
additional diminished cell viability, compared with IR
alone (P < 0.05; Fig. 3C and D).
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A
4 Gy + MK-1775
4 Gy
D
MK-1775
γ-H2AX
53BP1
DAPI
γ-H2AX
Merge
53BP1
DAPI
Merge
No IR
B
15 min
1.0
0.9
1.5
1.1
E98-FM
CDC2-pY15
30 min
β-Actin
1.0
0.7
1.5
0.8
VUMC-DIPG-A
1h
CDC2-pY15
β-Actin
C
24 h
E98-FM
Counts
48 h
72 h
50
E98-FM
CTRL
40
MK-1775
30
20
*** *** ***
10
MK-1775
30
***
20
***
***
10
h
h
h
h
72
48
24
1
in
in
m
m
30
h
h
72
h
h
Time after radiation
48
24
in
1
in
m
m
30
IR
o
N
2N
4N
4N
CTRL
0
0
2N
*
VUMC-DIPG-A
40
IR
G2–M: 22%
Counts
G2–M: 26%
IR+MK-1775
50
15
G2–M: 41%
o
Counts
IR
G2–M: 63%
Avg number of γ-H2AX foci/nucleus
E
N
G2–M: 19%
Avg number of γ-H2AX foci/nucleus
G2–M: 18%
15
MK-1775
G2–M: 23%
G2–M: 18%
Counts
CTRL
VUMC-DIPG-A
Time after radiation
Figure 2. MK-1775 inhibits WEE1-regulated CDC2 and IR-induced G2 arrest in E98-FM and DIPG cells. A, chemical structure of MK-1775. B, Western blot
analysis of phosphorylated CDC2 16 hours after treatment of E98-FM and VUMC-DIPG-A cells with 6 Gy and/or 0.1 mmol/L MK-1775. Numbers represent
relative phosphorylated CDC2 expression after normalization against control carried out using imageJ software. C, PI cell-cycle analysis of E98-FM
and VUMC-DIPG-A cells 16 hours after treatment and treated as in B. The percentage of cells in the G2–M phase are indicated. D, analysis of DNA damage
repair after treatment with IR at 4 Gy or IR and 0.1 mmol/L MK-1775, visualized by the DSB markers gH2AX and 53BP1. The panel shows representative
images of gH2AX foci, 53BP1 foci, DAPI-stained nuclei, and merged images in VUMC-DIPG-A cells at 15 minutes, 30 minutes, and 1, 24, 48, and 72 hours
after treatment. E, quantification of average number of gH2AX foci per nucleus in E98-FM and VUMC-DIPG-A cells at similar time points as in D ( , P < 0.05,
, P < 0.01; , P < 0.001 Student t test). At least 20 nuclei per time point were analyzed for this quantification.
MK-1775 enhances the radiation response in the
orthotopic E98-FM DIPG mouse model
We used the orthotopic E98-FM DIPG mouse model
(32) to study the radiation-enhancing effects of MK-1775
in vivo. E98-FM glioma cells were stereotactically injected
in the pons of nude mice. Tumor growth was monitored
twice a week using the BLI intensity of the photon activity
of firefly luciferase encoded by the E98-FM cells. Seven
days after intracranial injection of the cells, mice were
randomized into 4 groups (n ¼ 12 per group) on the basis
of BLI intensity. Two groups of mice received MK-1775
intraperitoneally (i.p.; 90 mg/kg) and 2 groups received
vehicle (DMSO diluted in PBS) i.p, at 7, 9, 11, 13, 15, and
17 days after injection of the cells. One group of MK-1775
and vehicle-treated mice was irradiated with 2 Gy at day
8 after injection of the cells. BLI revealed significant
tumor progression in both nonirradiated vehicle-treated
146
Mol Cancer Ther; 12(2) February 2013
and MK-1775-treated mice, while tumor growth was
significantly delayed in irradiated and irradiated MK1775-treated mice compared with the control group
(weeks 2 and 3, and weeks 2, 3, and 4, respectively; Fig.
4A and B). Moreover, the mean BLI signal of the irradiated
MK-1775-treated group was significantly decreased compared with the irradiated group at week 4 after injection
of the cells (P < 0.05). In addition, survival analysis
showed a significant advantage for combining IR with
MK-1775 over the vehicle-treated control group (log-rank,
P < 0.05), while the groups receiving only MK-1775 or IR
showed no significant effect on survival as compared with
the untreated mice (Fig. 4C). These results indicate that
pharmacologic targeting of WEE1 with MK-1775 in combination with IR delays the growth of E98-FM DIPG
tumors in vivo, although the effects observed under these
conditions were modest.
