Pharmacological Inhibition of the Protein Kinase MRK/ZAK ... · part of the conserved kinase active site domain. , unique Cys 22 in MRK. C, amino acid sequence alignment of the indicated

Small Molecule Therapeutics

Pharmacological Inhibition of the Protein KinaseMRK/ZAK Radiosensitizes MedulloblastomaDaniel Markowitz1, Caitlin Powell1, Nhan L. Tran2, Michael E. Berens2, Timothy C. Ryken3,Magimairajan Vanan4, Lisa Rosen5, Mingzhu He6, Shan Sun6, Marc Symons1,Yousef Al-Abed6, and Rosamaria Ruggieri1

Abstract

Medulloblastoma is a cerebellar tumor and the most com-mon pediatric brain malignancy. Radiotherapy is part of thestandard care for this tumor, but its effectiveness is accompa-nied by significant neurocognitive sequelae due to the delete-rious effects of radiation on the developing brain. We havepreviously shown that the protein kinase MRK/ZAK protectstumor cells from radiation-induced cell death by regulatingcell-cycle arrest after ionizing radiation. Here, we show thatsiRNA-mediated MRK depletion sensitizes medulloblastomaprimary cells to radiation. We have, therefore, designed andtested a specific small molecule inhibitor of MRK, M443, whichbinds to MRK in an irreversible fashion and inhibits its activity.We found that M443 strongly radiosensitizes UW228 medul-

loblastoma cells as well as UI226 patient–derived primary cells,whereas it does not affect the response to radiation of normalbrain cells. M443 also inhibits radiation-induced activationof both p38 and Chk2, two proteins that act downstream ofMRK and are involved in DNA damage–induced cell-cyclearrest. Importantly, in an animal model of medulloblastomathat employs orthotopic implantation of primary patient–derived UI226 cells in nude mice, M443 in combination withradiation achieved a synergistic increase in survival. Wehypothesize that combining radiotherapy with M443 will allowus to lower the radiation dose while maintaining therapeuticefficacy, thereby minimizing radiation-induced side effects.Mol Cancer Ther; 15(8); 1799–808. �2016 AACR.

IntroductionRadiotherapy is commonly used as part of cancer treat-

ment, and it is important in the management of 40% ofpatients who are cured of cancer (1). The therapeutic benefitsof radiotherapy are, however, accompanied by late toxicitythat severely affects quality of life, especially in pediatricpatients. Specifically, in the treatment of medulloblastoma,the most common malignant pediatric brain tumor, radia-

tion, together with high-dose chemotherapy, has improved 5-year survival rates in patients (2). However, it also causesserious deleterious effects that include neurocognitive deficits,developmental problems, and secondary malignancies in themajority of survivors (3–6). Identifying new approaches thatwould allow reduction of the total radiation dose in thesetreatments without compromising therapeutic efficacy is crit-ical for improving tumor management and quality of life.Radiosensitizers may offer this opportunity by increasing theefficacy of radiotherapy while reducing effects on the normaldeveloping brain.

The protein kinaseMRK (mixed lineage kinase–related kinase),also known as ZAK (sterile alpha motif and leucine zipper con-taining kinase AZK; referred to as MRK in this study; ref. 7),belongs to the family of MAPK kinase kinase (MAPKKK) and hasclose homology to the MLK proteins (7–10). MRK has two splicevariants, MRKa andMRKb, which share some functions but differin other regards. In particular, while MRKa overexpression pro-motes tumor growth (11), MRKb has been shown to have animportant role in the response to DNA damage and in the S andG2–M checkpoints (12). At the mRNA level, the two MRK spliceforms are ubiquitously expressed (7, 8), although the MRKbmRNA is more abundant than the MRKa mRNA. Recently,however, the MRKa mRNA has been shown to be abnormallyspliced in gastric tumors as well as in colorectal, bladder, andbreast cancers with consequent overexpression of the respectiveprotein (13). MRKb is activated by stress (9, 14–16), includingionizing radiation (IR; refs. 7, 12, 17) uponwhich it contributes toactivation of Chk2 and p38 proteins, which leads to IR-inducedcell-cycle arrest. MRK silencing caused both failure to arrest cell-cycle progression in response to IR and increased killing byradiation (12).

1Center of Oncology and Cell Biology, Feinstein Institute, Manhasset,New York. 2Translational Genomics Research Institute, Phoenix, Ari-zona. 3Department of Neurosurgery, Kansas University Medical Cen-ter, Kansas City, Kansas. 4Section of Pediatric Hematology/Oncology/BMT, University of Manitoba, Winnipeg, Canada. 5Biostatistic Unit,Feinstein Institute, Manhasset, New York. 6Center for Molecular Inno-vation, Feinstein Institute, Manhasset, New York.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

D. Markowitz and C. Powell are co-first authors of this article.

M. Symons, Y. Al-Abed, and R. Ruggieri are co-senior authors of this article.

The following authors, Yousef Al- Abed, Marc Symons and Rosamaria Ruggieri,have submitted a US patent application: Treatment of solid tumors by inhibitingMRK/ZAK (#50425/507) 2015. This application, however, does not alter theauthors' adherence to all the Molecular Cancer Therapeutics policies on sharingdata and materials.

