WEE1 Kinase Inhibition Enhances the Radiation Response of Diffuse Intrinsic Pontine Gliomas

Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0735

Molecular Cancer Chemical Therapeutics Therapeutics

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 cancers (7–9). Normal cells have functional cell-cycle Diffuse intrinsic pontine glioma (DIPG) is an almost checkpoints as compared with cancer cells, which often invariably fatal brain neoplasm affecting mainly children, have a deficient G1 arrest due to aberrant p53 signaling with a 2-year survival rate less than 10% (1, 2). Its hall- and, therefore, heavily rely on the G2 checkpoint to repair marks are the specific anatomic location from which it DNA damage caused by irradiation (IR; ref. 10). Abro- originates, the pons (3), its diffuse phenotype, often gation of the G2 checkpoint pushes glioma cells with spreading to the cerebellum and brain areas as far as the unrepaired DNA damage into mitotic catastrophe, result- cerebral hemispheres (4), and its bleak prognosis (3). ing in subsequent cell death (11). Interestingly, in DIPG, Despite various clinical trials, the standard treatment for recent genomic studies have revealed aberrations in genes DIPG patients remains conventional radiotherapy, which regulating the G1 checkpoint, including TP53, MDM2, provides transient neurologic improvement, resulting in a CDKN2A, and ATM (12–19), suggesting a dysfunctional better quality of life, but does not improve probability of G1 arrest in DIPG cells. Therefore, inhibition of WEE1 overall survival (1, 2, 5). Therefore, novel treatment strat- could be a potential strategy to enhance the response to IR egies to increase the efficacy of radiotherapy in DIPG are in DIPG cells. urgently needed. A number of small-molecule compounds that inhibit We have previously shown that inhibition of WEE1 WEE1 have been developed. These include PD0166285 (20, 21), PD0407824 (22, 23), WEE1 inhibitor II, and kinase, one of the main gatekeepers of the G2 cell-cycle checkpoint, is a potential therapeutic target for radio- PHCD (23–25). The most promising WEE1 inhibitor sensitization of adult gliomas (6) and of other type of 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 Authors' Affiliations: Departments of 1Pediatric Oncology, 2Neurosur- tumor growth reduction, increased survival, and gery, 3Radiation Oncology, 4Neuro-oncology Research Group, VU Univer- absence of significant toxicity in several studies using sity Medical Center, Amsterdam, the Netherlands; and 5Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General xenograft animal models (6, 9, 26–30). Moreover, pre- Hospital and Harvard Medical School, Boston, Massachusetts liminary results of a phase 1 study of oral MK-1775 as Both V. Caretti and L. Hiddingh contributed equally to this work. monotherapy and in combination with gemcitabine, cisplatin, or carboplatin reported good tolerance and Corresponding Author: Thomas Wurdinger, VU University Medical Cen- ter, CCA Room 3.36, De Boelelaan 1117, 1081 HV Amsterdam, the Nether- strong target engagement (31). lands. Phone: 31-24447909; Fax: 31-204442126; E-mail: We have previously shown that inhibition of WEE1 [email protected] could function as a potential radiosensitizer of adult doi: 10.1158/1535-7163.MCT-12-0735 gliomas, both in vitro and in vivo. As an extension to these 2012 American Association for Cancer Research. previous results, in this study, we investigated the

