The Novel ATP-Competitive Inhibitor of the MET...

Small Molecule Therapeutics

The Novel ATP-Competitive Inhibitor of the MET HepatocyteGrowth Factor Receptor EMD1214063 Displays InhibitoryActivity against Selected MET-Mutated Variants

Michaela Medov�a1,2, Benoît Pochon1,2,†, Bruno Streit1,2, Wieslawa Blank-Liss1,2, Paola Francica1,2, DeborahStroka2,3, Adrian Keogh2,3, Daniel M. Aebersold1, Andree Blaukat4, Friedhelm Bladt4, and Yitzhak Zimmer1,2

AbstractThe receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and

involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases,

primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors.

Here,we report the biologic activity of a novelMET inhibitor, EMD1214063, on cells that ectopically express the

mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results

show a dose-dependent decrease inMET autophosphorylation in response to EMD1214063 in five of the eight

cell lines (IC50 2–43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of

downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing

lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063

strongly influencedMET-drivenbiologic functions, such as cellularmorphology,MET-dependent cellmotility,

and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor

model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated

variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only.

Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-

derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-

treated animals.Collectively, the current data identifies EMD1214063 as apotentMETsmall-molecule inhibitor

with selective activity towards mutated MET variants. Mol Cancer Ther; 12(11); 1–10. �2013 AACR.

IntroductionDuring the last three decades, deregulated activity of

the hepatocyte growth factor (HGF)/HGF receptor METsystem has been strongly correlated with the pathogen-esis of numerous human malignancies (reviewed inrefs. 1–3). Consequently, MET is considered a significantmolecular target in cancer therapy. Current understand-

ing of the role of the MET receptor tyrosine kinase (RTK)signaling supports three main classes of therapeuticapproaches to inhibit its activity in clinical setups: (i)interventions on the receptor–ligand interaction level byHGF or MET monoclonal antibodies or use of HGFanalogs, (ii) inhibition of the tyrosine kinase activity bysmall-molecule inhibitors, and (iii) blockade of the recep-tor downstream association with its signaling adaptorsand effectors (4).

In addition to dysregulated paracrine/autocrine loopsandMET gene overexpression, which are among themostprevalent mechanisms that contribute to deregulatedfunction of the MET/HGF system, MET oncogenic activ-ity could be unleashed also by activating mutations in thetyrosine kinase domain of the receptor. Tyrosine kinasedomainmutations in theMET genewere first identified inhereditary renal papillary cell carcinoma (5) and subse-quently were described also in the sporadic form of thisdisease as well as in head and neck squamous cell carci-noma, hepatoblastoma, and glioma (reviewed in ref. 6).Generally, the presence of these mutations was shown tobe sufficient to increase MET tyrosine kinase activity in aligand-independent manner with subsequent increase incellular proliferation, motility, and in vivo metastaticpotential (5, 7). MET tyrosine kinase domain structural

Authors' Affiliations: 1Department of Radiation Oncology, Inselspital,Bern University Hospital, and University of Bern, 3010 Bern, Switzerland;2Department of Clinical Research, University of Bern, 3010 Bern, Switzer-land; 3Department of Visceral Surgery, Inselspital, BernUniversityHospital,and University of Bern, 3010 Bern, Switzerland; 4Merck Serono, an Affiliateof Merck Serono Research & Development, Merck KGaA, Darmstadt,Germany

†Deceased.

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

M. Medov�a and B. Pochon contributed equally to this work.

Corresponding Author: Michaela Medov�a, Radiation Oncology, Depart-ment of Clinical Research, University of Bern/Inselspital, MEM-E815,Murtenstrassse 35, 3010 Bern, Switzerland. Phone: 0041-3163-22643;Fax: 0041-3263-23297; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0151

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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studies indicate that activating mutations interfere withthe receptor autoinhibition (8) and that variousmutationshave different biologic effects, presumably due to cou-pling to distinct downstream effectors, consequently acti-vating discrete signaling cascades (9–11).