CorrespondingAuthors:Rosamaria Ruggieri, The Feinstein Institute for MedicalResearch, 350 Community Drive, Manhasset, NY 11030. Phone: 516-562-3410;Fax: 519-562-1022; E-mail: [email protected]; and Marc Symons,[email protected]

doi: 10.1158/1535-7163.MCT-15-0849

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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on July 15, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 20, 2016; DOI: 10.1158/1535-7163.MCT-15-0849

http://mct.aacrjournals.org/

In this study, we examine the role of MRK as a radiosensitiza-tion target in medulloblastoma. In addition, we describe a novelspecific small molecule that we have designed to inhibit MRKactivity and show that it works as an effective radiosensitizer in anorthotopic xenograft model of medulloblastoma.

Materials and MethodsCell lines and transfections

The humanmedulloblastoma cell line UW228 (18) was kindlyprovided in 2007 by Dr. James Rutka, Hospital for Sick Children,Toronto, Canada, and it was authenticated by PCR. Cells werecultured inDMEMcontaining 10%FBS, 1mmol/L glutamine and1% penicillin/streptomycin. UMDI cells are a derivative of theosteosarcoma cell line U2-OS, described in Korkina and collea-gues (19). In UMDI cells, expressing plasmid pC4-Fv1E-MRK,MRK can be activated by induced homodimerization with thedrug AP20187 (Clontech). The UMDI cells are cultured inMcCoymedia supplemented with 10% FBS, 0.6 mg/mL puromycin,50 IU/mL penicillin, 50 mg/mL streptomycin, and 2 mmol/LL-glutamine. The K-562 and 4T1 cell lines were purchased fromATCCand cultured according to distributor's protocol. The humanneural stem cells (NSC) were purchased from Invitrogen (ThermoFisher Scientific) and cultured in StemPro media (Life Technolo-gies), as directed by the manufacturer. Human astrocytes werepurchased from Gibco (Life Technologies) and cultured in Gibcoastrocyte medium.

The MRK and control luciferase siRNAs sequences were previ-ously described (12) and transfected at a concentration of10 nmol/L using Dharmafect #1 (Dharmacon) as recommendedby the manufacturer.

Human primary tumor cells and medulloblastoma xenograftmodel

The primary UI226 medulloblastoma cells are patient-derived xenografts that were established by Dr. Timothy Ryken,University of Kansas Medical Center (Kansas City, KS). UI226were largely propagated as flank cultures in nude mice andcultured in StemPro media (Life Technologies) for less than 3weeks before intracranial injections.

All animal work was carried out in accordance with NIHguidelines and was approved by the Institutional Animal Careand Use Committee of the Feinstein Institute in Manhasset, NY.Medulloblastoma cells (5.0 � 105 UI226 in 5 mL of StemPromedium) were injected over 5 minutes into the cerebellum of4-week-old athymic female mice (Taconic), using a stereotacticframe and the following coordinates: 2 mm to the right and1 mm posterior to the lambda, using a sterile dental drill. TheHamilton 10-mL syringe loaded with cells was lowered 3 mmunder the surface of the brain and then lifted for 0.5 mm tocreate a pocket for the cells. Three weeks after tumor cellimplantation and approximately 2 weeks before the animalsbecame moribund, treatment was started by implantation of anosmotic pump (Alzet, Durect) filled with either 0.05 mg/mLsolution of M443 or vehicle (0.01% DMSO in PBS) andimplanted in a subcutaneous pocket on the dorsal flank of theanimal. A catheter with attached cannula delivered the drugintracranially and directly into the tumor over a period of 2weeks, at a steady rate of 0.25 mL/hour. Irradiation of the micehead was initiated 2 days after pump implantation and con-ducted over 2 days (two fractions of 3 Gy each). It wasperformed using a biologic irradiator (RS2000, Rad SourceTechnologies), whereas the rest of the body was shielded.

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MRK downregulation radiosensitizesmedulloblastoma cells and inhibitsdownstream signals. UW228 cells weretransfectedwith luciferase control (Luc)or MRK siRNAs, exposed to theindicated doses of radiation and cellsurvival was determined by the MTTassay (A) or by the clonogenic assay (B)as described in Materials and Methods.The Western blot analysis shows atypical level of MRK silencing. Data arethe average � SEM of threeindependent experiments. P values forall treatment points were <0.005.C, forty-eight hours after transfectionwith the respective siRNAs, cells wereexposed to the indicated doses ofradiation. Cell lysates were processedfor Western blotting with the indicatedantibodies. Histograms showquantification of phospho-Chk2 signalsfrom 3 independent experiments � SE.Data represent Chk2 specific activity,that is, p-Chk2/Chk2 ration. As thevalues of p-Chk2 in the absence ofradiation are very low and in someexperiments undetectable by Westernblot analysis, the Chk2 specific activitydata at the 1-hour time point for theluciferase siRNA control cells werechosen to normalize the other data.