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potential radiation-enhancing effects of a more potent and WEE1 inhibitor and IR clinically relevant WEE1 inhibitor, MK-1775, in DIPG cells The WEE1 inhibitor MK-1775 (Axon Medchem) was in culture and in vivo using the E98-Fluc-mCherry (E98- resuspended in dimethyl sulfoxide (DMSO) to a concen- FM) DIPG mouse model, closely resembling the DIPG tration of 100 mmol/L and diluted in PBS for the in vivo phenotype in humans (32). experiments and in medium for the in vitro experiments. Cells were irradiated in a Gammacell 220 Research Irra- Materials and Methods diator (MDS Nordion). Ethics statement All animal experiments were conducted according to Western blotting the guidelines established by the European community Expression levels of WEE1 were assessed by Western and following a protocol (NCH10-05) approved by the blot analysis in both tissue samples and cell lines as institutional ethical committee on animal experiments described previously (6). In brief, after cell lysis (for of the VU University (Amsterdam, the Netherlands). All CDC2-pY15 a phospholysis buffer was used), 30 mg protein patient samples, including the de novo cell line VUMC- was transferred to a polyvinylidene difluoride (PVDF) DIPG-A, were used after appropriate written informed membrane and incubated with the primary antibodies: consent and under approval of the institutional medical mouse anti-WEE1 (1:1,000; Santa Cruz Biotechnology), ethical committee of the VU University Medical Center mouse anti-b-actin (1:10,000; Santa Cruz Biotechnology), (VUmc). The research described here has been con- and rabbit anti-CDC2-pY15 (1:2,000; Abcam), and subse- ducted according to the principles expressed in the quently incubated with horseradish peroxidase (HRP)- Declaration of Helsinki. labeled goat-anti-mouse or HRP-labeled goat-anti-rabbit immunoglobulins (DAKO). Protein detection and visual- In silico analysis of DIPG kinase expression ization was carried out using ECLþ Western Blotting R2, a microarray analysis and visualization platform, Detection Reagents (Pierce). provided by the Department of Oncogenomics of the Academic Medical Centre, Amsterdam, The Netherlands Immunohistochemistry (http://r2.amc.nl), was used to obtain an overview of Paraffin-embedded DIPG tissue samples and kinase mRNA expression in DIPG. A MAS5.0 normalized matched non-neoplastic brain samples were deparaffi- dataset of post-mortem DIPG tissues (ref. 17; n ¼ 27; GSE nized and rehydrated. Endogenous peroxidase was post-mortem GSE26576) was compared with normal brain inhibited by 30-minute incubation in 0.3% H2O2, diluted regions (ref. 33; n ¼ 172; GSE11882), consisting of the in methanol. Antigens were retrieved by boiling in Tris/ hippocampus, entorhinal cortex, superior frontal gyrus, EDTA buffer (pH 9.0) in a microwave for 10 minutes, and postcentral gyrus, and 2 samples of post-mortem followed by washing 3 times in PBS. Slides were incu- normal brainstem from the DIPG dataset (ref. 17; ref. bated with mouse anti-WEE1 (1:50; Cell Signaling Tech- GSE26576). To evaluate statistical significance, a false nology) overnight at 4C. Slides were washed 3 times in discovery rate (FDR)-corrected, moderated t test (Linear PBS and incubated with the secondary antibody, Models for Microarray Data - Limma) was used to com- Envisionþ Poly-HRP immunohistochemistry (IHC) Kit pare mean kinase mRNA expression levels between data- (Immunologic) for 30 minutes at room temperature. sets. An FDR P value less than 0.000005 was considered Positive reactions were visualized by incubation with significant. To compare WEE1 mRNA expression DAB chromogen solution. Slides were counterstained between groups within DIPG dataset and normal brain with hematoxylin, dehydrated, mounted, and analyzed tissue datasets, 1-sided ANOVA was used. by microscopy.

Cells and tissue samples Flow cytometry The primary low-passage VUMC-DIPG-A cells were At 16 hours after treatment, cells were washed twice derived from tumor tissue surgically removed from a with PBS containing 1% FBS and subsequently fixed in patient diagnosed with DIPG at the VUmc. E98 cells were 70% ethanol for 24 hours. Next, the cells were washed obtained from the Radboud University Nijmegen Medical once in PBS containing 1% FBS followed by RNAse A Centre (Nijmegen, the Netherlands; ref. 34) and trans- treatment (0.15 mg/mL) for 20 minutes and subsequent duced with a lentiviral vector containing Fluc and DNA staining with 50 mg/mL propidium iodide (PI) for mCherry at our institution (32). Cells used in this study 30 minutes. Cell-cycle distribution assessment was were not authenticated. The primary VUMC-DIPG-A and conducted using a FacsCalibur Flow Cytometer and E98-FM cells were cultured in Dulbecco’s Modified CellQuest Pro software (Becton-Dickinson). Subse- Eagle’s Medium (DMEM; PAA) containing 10% FBS and quently, data were analyzed using ModfitLT software antibiotics. DIPG tissue and control non-neoplastic brain (Verity Software House). tissue were obtained post-mortem from 5 DIPG patients (VUMC-DIPG-1, 2, 3, 4, and 5; ref. 35), while VUMC- Immunofluorescence staining DIPG-A cells were isolated from surgical specimen after Cells were fixed in 3% paraformaldehyde at different written informed consent. time points post irradiation (15 minutes, 30 minutes, and