Molecular Cancer Therapeutics
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Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735
WEE1 Inhibition Enhances the Radiation Response in DIPG
***
C
10
1
100
***
***
*
10
+M
K17
75
IR
D
IR
C
TR
L
+M
K17
75
IR
IR
B
M
K17
75
C
TR
L
1
M
K17
75
Relative % cell viability (log scale)
100
***
***
***
Relative % cell viability (log scale)
***
A
CTRL
MK-1775
CTRL
MK-1775
IR
IR+MK-1775
IR
IR+MK-1775
Figure 3. MK-1775 enhances the radiation response in E98-FM and DIPG cells. A and B, analysis of cell counts of E98-FM cells after treatment with IR at 4 Gy
and/or 0.1 mmol/L MK-1775. Relative percentages of cell counts were normalized to that of MK-1775-only treatment, as depicted in logarithmic scale.
The panel in B shows representative images of DAPI-stained cells for the different treatment conditions. C and D, similar analysis as in A and B using primary
VUMC-DIPG-A cells. Results are depicted as averages of an experiment conducted in triplicate; error bars indicate standard error of the mean ( , P < 0.05,
, P < 0.001, Student t test). Size bar, 20 mm.
Discussion
DIPG is the most deadly pediatric malignancy. Radiotherapy remains the standard treatment, although this
only causes temporary tumor regression. Despite
numerous clinical trials, no therapeutic agent has, thus
far, shown a survival benefit for DIPG patients (2).
Nonetheless, the clinical use of these agents was not
based on translational studies due to the lack of DIPG
tissue and in vitro and in vivo models available for
preclinical research. Here, we present a translational
study with WEE1 target assessment on DIPG tissue,
functional experiments using primary DIPG cells in
vitro and the orthotopic E98-FM DIPG model (32) to
show the radiation-enhancing effects of WEE1 inhibition in vivo.
www.aacrjournals.org
Kinase expression data analysis showed WEE1 kinase
to be in the top-10 overexpressed kinases in a dataset of
DIPG samples compared with normal brain tissues.
Although the other overexpressed kinases could also be
of value for investigating therapeutic targeting in DIPG,
we analyzed WEE1 kinase, because it was previously
shown to be a potential target for the radiosensitization
of adult glioma cells (6), and because a clinically relevant
WEE1 inhibitor was available (31). We found WEE1 to be
highly overexpressed in the DIPG tissues analyzed. Interestingly, WEE1 was also overexpressed in tumor tissue
invading brain areas beyond the brainstem (including
cerebellum, insula, and frontal lobe), indicating that
the infiltrative DIPG components can also be potentially
targeted by WEE1 inhibitors. IHC confirmed WEE1
Mol Cancer Ther; 12(2) February 2013
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147
Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735
Caretti et al.
A
***
**
Log10 fluc activity
7
***
***
*
*
6
5
4
C
M TRL
K17
75
IR
+M I
K- R
17
75
C
M TRL
K17
75
IR
+M I
K- R
17
75
C
M TRL
K17
75
IR
+M
K- IR
17
75
C
M TRL
K17
75
IR
+M I
K- R
17
75
3
Week 1
Week 2
Week 3
Week 4
B
1
2
3
4
C
100
CTRL
80
Survival
MK-1775
CTRL
MK-1775
IR
IR+MK-1775
60
40
IR
20
IR+MK-1775
0
15
CTRL vs. IR+MK -1775
P < 0.05
20
25
30
Days after surgery
35
40
Figure 4. In vivo analysis of MK-1775 using the orthotopic E98-FM DIPG model. A and B, log10-transformed BLI values of individual mice per treatment group
(n, 12 per group) at different time points after injection of the E98-FM cells in the pons (A) and representative bioluminescence images (B). The Fluc signal
activity is calculated as photons/second/cm2. The horizontal bar indicates the median and error bars indicate range ( , P < 0.05; , P < 0.01; , P < 0.001,
Student t test). C, Kaplan–Meier survival curves of the different treatment groups. Only mice treated with IR and MK-1775 showed a significantly longer survival
as compared with untreated mice, in contrast to mice treated with MK-1775 or IR alone (log-rank, P < 0.05).
overexpression in the bulk of the pontine tumor as well as
in areas of the pons infiltrated by tumor cells, for example,
in the invasive tumor front surrounding blood vessels.
Finally, WEE1 expression level was found to be consistently lower in brain tissue not affected by the disease,
indicating a therapeutic index and possible tumor-specific
action of drugs inhibiting WEE1.
WEE1 inhibitors can function as enhancers of radiation
responses because they abrogate the G2 checkpoint, thus
preventing IR-induced DNA damage repair (41). To
investigate the possible radiation-enhancing effects of
WEE1 inhibition in DIPG, MK-1775 was selected, given
its proven selective action on WEE1 and its clinical relevance (31, 41). In addition, a previous study reported that
MK-1775 had no toxic effects on normal human astrocytes
(30), and results from a clinical trial indicate that MK-1775
is tolerated by adult patients (31). Recently, preclinical
studies have showed the radiosensitizing effects of MK-
148
Mol Cancer Ther; 12(2) February 2013
1775 in lung cancer (9), pancreatic cancer (29), and glioblastoma cell lines (30). Here, MK-1775 treatment resulted
in an enhancement of the radiation response in primary
DIPG cells, although less pronounced than on E98-FM
glioma cells, which may be at least partly attributable to a
difference in proliferation rate (doubling time of 72 hours
for VUMC-DIPG-A vs. 24 hours for E98-FM; ref. 30).