Markowitz et al.

Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1800




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*sp|Q9NYL2_MRK|16-48 LQFFENCGGG SFGSVYRAK-sp|P00519_ABL|242-274 ITMKHKLGGG QYGEVYE---sp|P15056_BRAF|457-486 ITVGQRIGSG SFGTVYKGK-sp|P16234_PGFAR|593-630 LVLGRVLGSG AFGKVVEGTAsp|P09619_PGFRB|600-637 LVLGRTLGSG AFGQVVEATAsp|P04049_RAF1|349-378 VMLSTRIGSG SFGTVYKGK-sp|P12931_SRC|270-301 LRLEVKLGQG CFGEVWM---sp|P35968_VGFR2|834-871 LKLGKPLGRG AFGQVIEADAsp|Q08345_DDR1|610-658 LRFKEKLGEG QFGEVHLCEVsp|Q16832_DDR2|563-611 LTFKEKLGEG QFGEVHLCEVsp|P17948_FLT1|827-864 LKLGKSLGRG AFGKVVQASAsp|P36888_FLT|610-647 LEFGKVLGSG AFGKVMNATAsp|P35916_FLT4|845-882 LHLGRVLGYG AFGKVVEASAsp|P10721_cKIT|589-626 LSFGKTLGAG AFGKVVEATAsp|O94804_LOK|36-68 WEIVGELGDG AFGKVYKAK-sp|P35590_TIE1|839-873 ITFEDLIGEG NFGQVIRAM-

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Figure 2.

Design of the inhibitor of MRK, M443.A,UMDI cells that express homodimerizable MRKwere pretreated for 1 hour with 0.5 mmol/L of the indicated inhibitors (top) orat the indicated concentration (bottom) in the serum-free medium and then subjected to MRK activation with the homodimerizer AP20187 (AP) for 1 hourbefore harvesting forWestern blotting with the indicated antibodies. B, amino acid sequence alignment of the indicated proteins (known targets of nilotinib) acrosspart of the conserved kinase active site domain. � , unique Cys 22 in MRK. C, amino acid sequence alignment of the indicated proteins (MRK homologs).D, ribbon representation of the structure of MRK homology model in complex with compound M443 (magenta). E, close-up view of the binding site of M443 incomparison of nilotinib (green). M443 is illustrated after forming a covalent bond with cysteine 22. The nilotinib structure was extracted from c-Abl crystalstructure (PDB:3CS9). MRK residues are shown in gray. Nonpolar hydrogens are hidden. F, nilotinib structure. G, M443 structure.

Radiosensitization of Medulloblastoma by Inhibition of MRK

www.aacrjournals.org Mol Cancer Ther; 15(8) August 2016 1801




Western blot analysis and antibodiesCells were processed for Western blotting as previously

described (19). The MRK mAb, 4–23, was previously described(7). The MRK phospho-specific mAb, A12, was generated againstthe following di-phospho MRK peptide: FHNHpTTHMpS-LVGTFP. The b-tubulin antibody was from Millipore; the Chk2,P-Chk2, c-Abl, and P-cAbl antibodies were from Cell SignalingTechnology. The Hu-HLA antibody was from Abcam. TheP-MPM2 antibody was purchased from Millipore.

Cell viability and colony formation assayTwo days after transfection with siRNAs, medulloblastoma

cells were seeded in a 96-well plate, and the following day, theywere exposed to different doses of radiation. Cell viability wasdetermined 72 hours after radiation treatment by MTT (Sigma)absorbance at 595 nm. For cells that were treated with M443, 6hours after treatment with different concentrations of the drug orvehicle control, they were exposed to radiation and processed forthe MTT assay 72 hours later as above.

Clonogenic survival was determined using a colony formationassay. Five hundred cells were seeded in 6-cm dishes 2 days aftersiRNA transfections. The following day, cells were exposed to thedifferent doses of radiation using a biologic irradiator (RS2000,Rad Source Technologies) and cultured for 7 days with mediachanges every other day. Subsequently, cells were fixed in 3.7%formaldehyde in PBS and stained with 0.2% (w/v) sulforhoda-mine B (SRB) dye in 1% acetic acid for 20 minutes. The disheswere washed with 1% glacial acetic acid and allowed to dry.Colonies containing more than 50 cells were counted, and theresults were used to calculate the surviving fractions. For thetreatment with M443 or vehicle control, 24 hours after seeding,cells were exposed to the drug for 6 hours and subsequently toradiation. Colonies were fixed and processed as described above.The dose enhancement factor (DEF) was calculated from thedose–response curves, as the ratio between the dose reducingsurvival to 10% for radiation alone and that for radiation of MRKsiRNA–transfected or M443-treated cells.