In the last 10 years, the development of specific small-molecule tyrosine kinase inhibitors (TKI),which target thecatalytic activity of the receptor primarily, but not only,via competing with ATP on its binding to the ATP pocketof the kinase domain, is critical for successful treatment ofMET-driven tumors. First attempts to identify ATP-com-petitive MET inhibitors led to the characterization of thestaurosporine analog K252a, a broad-spectrum kinaseinhibitor that blocks MET kinase activity at submicromo-lar concentrations (12). Later, the highly selective indo-line-2-one core anti-MET small-molecule compoundsSU11274 andPHA665752were discovered anddevelopedby SUGEN, but their limited pharmacokinetic propertiesrestricted the use of these inhibitors to in vitro and limitedin vivo studies (13–16). However, the encouraging obser-vations that were made with these two prototypic toolmolecules stimulated rapid development of new genera-tion available anti-MET compounds whose integration inclinical practice is highly awaited.

Over the last decade, accumulating clinical experiencewith TKIs that target primarily BCR-ABL, KIT, and EGFreceptor (EGFR) have proven that the efficacy of theseinhibitors in imposing tumor growth control could besignificantly compromised by primary or secondarymutations, leading respectively either to resistance toinitial treatment response or to tumor relapse (17–19).These observations have important implications in thecase that similar resistance would be also observed withMET inhibitors, which are currently evaluated in clinicaltrials.

Approximately 20 different selective as well as nonspe-cific MET TKIs have been reported so far (6, 20), of whichabout 10 are currently in different phases of clinical trials(21). In that category, EMD1214063 and EMD1204831 areselective, reversible, and highly potent indoline-2-onecore oral ATP-competitive inhibitors of the MET kinasedomain, which have recently completed phase I clinicaltrial (22–24). As a recent study by Bladt and colleaguesreported the impact of this novel and highly potent METTKI on tumor cells expressing wild-type MET (22) andsince tyrosine kinase point mutations emerge as criticaldeterminants of resistance to TKIs, in the present study,we aimed at evaluating and comparing the responses ofvarious mutated forms of the MET receptor toEMD1214063 in vitro and in an in vivo xenograft model.

Our results identify five of the eight MET variants asresponsive to EMD1214063 based on its ability to blockMET autophophorylation with IC50 values rangingbetween 2.2 and 43 nmol/L. MET kinase inhibition wasassociated with reduction of downstream signaling andMET-driven biologic activities. Three of the MET mutat-ed variants, Y1248H, L1213V, and V1206L did notexhibit kinase inhibition even at a concentration of 75

nmol/L. An in vivo mouse subcutaneous model con-firmed the in vitro observations with cells expressing theL1213V variant that maintained growth when com-pared with H1112L-derived tumors, which displayeda dramatic shrinkage following only five sessions ofdaily 50 mg/kg EMD1214063 administration.

Materials and MethodsCell lines

The construction of the vectors expressing the METmutations M1268T, Y1248H, H1112Y, L1213V, H1112L,V1110I, V1206L, and V1238I was previously describedand their identities were verified by sequencing of bothstrands of DNA in the region of interest (25). NIH3T3 celllines that stably express theseMET-mutatedvariantswereprovided by Dr. Laura Schmidt (National Cancer Insti-tute, Frederick,MD). Cellsweremaintained inDulbecco’sModified Eagle Medium (GIBCO, Invitrogen Corp.) sup-plemented with 10% fetal calf serum (Sigma), antibiotic–antimycotic, and 0.5 mg/mL Geneticin/G-418 sulfate(GIBCO, Invitrogen Corp.) and used for experimentswithin 2 months being in culture.

EMD1214063EMD1214063 (3-(1-(3-(5-(1-Methylpiperidin-4-ylmethoxy)-

pyrimidin-2-yl)-benzyl)-1,6-dihydro-6-oxo-pyridazin-3-yl)-benzonitrile) was obtained from Merck, diluted in DMSOand kept at�20�C.Working solutionswere prepared freshlyin the corresponding media.

Western blot analysis and antibodiesCells were treated with EMD1214063 as indicated in the

legends to the figures and lysed in a lysis buffer containing1% Triton X-100, 0.5%NP-40, 1 mmol/L EGTA, 1 mmol/LEDTA, 150mmol/LNaCl, 10mmol/LTris-HCl, 1mmol/LNa3VO4, 10 mmol/L NaF, 1 mmol/L ZnCl2, 50 mmol/LNa2MoO4, and a cocktail of proteases inhibitors (Com-plete Mini, Roche). Cell extracts were cleared by centri-fugation and total protein concentration was determinedby using the Bio-Rad protein quantification reagent (Bio-Rad Laboratories, Inc.). In all, 100 mg of total proteins wereresolved by SDS-PAGE on a 7% gel under reducing con-ditions, transferred onto a polyvinylidene difluoridemem-brane and analyzed byWestern Blotting (InvitrogenCorp).Detection of secondary antibodies was conducted usingtheECLkit (AmershamPharmaciaBiotech).QuantificationofECLsignalswas conductedusingQuantityOne software(Bio-Rad Laboratories, Inc.) and data were processed byExcel (Microsoft) to obtain IC50 values for MET kinaseinhibition by EMD1214063.