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1, 24, 48, and 72 hours). Before staining, the cells were Results rinsed in PBS and permeabilized in PBS containing 0.1% In silico analysis of kinase mRNA expression Trition X-100 for 30 minutes at room temperature and identifies WEE1 as a potential drug target in DIPG blocked in PBS containing 5% FCS. Coverslips were incu- Using the publicly available microarray analysis and bated with both mouse-anti-g-histone-H2AX (1:100; Milli- visualization platform R2 (http://r2.amc.nl), we ana- pore) and rabbit-anti-53BP1 (1:100; Novus Biologicals) in lyzed a DIPG gene expression data set (17) and compared PBS containing 5% FCS overnight at 4C. This was fol- it with nonmalignant brain tissue (33, 36), including 2 lowed by secondary antibody incubation with goat-anti- tissue samples of normal brainstem (17), to identify the top rabbit/Alexa568 immunoglobulins (1:100; Invitrogen) upregulated kinases for this malignancy. To compare the and, subsequently, with rabbit-anti-mouse/fluorescein differential kinase gene expression of cancer versus nor- isothiocyanate (FITC) immunoglobulins (1:100; DAKO) mal samples, the fold-change to nonmalignant brain tis- both in PBS containing 5% FCS for 30 minutes at room sue was determined for all kinases within the dataset. A temperature in the dark. Slips were rinsed in PBS 3 times FDR-corrected moderated t test was used to compare and nuclei were stained with 40, 6-diamidino-2-phenylin- means of kinase gene expression levels between the data- dole (DAPI; 1:10,000) in PBS at room temperature in the sets and a P value less than 0.000005 was considered dark. This was followed by successive rinses in PBS and significant. Analysis of the kinase expression data showed sterile water. The slips were then mounted on glass slides that WEE1 ranked seventh among the overexpressed using Vectashield (Vector Laboratories) and visualized kinases (Table 1). Given our previous experience on the with a Carl Zeiss Axioskop 20 microscope at 100 radiosensitizing effect of WEE1 inhibition in adult glioma objective. and the existence of a selective inhibitor, MK-1775, cur- rently being tested in clinical trials, we decided to further Cell counts investigate WEE1 as a potential target to enhance the IR At 4 days after treatment, cells were fixed with 3.7% response in DIPG cells. Using the R2 platform, we spe- formaldehyde and DNA was stained with DAPI cifically investigated WEE1 mRNA expression in DIPG, (0.3 mg/mL). Cell numbers were assessed by counting low-grade glioma (LGG) in the brainstem, normal brain- the number of DAPI-stained cells using the Acumen Ex3 stem, nonmalignant brain regions, and normal cerebellum laser scanning cytometer (TTP LabTech). (Fig. 1A). Using 1-way ANOVA, WEE1 mRNA levels were determined to be significantly higher in DIPG than In vivo analysis using the orthotopic E98-FM DIPG in different regions of normal control brain. Interestingly, mouse model WEE1 mRNA levels were significantly higher in DIPG Female athymic nude mice (age 6–8 weeks; Harlan, compared with LGG of the brainstem, as we previously Zeist, the Netherlands) were kept under specific path- observed in adult low- and high-grade gliomas (6). ogen-free conditions in air-filtered cages and received food and water ad libitum. E98-FM cells were injected as WEE1 protein is overexpressed in DIPG patient described previously (32). In short, tumors were gen- material and cell lines erated via stereotactic injection of 0.5 106 E98-FM cells Next, we analyzed the protein expression of WEE1 in a volume of 5 mL into the murine pons (coordinates kinase by Western blot analysis in 5 different post-mortem from lambda: 1.0 mm X, 0.8 mm Y, 5.0 mm Z). Tumor DIPG tissues (35) isolated from the pons and matched these growth was monitored twice-weekly by biolumines- to non-neoplastic brain tissues of the same patient (Fig. 1B). cence imaging (BLI), as previously reported (32). At We observed significant overexpression of WEE1 protein in day 7 after intracranial injection, mice were stratified 4 out of 5 tumors compared with the non-neoplastic brain on the basis of BLI signal intensities into 4 treatment tissues. For patient VUMC-DIPG-1, we were not able to groups (n ¼ 12) with comparable mean Fluc activity detect WEE1 protein overexpression in the tumor in the and, then, received MK-1775 (90 mg/kg) or vehicle (PBS pons region, which may be due to the presence of tumor containing 18% DMSO) in a total volume of 200 mLvia necrosis in the sample used. However, we detected signif- intraperitoneal injection every other day for a total of 6 icant WEE1 protein expression in VUMC-DIPG-1 tissue injections per mouse. At day 8, mice received head-only isolated fromthefrontal lobeofthe samepatient(Fig.1B).In IR with a single dose of 2 Gy using Clinac D/E (Varian addition, we analyzed WEE1 protein expression by West- Medical Systems), as described previously (32). Mice ern blot analysis in a primary DIPG cell line and in E98-FM were sacrificed after humane endpoints were reached glioma cells (32), again showing significant WEE1 protein via sedation (1.5 L O2/min and 2.5% isoflurane), fol- levels (Fig. 1C). Furthermore, by IHC we found WEE1 lowed by cervical dislocation. To assess significant dif- protein overexpression, as shown by a clear nuclear stain- ferences between treatment groups mice with BLI ing, in DIPG tumors located in the pons (Fig. 1D–G and I–K, values within each treatment group outside the mean left) as compared with matched nonneoplastic brain tissue 2 SDrangewereexcludedfromfurtheranalysis. (Fig. 1L). In particular, high levels ofWEE1 were detectedin Next, BLI signals were log10-transformed and unpaired the tumor bulk (Fig. 1D, asterisk), as well as in the infiltra- Student t test was carried out. A difference was con- tive DIPG component (Fig. 1E), and in tumor cells sur- sidered significant when P < 0.05. rounding the blood vessels (Fig. 1F). Nuclei in the