M443 synthesisM443 was synthesized in 7 steps with a total yield of 3%.

Methyl 3-amino-4-methylbenzoate (Aldrich CAS :18595-18-1)was reacted with cyanamide to obtain the guanidine M439 at90% yield; the reaction of compound A (CAS: 858643-92-2)with N,N-dimethylformamide dimethyl acetal yielded M422.By refluxing M422 with M439 in absolute ethanol, M440 wasobtained with a yield of 75%. After saponification of ethyl esterwith NaOH in hot aqueous ethanol, M441 was obtained andcoupled with compound C (CAS-641571-11-1) employingDECP (diethyl cyanophosphate) to provide the amide M442;after deprotection with acid, coupling with acid chloridescompleted the synthesis of M443.

MRK homology model and M443 covalent dockingThe MRK sequence was obtained from UniProt (ID: Q9NYL2).

A homology model was produced for MRK by alignment with thecAbl crystal structure (PDB: 3CS9) followedby three-dimensionalmodel building and energy minimization using MOE (MolecularOperating Environment, 2014.0901; Chemical ComputingGroup Inc.). A database of 10 distinct protein models was gen-erated and showed good overall alignment with the a-carbon

backbone of cAbl template (rmsdeviation from0.37–0.43Å). Thephi/psi backbone angles and side chain rotamers were evaluatedto discard structures with high-energy, disallowed bonds and thebest model was selected for the following studies.

The MRK homology model obtained from the previous stepwas subjected inMaestro (v. 10.2, Schr€odinger, LLC, 2015).M443structure was constructed in 2D Sketcher and subsequently pre-pared by LigPrepmodule (version 3.4, Schr€odinger, LLC, 2015) inMaestro. The CovDock docking program (20) within theSchr€odinger suite was employed for M443 and MRK covalentdocking. Cysteine 22 on MRK was targeted as the reactive residueto undergo Michael addition with compoundM443. The rankingof poses by affinity score calculates the average of the pre-reactedand post-reacted Glide score for the given pose (20). The topranking CovDock affinity pose was chosen as the final pose ofM443.

ImmunofluorescenceUW228 cells were seeded at 70,000 cells/well on coverslips in a

24-well plate. The following day, cells were treated with M443 orvehicle and 3 hours later exposed to 6 Gy of radiation. Cells weresubsequently fixed at different time points and processed forimmunofluorescence as previously described (21) with the P-MPM2 antibody (Millipore) and counterstained with DAPI.Images were collected using a Zeiss axiovert 200M invertedmicroscope, equipped with a 20� objective (0.3 NA) and a ZeissAxiocamMR camera running on Axiovision software.

Statistical analysisTwo-way ANOVA was used to compare the mean of each

outcome between groups of interest and either radiation dose ortime. The interaction term between group and dose/time was alsoexamined. If a significant difference between means was found,then multiple comparisons between pairs were computed. TheTukey–Kramer method was used to adjust for multiple compar-isons. Results were considered significant at a significance level ofP < 0.05. Analyses were conducted using SAS version 9.4 (SASInstitute, Inc.). The Kaplan–Meier estimate and a log-rank (Man-tel–Cox) test were used to generate survival curves with theGraphPad Prism 6.0 software. To determine whether there wasa synergistic effect between M443 and radiation treatment (IR),the null hypothesis of an M443 � IR interaction was tested in aCoxmodel applied to a 2�2 factorial design [M443 (no/yes)� IR(no/yes)]. Results were considered significant at a significancelevel of P < 0.05. Analyses were conducted using SAS version 9.3(SAS Institute, Inc.).

ResultsDownregulation of MRK radiosensitizes medulloblastomacells

We have previously shown that MRK is activated by IR andcontrols the cellular response to DNA damage in osteosarcomaand colorectal cancer cells (7, 12). To test whether MRK has asimilar role in medulloblastoma cells, we asked whether down-regulation of MRK expression can sensitize these cells to killingby radiation. UW228 medulloblastoma cells were transfectedwith control (luciferase) or specific MRK siRNAs and subsequent-ly tested for viability after exposure to different doses of IR withthe MTT and clonogenic assays. Figure 1A shows that MRKdepletion decreased cell viability after IR by 33% of control at

Markowitz et al.