Rabbit polyclonal anti-phospho-MET (Tyr1234/1235)and mouse monoclonal anti-phospho-AKT (Ser473) anti-bodies were from Cell Signaling (Cell Signaling Technol-ogy, Inc.). The rabbit polyclonal anti-MET and anti-phos-pho-PLCg1 (Tyr783) antibodies were purchased fromSanta Cruz (Santa Cruz Biotechnology, Inc.). The mousemonoclonal anti-b-Actin and the rabbit polyclonal anti-

Medov�a et al.

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phospho-ERK1/2 antibodies were purchased from Milli-pore (Chemicon and Upstate, respectively).

Cell-cycle analysis by flow cytometryCells were plated in 6-well plates and the next day

treated overnight with 30 nmol/L EMD1214063. Aftertreatment, cells were trypsinized, washed, fixed with70% ethanol in PBS, pH 7.4, and stored at þ4�C. On theday ofmeasurements, fixed cells were treatedwith 100 mLof RNaseAþPropidium iodide (PI) cocktail (0.1%Triton-X100, 20 mg/mLPI, 400 mg/mLRNaseA in PBS) at 4�C for60 minutes and subsequently analyzed with a FACSscan(BectonDickinson). The results shown represent themeanof three independent experiments, � SEM, and weredetermined using the FlowJo software.

Wound healing assayCells were seeded in a 6-well plate (2 � 105 cells/well)

and treated with 50 nmol/L EMD1214063 as indicated inthe legend to the figure. After 4 hours of treatment, aportion of themonolayer was scratchedwith a pipette tip.Cells were examined for resealing of the "wounded"monolayer 24 hours later by staining with 0.005% crystalviolet in PBS, pH 7.4, for 3 minutes before photodocu-mentation with a Leica DC 300F camera (4� objective).Analysis was done using a Leica IM-50 Software. Inaddition, EMD1214063-mediated changes in cellularmor-phologywere observed bymicroscopy, documentedwitha Leica DC 300F camera (10� objective), and processedwith the Leica IM-50 software at the same time-points.

Growth in soft agaroseCells were plated in 6-well plates and 24 hours later,

15 nmol/L of EMD1214063 or DMSO as a negative con-trol was added. One day after addition of EMD1214063or DMSO, the cells were harvested by trypsiniza-tion, counted, and plated on seaplaque agarose (1% forbottom layers and 0.55% for top layers, � 15 nmol/LEMD1214063). After solidification, plates were incubateduntilmacroscopic colonieswere visible (average period of18 days) and gels were further supplemented with 15nmol/L of EMD1214063 or DMSO every 7 days. Colonieswere stained by adding 0.5mL of 0.005% crystal violet perwell for overnight, excess solution was aspirated andpictures taken with a digital camera. Colonies werecounted using the Bio-Rad Quantity One software.

In vivo studiesNIH3T3 expressing MET-mutated variants were trans-

duced by the lentivirus pCR-XL-FLUL that expresses thefirefly luciferase (Luc), which was constructed by Dr.Mario Tschan, Department of Pathology, University ofBern, Bern, Switzerland.A total of 20,000 Luc-expressing NIH3T3 MET H1112L

or L1213V cells were injected subcutaneously into theright flank of 16-week-old female Rag2 common gam-ma-null mice and the growth of the tumors was regularlymonitored by luminescence using the NightOwl and by

caliper measurements. As soon as all the tumors becamevisible and palpable, mice were randomly divided intothe treatment by TKI (50 mg/kg of EMD1214063 per day)or the control (vehicle only, Solutol HS 15, BASF Chem-Trade GmbH) group, treated per os for 5 consecutive daysand following 2 days without treatment. After 5 daysof treatment, tumors were visualized and measured asaforementioned.