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Table 1. Top 20 upregulated kinases in DIPG

Expression Fold normal Expression increase P value Rank Gene Name Probeset brain (log2) DIPG (log2) (log2) (FDR) 1 TOP2A Topoisomerase (DNA) II alpha 201292_at 1,05 7,85 7,45 3,30E-68 2 MELK Maternal embryonic leucine 204825_at 1,61 7,49 4,66 6,64E-62 zipper kinase 3 BUB1 Budding uninhibited by 209642_at 1,98 6,69 3,38 3,70E-44 benzimidazoles 1 homolog 4 TTK TTK protein kinase 204822_at 2,12 6,83 3,23 2,78E-51 5 PBK PDZ binding kinase 219148_at 2,46 7,61 3,10 5,90E-61 6 OSR1 Odd-skipped related 1 (Drosophila) 228399_at 1,87 5,66 3,03 7,03E-22 7 WEE1 WEE1 homolog 212533_at 3,60 8,81 2,45 2,60E-46 8 NEK2 NIMA (never in mitosis 204641_at 3,44 6,74 1,96 8,43E-24 gene a)-related kinase 2 9 STK33 Serine/threonine kinase 33 228035_at 3,18 6,14 1,93 2,03E-19 10 TEX14 Testis expressed 14 221035_s_at 2,92 5,50 1,88 5,24E-14 11 CHEK1 CHK1 checkpoint homolog (S. pombe) 205394_at 2,90 5,37 1,85 2,57E-30 12 AURKB Aurora kinase B 209464_at 3,05 5,32 1,74 2,45E-20 13 BUB1B Budding uninhibited by benzimidazoles 203755_at 3,98 6,84 1,72 4,08E-32 1 homolog beta (yeast) 14 HK2 Hexokinase 2 202934_at 5,54 8,95 1,62 2,81E-32 15 CHEK2 CHK2 checkpoint homolog (S. pombe) 210416_s_at 3,51 5,43 1,55 3,41E-15 16 AURKA Aurora kinase A 204092_s_at 4,26 6,57 1,54 4,37E-33 17 ROR2 Receptor tyrosine kinase-like 205578_at 2,41 3,71 1,54 3,62E-06 orphan receptor 2 18 PLAU Plasminogen activator, urokinase 205479_s_at 3,52 5,41 1,54 2,29E-14 19 DYRK3 Dual-speciﬁcity tyrosine-(Y)- 210151_s_at 4,28 6,32 1,48 6,32E-15 phosphorylation regulated kinase 3 20 MASTL Microtubule-associated 228468_at 4,21 6,13 1,46 3,76E-47 serine/threonine kinase-like