We also investigated the radiation-enhancing effects of
MK-1775 in the E98-FM DIPG mouse model in vivo. After
injection into the mouse pons, the human E98-FM glioma
cells gave rise to highly infiltrative tumors, accurately
reproducing the invasive phenotype of DIPG, which
represents one of the major challenges in the treatment
of this disease (32). The mean BLI signal, indicative for
E98-FM tumor size, was consistently lower in both the
combined treatment and the irradiated treatment group
as compared with the control group. This effect only
persisted in the combination treatment group over time,
Molecular Cancer Therapeutics
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Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735
WEE1 Inhibition Enhances the Radiation Response in DIPG
resulting in a significant difference in mean BLI between
the irradiated and the combination treatment group at
week 4 after injection of the cells. The group receiving both
IR and MK-1775 showed a significant longer survival than
the nontreated control group, while IR alone did not yield
such a significant survival advantage. This indicates that
combining IR with MK-1775 improves the antitumor
effect of IR alone. Finally, while treatment with MK1775 alone showed lower cell counts in vitro, monotherapy
with MK-1775 in vivo did not result in a significant antitumor effect. In previous in vivo studies, MK-1775 monotherapy resulted in decreased tumor burden (9, 27–30).
Thus, further studies using additional DIPG preclinical
models and optimization of MK-1775 and IR dosing are
needed. Moreover, supratentorial pediatric gliomas,
although potentially representing a separate disease, may
also respond to treatment with WEE1 inhibitors in combination with IR, and a comparison between infratentorial
DIPG and supratentorial pediatric gliomas could be of
importance in the context of drug delivery to these different regions of the brain. The route of administration
may also influence the therapeutic outcome, and it would
be of interest to compare i.p. versus oral administration of
MK-1775 in combination with pharmacodynamic and
pharmacokinetic analyses, because MK-1775 blood level
was found to be undetectable by 10 hours (30). Finally,
previous studies have used subcutaneous tumors to
assess the effects of MK-1775 in vivo (9, 27–30). Therefore,
the limited radiation-enhancing effect observed on DIPG
in vivo may also be attributable to a possible inability of
MK-1775 to cross the blood–brain barrier, which may be
intact in large parts of the DIPG tumor (32).
In conclusion, in this study we showed that WEE1 is
overexpressed in DIPG and its inhibition in vitro and in
vivo resulted in additional antitumor effects when combined with IR. Ultimately, DIPG is a very aggressive and
heterogeneous tumor and the development of a clinical
multitarget approach will be necessary. In this context,
WEE1 inhibition in conjunction with radiotherapy may be
one of the therapeutic strategies used in the treatment
armamentarium needed to treat this at present still fatal
disease.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: V. Caretti, L. Hiddingh, G.J.L. Kaspers, T.
Wurdinger
Development of methodology: V. Caretti, L. Hiddingh, P.W. Koken, T.
Wurdinger
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): V. Caretti, L. Hiddingh, T. Lagerweij, P. Schellen,
P.W. Koken, T. Wurdinger
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V. Caretti, L. Hiddingh, E. Hulleman, D.G.
van Vuurden, G.J.L. Kaspers, T. Wurdinger
Writing, review, and/or revision of the manuscript: V. Caretti, L. Hiddingh, P.W. Koken, E. Hulleman, D.G. van Vuurden, W.P. Vandertop, G.J.
L. Kaspers, D.P. Noske, T. Wurdinger
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Wurdinger
Study supervision: G.J.L. Kaspers, D.P. Noske, T. Wurdinger
Acknowledgments
The authors thank Fred Buijs (Department of Nuclear Medicine & PET
Research), Laurine Wedekind, and Petra van der Stoop (Neuro-oncology
Research Group) for skilled technical support, all from the VU University
Medical Center, Amsterdam, the Netherlands. The authors also kindly
acknowledge Jan Koster (AMC, Amsterdam, the Netherlands) for the
adaptations made in R2.
Grant Support
This research was financially supported by the Semmy foundation (G.J.
L. Kaspers).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received July 16, 2012; revised November 19, 2012; accepted December
6, 2012; published OnlineFirst December 27, 2012.
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Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 14, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735
WEE1 Kinase Inhibition Enhances the Radiation Response of
Diffuse Intrinsic Pontine Gliomas
Viola Caretti, Lotte Hiddingh, Tonny Lagerweij, et al.
Mol Cancer Ther 2013;12:141-150. Published OnlineFirst December 27, 2012.
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