3Gy. Similarly, the clonogenic assay showed a significant decreasein survival with a dose enhancement factor (DEF) of 1.6 at 10%viability (Fig. 1B). In both assays, downregulation ofMRK expres-sion alone did not significantly affect viability by more than 5%(R. Ruggieri, unpublished observations). Analysis of the sig-naling elements known to be activated downstream of MRK(12) demonstrated a significant attenuation of IR-inducedChk2 phosphorylation at 1 and 2 hours after IR and effectivereduction in p38 phosphorylation (Fig. 1C). The lack ofcomplete Chk2 inhibition following MRK downregulationcould be explained by the fact that Chk2 is known to beactivated by multiple upstream factors that include DNA-dependent protein kinase and Polo-like kinase-3 (22). Thus,MRK influences the DNA damage response in medulloblasto-ma cells, and MRK downregulation sensitizes medulloblasto-ma cells to radiation.

Design of an irreversible inhibitor of MRKTo identifyMRK-specific smallmolecule inhibitors, we selected

a number of known kinase inhibitors that had been tested againstthe kinome and found to bind to MRK (23). We assessed theirability to inhibit MRK kinase activity using a cell-based assay thatwe previously described (19). Briefly, we made use of the M28osteosarcoma cell derivative (UMDI) that expresses a recombi-

nant form of MRK, which can be activated by homodimerizationwith the drug AP20187. Figure 2A shows that preincubation ofthese cells with the different drugs led to various levels of MRKinhibition. Among the inhibitors, we selected nilotinib, whichhad been previously shown by Manley and colleagues to bindwith very high affinity to MRK (24).This study also included amodel of the MRK active site bound to nilotinib, based on thecorresponding structure of nilotinib bound to its original target c-Abl. We noticed that the pyridine ring in nilotinib was located inclose proximity to cysteine 22 in MRK, suggesting the possibilityof designing anilotinib derivative that can covalently interactwithMRK. Importantly, c-Abl has a leucine at the correspondingposition. In addition, none of the known off-targets of nilotinib(Fig. 2B) and none of the MRK family members or related kinases(Fig. 2C) have a cysteine at this position. Thus, in an effort toincrease the specificity of nilotinib for inhibitingMRK,wedecidedto derivatize nilotinib by modifying the pyridine ring to a piper-idine ring with the addition of an ab unsaturated amide to createcompound M443 (see Materials and Methods, Fig. 2D–G andSupplementary Fig. S1).

We anticipated that M443 would bind covalently to MRK viacysteine 22 and irreversibly inhibit MRK activity (Fig. 2D andE). To examine whether M443 indeed inhibits MRK in anirreversible manner, we pretreated UMDI cells with M443 for

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M443 inhibitsMRK in an irreversible andspecific fashion. A, time course of MRKinhibition by M443. UMDI cells werepretreated with 500 nmol/L M443 (M)or treated with DMSO for the indicatedtime. Then, cells were washed 3 timeswith fresh medium (þ), medium wasrefreshed after 15 minutes twice, andAP was added for 30 minutes beforeharvesting and processing cells forWestern blotting. B, UMDI cells weretreated as in Awith 100 nmol/L nilotinibor 500 nmol/L M443 for 6 hours.Histogram shows quantification of datafrom three independent experiments,expressed as mean� SD. C, K-562 cellswere treated with 500 nmol/L M443,nilotinib, or vehicle control for 6 hoursand cell lysates were processed forWestern blotting with the indicatedantibodies. GAPDH was used as aloading control. The blot is arepresentative of three independentexperiments.






different times. After extensive washes, we treated cells with thehomodimerizing AP compound to activate MRK. We first notedthat the extent of MRK inhibition by M443 was time-dependentand not reversed by washing out the inhibitor (Fig. 3A). AsMRK inhibition was optimal after 6 hours of exposure to M443(Fig. 3A), we chose this time point to compare M443 withnilotinib. Figure 3B shows that in contrast to M443, inhibitionby nilotinib could be totally reversed after washing out thedrug. These results provide strong evidence that M443 is anirreversible inhibitor of MRK.

Specificity of M443To assess the specificity of M443, we compared the degree to

which nilotinib and M443 inhibit the activity of BCR-Abl, theoriginal target of nilotinib. To this end, we used the chronicmyelogenous leukemia cell line K-562, which expresses the con-stitutively active BCR-Abl protein. Figure 3C shows that whileboth drugs inhibit MRK activity, nilotinib and M443 clearlydiverge in their effect on BCR-Abl: nilotinib efficiently inhibitsBCR-Abl whereas M443 does not. Thus, the nilotinib derivativeM443 selectively inhibits MRK.