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 WEE1 kinase activity in these cells. The effect of MK-1775- nuclear staining (Fig. 1G). Notably, WEE1 was found to mediated WEE1 inhibition on the IR-induced G2 arrest be overexpressed in tumor cells invading brain areas was determined by cell-cycle analysis using ﬂow cytome- beyond the brainstem (Fig. 1H and I–K, right). Finally, try (Fig. 2C). In both cell lines, IR at 6 Gy induced a G2–M clear WEE1 nuclear staining was also detected in tumor arrest after 16 hours, indicated by an accumulation of cells tissue derived from the orthotopic E98-FM DIPG mouse with doubled DNA content and indicative for a dysfunc- model (Fig. 1M, arrow). tional G1 arrest, which was reduced on treatment with 0.1 mmol/L MK-1775. Subsequently, we examined the effect MK-1775 inhibits WEE1-regulated CDC2, IR- of WEE1 inhibition on the repair of IR-induced DNA induced G2 arrest, and repair of IR-induced DNA damage within these cells (Fig. 2D and E). The IR-induced damage in DIPG cells DNA damage was visualized using the DNA double- E98-FM and VUMC-DIPG-A cells were used to study strand break (DSB) markers gH2AX and 53BP1 at 15 the effect of the WEE1 kinase inhibitor MK-1775 in vitro. minutes, 30 minutes, and 1, 24, 48, and 72 hours after Because WEE1 phosphorylates CDC2 on being subjected treatment with IR at 4 Gy in the presence or absence of 0.1 to IR (37–39), we conducted Western blot analysis for mmol/L MK-1775. As shown in Fig. 2D gH2AX and 53BP1 phosphorylated CDC2 (Y15) 16 hours after treatment with colocalize. These results indicate DNA damage in both 0.1 mmol/L MK-1775 and IR (Fig. 2B). IR at 6 Gy resulted in cell lines after IR treatment as compared with untreated increased CDC2-pY15 levels, which were reduced on treat- cells. Cells treated with IR showed fast onset of DSBs (as ment with MK-1775, indicating functional inhibition of early as 15 minutes after IR); however, a decreased

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WEE1 Inhibition Enhances the Radiation Response in DIPG

AB C 1,400 1,200 1,000 800 600 400 Frontal Lobe Frontal Healthy brain Healthy VUMC-DIPG-1 VUMC-DIPG-2 VUMC-DIPG-4 VUMC-DIPG-5 Matched CTRL-1 VUMC-DIPG-3 Matched CTRL-4 Matched CTRL-2 Matched CTRL-3 Matched CTRL-5 VUMC-DIPG-1 E98-FM VUMC-DIPG-A Healthy brain Healthy 200 WEE1 mRNA Expression WEE1 0 β

LGG -Actin DIPG ellum Brain C.b Brainstem

D EF GH *

I JKLM

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 cells in vitro. A clonogenic survival assay is the gold in cells treated only with IR, indicating the occurrence of standard for this purpose (40), but because these cells do DNA damage repair within these cells (Fig. 2D and E). not form appreciable colonies, we monitored cell viability Notably, although no differences in the number of IR- by counting DAPI-stained cells using a laser scanning induced DSB foci were observed at the early time points cytometer. We treated E98-FM cells with IR at 4 Gy in after IR, the cells treated with both IR and MK-1775 the presence or absence of 0.1 mmol/L MK-1775. After 4 showed persistence of DNA damage over time, as indi- days, cells were stained with DAPI and cell numbers were cated by a higher number of gH2AX and 53BP1 foci in counted. Treatment of the E98-FM cells with 0.1 mmol/L these cells compared with cells treated with IR at 4 Gy MK-1775 or IR monotherapy resulted in a signiﬁcant but alone (Fig. 2D and E; E98-FM and VUMC-DIPG-A, P < moderate decrease in cell viability compared with 0.001). These results show that MK-1775 is capable of untreated cells. Treatment with MK-1775 in combination functional WEE1 inhibition and, thereby, of attenuating with IR resulted in radiosensitization of E98-FM cells (P < the IR-induced G2 arrest in G2-dependent E98-FM and 0.001; Fig. 3A and B). Next, we analyzed the effect of 0.1 VUMC-DIPG-A cells and, subsequently, of inhibiting the mmol/L MK-1775, in combination with IR at 4 Gy, on the repair of IR-induced DNA damage in these cells. cell viability of primary VUMC-DIPG-A cells. Both 0.1 mmol/L MK-1775 and IR monotherapy show only a lim- MK-1775 enhances the radiation response in ited effect on cell viability of VUMC-DIPG-A. However, cultured DIPG cells combined treatment with MK-1775 and IR resulted in Next, we determined whether the attenuation of IR- additional diminished cell viability, compared with IR P induced G2 arrest by MK-1775 reduces viability of these alone ( < 0.05; Fig. 3C and D).

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4 Gy 4 Gy + MK-1775 γ γ A MK-1775 D -H2AX 53BP1 DAPI Merge -H2AX 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 CDC2-pY15 1 h

β-Actin C 24 h E98-FM VUMC-DIPG-A

G2–M: 18% G2–M: 23% CTRL 48 h Counts

G2–M: 18% G2–M: 19% 72 h MK-1775 Counts

E 50 50 E98-FM VUMC-DIPG-A * CTRL CTRL G2–M: 63% G2–M: 41% 40 40 IR MK-1775 MK-1775

Counts 30 30 H2AX foci/nucleus -H2AX foci/nucleus - γ γ *** *** 20 *** 20 *** G –M: 26% G –M: 22% *** *** 2 2 10 10 Avg number of IR+MK-1775 Avg number of

Counts 0 0

1 h 1 h 24 h 48 h 72 h 24 h 48 h 72 h No IR No IR 15 min 30 min 15 min 30 min 2N 2N 4N 4N Time after radiation 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 and MK-1775-treated mice, while tumor growth was orthotopic E98-FM DIPG mouse model significantly delayed in irradiated and irradiated MK- We used the orthotopic E98-FM DIPG mouse model 1775-treated mice compared with the control group (32) to study the radiation-enhancing effects of MK-1775 (weeks 2 and 3, and weeks 2, 3, and 4, respectively; Fig. in vivo. E98-FM glioma cells were stereotactically injected 4A and B). Moreover, the mean BLI signal of the irradiated in the pons of nude mice. Tumor growth was monitored MK-1775-treated group was significantly decreased com- twice a week using the BLI intensity of the photon activity pared with the irradiated group at week 4 after injection of firefly luciferase encoded by the E98-FM cells. Seven of the cells (P < 0.05). In addition, survival analysis days after intracranial injection of the cells, mice were showed a significant advantage for combining IR with randomized into 4 groups (n ¼ 12 per group) on the basis MK-1775 over the vehicle-treated control group (log-rank, of BLI intensity. Two groups of mice received MK-1775 P < 0.05), while the groups receiving only MK-1775 or IR intraperitoneally (i.p.; 90 mg/kg) and 2 groups received showed no significant effect on survival as compared with vehicle (DMSO diluted in PBS) i.p, at 7, 9, 11, 13, 15, and the untreated mice (Fig. 4C). These results indicate that 17 days after injection of the cells. One group of MK-1775 pharmacologic targeting of WEE1 with MK-1775 in com- and vehicle-treated mice was irradiated with 2 Gy at day bination with IR delays the growth of E98-FM DIPG 8 after injection of the cells. BLI revealed significant tumors in vivo, although the effects observed under these tumor progression in both nonirradiated vehicle-treated conditions were modest.