M443 effectively inhibits MRK downstream signals inprimary medulloblastoma cells and prevents cell-cyclearrest induced by IR

To examine whether M443 inhibits signaling downstream ofMRK, we tested both the UW228 cell line as well as the UI226primary patient–derivedmedulloblastoma cells for their responseto IR in the presence and absence of M443. Figure 4A shows thatMRK activation by IR is maximal at 30 minutes after exposure toradiation. Therefore, for subsequent analysis, this time point wasused. In both cell cultures, the IR-stimulated activation of MRK,Chk2, and p38 was greatly inhibited by 500 nmol/L M443. AsChk2 activity is important for the S and G2–M checkpoints, wealso examined the effect of M443 on cell-cycle arrest after IR. Tothis end, we measured the mitotic index in UW228 cells aftertreatment with M443 and exposure to IR. Cells were seeded oncoverslips, pretreated with 500 nmol/L M443 or vehicle, exposedto 6 Gy of IR, fixed at different times after IR and processed forimmunofluorescence with the MPM2 phospho-specific antibodythat specifically stains mitotic cells. Figure 4B shows that incontrast to control cells, the M443-treated cells failed to arrestafter IR and maintained a similar mitotic index as the nonirradi-ated cells. Thus, inhibition of MRK leads to inhibition of Chk2and failure to arrest in the cell cycle in response to IR-inducedDNA damage.

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Figure 4.M443 inhibits MRK downstream signaling and prevents radiation-induced cell-cycle arrest. A, top, time course of MRK activation by IR. UW228 cells wereexposed to 3 Gy of IR, harvested at the indicated time points, and processedfor Western blotting with the p-MRK antibody. Tubulin was used as loadingcontrol. Bottom, UW228 cells and primary UI226 cells were pretreated with500 nmol/L M443 for 6 hours and then they were exposed to 3 Gy of radiation,and, 30 minutes later, they were harvested and processed for Western blottingwith the indicated antibodies. B, UW228 cells, seeded on coverslips, werepretreated with 500 nmol/L M443 for 3 hours, exposed to 6 Gy of radiation, andthen processed for immunofluorescence with the phospho-specific MPM2antibody as described in Materials and Methods. Mitotic cells from 10 fields percondition were calculated from 3 independent experiments and plotted in thegraph below. Bars, 50 mm.

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M443 radiosensitizes medulloblastoma cellsAs a consequence of failed cell-cycle arrest in M443-treated

medulloblastoma cells, we would expect to see increased sensi-tivity to IR. To confirm this, we used both UI226 and UW228cells. As the UI226 medulloblastoma primary cells do not formcolonies when plated sparsely, we tested colony formation bythe UW228 cell line and used the MTT assay for the UI226cells. Figure 5 shows that M443 effectively radiosensitizes bothcell cultures to radiation. The clonogenic assay in UW228 cellsshowed a DEF at 10% viability of 1.6, which is the same observedupon silencing of MRK in these cells (Fig. 1B). A similar responsewas observed with the parental compound, nilotinib (Fig. 5B).TheMTT assay showed thatM443 in conjunctionwith IR caused a48% reduction in viability of the primary medulloblastomacells in comparison to IR alone. Interestingly, these cellsrespondedmaximally already to 125 nmol/L of M443, the lowestdose used, indicating that the IC50 for the radiosensitization effectis lower than 125 nmol/L. Similar results were obtained with asecond medulloblastoma primary line (R. Ruggieri, unpublishedobservations). Importantly, M443 did not show any toxicity inthe absence of radiation and failed to radiosensitize normalneuronal stem cells and astrocytes (Supplementary Fig. S2). Thus,these data indicate that M443 can be used to radiosensitizemedulloblastoma tumors without compromising the surround-ing parenchyma.

To test the extent to which M443 radiosensitizes tumors otherthan medulloblastoma, we tested breast cancer 4T1 cells for theirresponse to the drug in conjunction with IR. SupplementaryFigure S3 shows that M443 can radiosensitize breast cancer cellsas well.

M443 synergizes with IR to extend survival in an orthotopicmedulloblastoma xenograft model

To examine whether M443 can act as a radiosensitizer in amouse model of medulloblastoma, we used the primary UI226cells to generate an orthotopic medulloblastoma tumor innude mice. Previous work had established that approximately3 weeks after tumor cell implantation, mice become symp-tomatic within a relatively narrow time frame, just over a week,indicating similar tumor growth rates among the animals. Twoweeks before mice became moribund, they were randomizedinto four groups: control, treated with M443 alone, treatedwith 6 Gy of IR (2 � 3 Gy over 2 days), and with thecombination of M443 and IR. M443 was delivered directlyinto the tumor via an osmotic pump, and 2 days later, micewere exposed to IR.

Controlmice survivedwith amedian of 32 days after tumor cellimplantation. Treatment with M443 alone added 5.5 days to thissurvival, whereas the chosen low dose of radiation did notsignificantly increase survival (medianof 1day additional survivalover that of control). In contrast, the combination ofM443 and IRextended survival with a median of 16 days longer than control.Statistical analysis of the data demonstrated a synergistic effect ofthe two combined treatments (Fig. 6A). Supporting the safety datashown in Supplementary Fig. S2, treatment with M443 did notaffect the animal weight, as theweight loss observedwas observedin all groups just a few days before the animals becamemoribund(Supplementary Fig. S4).