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A *** *** C *** *** *** ***

100 *** 100 *

10 10 Relative % cell viability (log scale) Relative % cell (logviability scale) 1 1

5 IR IR CTRL CTRL MK-177 MK-1775 +MK-1775 BDIR IR+MK-1775

CTRL MK-1775 CTRL MK-1775

RI +RI M -K 71 75 IR IR+ KM -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 Kinase expression data analysis showed WEE1 kinase DIPG is the most deadly pediatric malignancy. Radio- to be in the top-10 overexpressed kinases in a dataset of therapy remains the standard treatment, although this DIPG samples compared with normal brain tissues. only causes temporary tumor regression. Despite Although the other overexpressed kinases could also be numerous clinical trials, no therapeutic agent has, thus of value for investigating therapeutic targeting in DIPG, far, shown a survival benefit for DIPG patients (2). we analyzed WEE1 kinase, because it was previously Nonetheless, the clinical use of these agents was not shown to be a potential target for the radiosensitization based on translational studies due to the lack of DIPG of adult glioma cells (6), and because a clinically relevant tissue and in vitro and in vivo models available for WEE1 inhibitor was available (31). We found WEE1 to be preclinical research. Here, we present a translational highly overexpressed in the DIPG tissues analyzed. Inter- study with WEE1 target assessment on DIPG tissue, estingly, WEE1 was also overexpressed in tumor tissue functional experiments using primary DIPG cells in invading brain areas beyond the brainstem (including vitro and the orthotopic E98-FM DIPG model (32) to cerebellum, insula, and frontal lobe), indicating that show the radiation-enhancing effects of WEE1 inhibi- the infiltrative DIPG components can also be potentially tion in vivo. targeted by WEE1 inhibitors. IHC confirmed WEE1

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A *** *** * 7 ** *** *

5 fluc activity fluc 10 4 Log

IR IR IR 775 IR CTRL CTRL 1 CTRL CTRL MK-1775 MK-1775 MK-1775 MK-1775 IR+MK-1775 IR+MK- IR+MK-1775 IR+MK-1775

Week 1 Week 2 Week 3 Week 4 B C 123 4 100 CTRL MK-1775 CTRL 80 IR IR+MK-1775

MK-1775 60

Survival 40 IR 20 CTRL vs. IR+MK -1775 P < 0.05 IR+MK-1775 0 15 20 25 30 35 40 Days after surgery

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 1775 in lung cancer (9), pancreatic cancer (29), and glio- in areas of the pons infiltrated by tumor cells, for example, blastoma cell lines (30). Here, MK-1775 treatment resulted in the invasive tumor front surrounding blood vessels. in an enhancement of the radiation response in primary Finally, WEE1 expression level was found to be consis- DIPG cells, although less pronounced than on E98-FM tently lower in brain tissue not affected by the disease, glioma cells, which may be at least partly attributable to a indicating a therapeutic index and possible tumor-specific difference in proliferation rate (doubling time of 72 hours action of drugs inhibiting WEE1. for VUMC-DIPG-A vs. 24 hours for E98-FM; ref. 30). WEE1 inhibitors can function as enhancers of radiation We also investigated the radiation-enhancing effects of in vivo responses because they abrogate the G2 checkpoint, thus MK-1775 in the E98-FM DIPG mouse model . After preventing IR-induced DNA damage repair (41). To injection into the mouse pons, the human E98-FM glioma investigate the possible radiation-enhancing effects of cells gave rise to highly infiltrative tumors, accurately WEE1 inhibition in DIPG, MK-1775 was selected, given reproducing the invasive phenotype of DIPG, which its proven selective action on WEE1 and its clinical rele- represents one of the major challenges in the treatment vance (31, 41). In addition, a previous study reported that of this disease (32). The mean BLI signal, indicative for MK-1775 had no toxic effects on normal human astrocytes E98-FM tumor size, was consistently lower in both the (30), and results from a clinical trial indicate that MK-1775 combined treatment and the irradiated treatment group is tolerated by adult patients (31). Recently, preclinical as compared with the control group. This effect only studies have showed the radiosensitizing effects of MK- persisted in the combination treatment group over time,