To confirm in vivo target inhibition by M443, we tested MRKlevels and activity in brains from treated and untreated mice 18hours after exposure to the drug. For this assay, the brains were

harvested and their cerebella were divided into four sections thatwere then examined by Western blotting with phospho-specificand total MRK antibodies. The tumor-containing sections of thecerebellum were identified by the human anti-HLA antibody,because despite performing the tumor cell injections in a standardfashion, it is difficult to predict the invasion and growth path of

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Figure 5.

M443 radiosensitizes medulloblastoma cells in vitro. UW228 cells werepretreated with the indicated doses of M443 (A) or nilotinib (B), exposed to theindicated doses of radiation, and then processed for the clonogenic assay asdescribed in Materials and Methods. C, UI226 primary medulloblastoma cellswere pretreated with the indicated doses of M443, exposed to various doses ofradiation, and processed for the MTT assay 72 hours later.






the tumor. Figure 6B shows that the tumor-containing fractionhad elevated levels of both total and active MRK (lane RB in thevehicle-treated brain). In contrast, the tumor-containing fractionfrom the M443-treated brain showed total loss of MRK activity.Interestingly, the MRK protein levels were also reduced in thetreated fractions. This result is consistent with our previousobservation that sustained inhibition of MRK leads to its insta-bility (R. Ruggieri, unpublished observation). Interestingly, thefractions containing normal brain, which in the control brainshowed some level of MRK protein, in the treated brain also hadlost MRK, suggesting that diffusion of M443 across the wholecerebellum inhibited normal MRK as well.

Taken together, our study demonstrates that MRK is a suitabletarget for radiosensitization of medulloblastoma tumors andlikely additional types of tumors.

DiscussionIn this study, we have identified the protein kinaseMRK/ZAK as

a promising novel target for radiosensitization in medulloblas-

toma and designed a small molecule inhibitor M443 that irre-versibly blocks its activity. Importantly, administration of M443in vivo effectively synergizes with radiation in a mouse model ofmedulloblastoma, where it increases the survival time by 50%.This result provides a proof of principle that MRK is a target forradiosensitization in medulloblastoma.

We have developed an irreversible inhibitor of MRK, M443,based on the close proximity of the M443 parental compoundnilotinib to cysteine 22 in the model structure of the MRK ATP-binding domain (24). The unique presence of this cysteineat this position in MRK, compared with other nilotinib targetsand other MRK-related proteins, suggested that an irreversibleinhibitor of MRK could provide high selectivity toward MRK,which is likely to result in reduced off-target effects of atreatment based on this molecule. Our initial investigation onthe effects of M443 on BRC-Abl, the original target of nilotinib,indicates that M443 selectively inhibits MRK, but not BCR-Abl.Although a wider specificity screen is needed, this result sug-gests good selectivity for this compound.

The past reservations about irreversible inhibitors that werebased on safety concerns recently have been mitigated by severalclinical successes. In fact, irreversible kinase inhibitors for EGFreceptor (EGFR), Bruton tyrosine kinase (BTK), and MEK1 arebeing tested in the clinic as anticancer agents (25). Importantly,this type of inhibitors is expected to have extended pharmaco-dynamic duration of inhibition, which could allow for lowercirculating doses with less potential side effects. Although thetoxicity profile of M443 needs to be assessed after systemicadministration in vivo, it is interesting to note that we did notobserve any apparent toxicity in tumor cells treated with M443(R. Ruggieri, unpublished observations), nor in animals based onlack of weight loss following drug administration. In addition,no effect was observed on normal brain cells (SupplementaryFig. S2). We believe that this characteristic of the MRK inhibitor isparticularly important in the context of pediatric brain tumors forwhich safer therapeutic approaches are needed. Future character-ization of the pharmacokinetic properties of M443 will establishthe extent to which this compound crosses the blood–brainbarrier.

Analysis of the tumor tissue after convection delivery revealed aclear in vivo inhibition of MRK activity by M443. The phospho-specific MRK antibody should be a very valuable tool to identifypatients that might have elevated activation levels of MRK andtherefore may be more responsive to a treatment that includesMRK inhibition.

M443 inhibits MRK downstream pathways that include IR-induced activation of Chk2 and the consequent cell-cycle arrest.The fact that these effects of MRK silencing were replicated byM443 supports the specificity of these phenotypes and strength-ens a role for the MRK-b isoform in the DNA damage response,previously observed in other cell types (12). Both Chk2 and p38have been shown to control cell-cycle arrest after DNA damage byregulating the activity of the CDC25 family of proteins and thusthe activity of the CDKproteins (26). The role of p38a in theDNAdamage response has, however, been demonstrated only inresponse to UV-induced damage (27). The activation of p38a byIR inmedulloblastoma is clearly dependent onMRKactivity and itmay point to either a role for p38a in IR-inducedDNAdamage, assuggested for the response to UV, or to additional roles thatMRK may have in response to IR. As we have shown previously,IR-inducedMRKactivation is dependent onNbs1 andpartially on

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Figure 6.