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WEE1 Inhibition Enhances the Radiation Response in DIPG

resulting in a significant difference in mean BLI between multitarget approach will be necessary. In this context, the irradiated and the combination treatment group at WEE1 inhibition in conjunction with radiotherapy may be week 4 after injection of the cells. The group receiving both one of the therapeutic strategies used in the treatment IR and MK-1775 showed a significant longer survival than armamentarium needed to treat this at present still fatal the nontreated control group, while IR alone did not yield disease. such a significant survival advantage. This indicates that combining IR with MK-1775 improves the antitumor Disclosure of Potential Conflicts of Interest effect of IR alone. Finally, while treatment with MK- No potential conflicts of interest were disclosed. 1775 alone showed lower cell counts in vitro, monotherapy with MK-1775 in vivo did not result in a significant anti- Authors' Contributions tumor effect. In previous in vivo studies, MK-1775 mono- Conception and design: V. Caretti, L. Hiddingh, G.J.L. Kaspers, T. Wurdinger therapy resulted in decreased tumor burden (9, 27–30). Development of methodology: V. Caretti, L. Hiddingh, P.W. Koken, T. Thus, further studies using additional DIPG preclinical Wurdinger Acquisition of data (provided animals, acquired and managed patients, models and optimization of MK-1775 and IR dosing are provided facilities, etc.): V. Caretti, L. Hiddingh, T. Lagerweij, P. Schellen, needed. Moreover, supratentorial pediatric gliomas, P.W. Koken, T. Wurdinger although potentially representing a separate disease, may Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): V. Caretti, L. Hiddingh, E. Hulleman, D.G. also respond to treatment with WEE1 inhibitors in com- van Vuurden, G.J.L. Kaspers, T. Wurdinger bination with IR, and a comparison between infratentorial Writing, review, and/or revision of the manuscript: V. Caretti, L. Hid- DIPG and supratentorial pediatric gliomas could be of dingh, P.W. Koken, E. Hulleman, D.G. van Vuurden, W.P. Vandertop, G.J. L. Kaspers, D.P. Noske, T. Wurdinger importance in the context of drug delivery to these dif- Administrative, technical, or material support (i.e., reporting or orga- ferent regions of the brain. The route of administration nizing data, constructing databases): T. Wurdinger may also influence the therapeutic outcome, and it would Study supervision: G.J.L. Kaspers, D.P. Noske, T. Wurdinger be of interest to compare i.p. versus oral administration of MK-1775 in combination with pharmacodynamic and Acknowledgments The authors thank Fred Buijs (Department of Nuclear Medicine & PET pharmacokinetic analyses, because MK-1775 blood level Research), Laurine Wedekind, and Petra van der Stoop (Neuro-oncology was found to be undetectable by 10 hours (30). Finally, Research Group) for skilled technical support, all from the VU University Medical Center, Amsterdam, the Netherlands. The authors also kindly previous studies have used subcutaneous tumors to acknowledge Jan Koster (AMC, Amsterdam, the Netherlands) for the assess the effects of MK-1775 in vivo (9, 27–30). Therefore, adaptations made in R2. the limited radiation-enhancing effect observed on DIPG in vivo may also be attributable to a possible inability of Grant Support MK-1775 to cross the blood–brain barrier, which may be This research was financially supported by the Semmy foundation (G.J. L. Kaspers). intact in large parts of the DIPG tumor (32). The costs of publication of this article were defrayed in part by the In conclusion, in this study we showed that WEE1 is payment of page charges. This article must therefore be hereby marked overexpressed in DIPG and its inhibition in vitro and in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vivo resulted in additional antitumor effects when combined with IR. Ultimately, DIPG is a very aggressive and Received July 16, 2012; revised November 19, 2012; accepted December heterogeneous tumor and the development of a clinical 6, 2012; published OnlineFirst December 27, 2012.

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Viola Caretti, Lotte Hiddingh, Tonny Lagerweij, et al.

Mol Cancer Ther 2013;12:141-150. Published OnlineFirst December 27, 2012.

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