M443 synergizes with radiation to prolong survival in a murine model ofmedulloblastoma. A, Kaplan–Meier survival curves demonstrating synergismbetween M443 and radiation treatment. n ¼ 10–12 mice/group. Time refers todays after intracranial tumor cell injections. The bar and asterisks above theKaplan–Meier curves indicate the respective treatment periods (2 weeks ofcontinues exposure to the drug via the osmotic pump and 2 doses of IR indicatedby the asterisks). B, in vivo target inhibition and specificity of M443. Eighteenhours after pump implantation, brains were harvested and each cerebellumwassectioned in four parts: right top (RT), right bottom (RB), left top (LT), and leftbottom (LB). Tissue extracts were processed for Western blotting with theindicated antibodies. The hu-HLA antibody identifies the tumor-containingfractions.


Markowitz et al.




ATM (17), two proteins involved in the DNA damage responseand in cell-cycle checkpoint regulation (28, 29). In turn, MRKactivates Chk2, a checkpoint protein that, together with Chk1, isconsidered a good target for radiosensitization in cancer and forwhich inhibitors are actively being evaluated in the clinic (30, 31).Thus, M443 represents an additional novel approach to target theDNA damage response in cancer.

As the two splice forms of MRK share the same kinasedomain, M443 is expected also to inhibit MRKa. Thus, becauseMRKa is aberrantly expressed in cancers like gastric, colorectal,bladder, and breast tumors, where it plays a critical role intumor cell proliferation, M443 also may have therapeuticeffects in these cancers (13). Interestingly, we found that inaddition to medulloblastoma cells, breast cancer cells are alsosensitized to radiation following MRK inhibition by M443(Supplementary Fig. S2). In addition, we found that siRNA-mediated MRK silencing radiosensitizes several types of cancercells including osteosarcoma and glioblastoma cells (ref. 12and data not shown). Thus, inhibition of MRK may be a usefulapproach to radiosensitize different types of solid tumors.Future studies will investigate this possibility.

In summary, we have developed a specific and irreversibleinhibitor of MRK that effectively radiosensitizes medulloblasto-ma tumors and potentially other cancers as well. Future devel-opment of this small molecule may provide a new therapeuticapproach for radiosensitization in cancer.

Disclosure of Potential Conflicts of InterestR. Ruggieri has ownership interest as co-inventor on patent application

for treatment of solid tumors by inhibiting MRK/ZAK (2015). Y. Al-Abedownership interest as co-inventor on patent application for treatment ofsolid tumors by inhibiting MRK/ZAK (2015) and is president and co-founder of Fatimo Innovation LLC. M. Symons has ownership interest asco-inventor on patent application for treatment of solid tumors by inhibit-

ing MRK/ZAK (2015). No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: D. Markowitz, M.E. Berens, S. Sun, M. Symons,Y. Al-Abed, R. RuggieriDevelopment ofmethodology:N.L. Tran,M.He, S. Sun, Y. Al-Abed, R. RuggieriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Markowitz, C. Powell, T.C. Ryken, M. Vanan,R. RuggieriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.Markowitz, C. Powell, M. Vanan, L. Rosen, S. Sun,M. Symons, R. RuggieriWriting, review, and/or revision of the manuscript: N.L. Tran, T.C. Ryken,M. Vanan, L. Rosen, S. Sun, M. Symons, R. RuggieriAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Powell, M. Vanan, M. SymonsStudy supervision: M. Symons, R. RuggieriOther (synthesized the MRK inhibitor/M443): M. HeOther (design and synthesis of the pharmacological inhibitor M443):Y. Al-Abed

AcknowledgmentsThe authors thank James Rutka (The Hospital for Sick Children) for kindly

providing the UW228 cells and Amanda Chan (The Feinstein Institute) for herhelp with microscopy data acquisition.

Grant SupportThis study was supported by the following fund sources: Swim Across

America (SAA) Foundation and Project to Cure (PTC) Foundation (to M.Symons), the Bradley Foundation (to R. Ruggieri), and The Ben & CatherineIvy Foundation (to M.E. Berens).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 9, 2015; revised May 5, 2016; accepted May 10, 2016;published OnlineFirst May 20, 2016.

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2016;15:1799-1808. Published OnlineFirst May 20, 2016.Mol Cancer Ther Daniel Markowitz, Caitlin Powell, Nhan L. Tran, et al. Radiosensitizes MedulloblastomaPharmacological Inhibition of the Protein Kinase MRK/ZAK

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Pharmacological Inhibition of the Protein Kinase MRK/ZAK ... · part of the conserved kinase active site domain. , unique Cys 22 in MRK. C, amino acid sequence alignment of the indicated