IC50 valuesThe inhibitory effects of EMD1214063 (0–1 mmol/L) on

MET autophosphorylation (Tyr1234/1235; pMET) wereinferred fromWestern blot analysis. The IC50 values wereobtained following densitometry of pMET bands normal-ized to b-Actin expression. A least-squares fit of theobserved data with the response function

rðcÞ ¼ r0 þ ð1� r0Þ=ð1þ ðc=IC50ÞkÞ

where r0 is the baseline relative autophosphorylation, IC50

is the half maximal effective concentration, and k is theHill coefficient, was conducted.

Statistical analysisStatistical significancewas calculated using the Student

t test, P values < 0.05 were considered significant. Wherenot indicated, the data represent themean of at least threeindependent experiments � SEM.

ResultsEffect of EMD1214063 onMET phosphorylation andits downstream signaling

Initially, we tested the capacity of the specific METsmall-molecule TKI EMD1214063 (Fig. 1A) to reduce thereceptor Tyr1234/1235 autophosphorylation in NIH3T3cell lines, which stably express the M1268T, Y1248H,H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238IMET-mutated variants (Fig. 1B). Calculation of the corre-sponding IC50s yielded values of 2.2, 6.9, 17.3, 26.1, and42.6 nmol/L for H1112L, H1112Y, M1268T, V1238Y, andV1110I, respectively (Fig. 1C and Table 1). These valuesare in good agreement with data from a recent studyby Bladt and colleagues who reported an average IC50

of 23 nmol/L for inhibition of tyrosine autophosphoryla-tion of thewild-type form ofMET (22). On the other hand,the V1206L, L1213V, and Y1248H variants displayedconsiderably higher IC50 values of 224.0, 270.1, and>1,000 nmol/L, respectively (Fig. 1C and Table 1).

The decrease inMET activation by EMD1214063withina range of up to 75 nmol/Lwas further associatedwith aninhibition of the activation state of the MET downstreamsignaling molecules AKT, ERK, and PLCg (Fig. 2), albeitwith particular differences between the highly sensitivevariants. Thus, while reduction in ERK1/2 phosphoryla-tion was most evident in the case of variants M1268T andH1112L, a rathermoremoderate decrease was detected incells expressing the H1112Y variant. Similar observations

Impact of EMD1214063 on MET-Mutated Variants

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were made in respect with the phosphorylation status ofPLCg . Although themolecular basis for this phenomenonhas yet to be determined, similar findings have been

previously reported with respect to the effect of the METtool TKI SU11274 on the phosphorylation status of PLCgY783 in different MET variants (26).

Figure 1. A, structures of EMD1214063 (MSC2156119) and EMD1204831 (MSC2156996). B, impact of increasing concentrations of EMD1214063 (16 hours)on MET autophosphorylation (Tyr1234/1235) levels in NIH3T3 mouse fibroblast cells stably expressing MET-activating point mutations M1268T, Y1248H,H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. C, impact of EMD1214063 treatment (0–1 mmol/L; for 16 hours) on MET autophosphorylation(Tyr1234/1235) inferred from Western blotting analysis (Fig. 1B) and the least-squares fit of the values at different concentrations with the dose responsefunction. Resulting values for IC50 are reported in Table 1.

Table 1. IC50 values for MET autophosphorylation (Tyr1234/1235) in eight studied MET-mutated variants

MET-mutated variant M1268T Y1248H H1112Y L1213V H1112L V1110I V1206L V1238I

IC50 (nmol/L) 17.3 >1000 6.9 270.1 2.2 42.6 224.0 26.1

NOTE: The values are based on least-squares fits shown in Fig. 1C.

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Contrary to the observations with M1268T, H1112Y,H1112L, V1238I, and V1110I, EMD1214063 doses up to 75nmol/L did not display any inhibition of the MET enzy-matic activity in cells expressing theY1248H, L1213V, andV1206L mutants as judged from the levels of phosphor-ylated MET. Consequently, EMD1214063 also did notalter the activation of the aforementioned MET down-stream signaling effectors in these three cell lines, suggest-ing their resistance to this drug.Determination of the IC50sof EMD1214063 for autophosphorylation blocking ofthese variants resulted in values of 224.0, 270.1, and>1,000 nmol/L for V1206L, L1213V, and Y1248H, respec-tively (Fig. 1C and Table 1).

EMD1214063 selectively attenuates MET-dependent cell-cycle progression of cell linesexpressing drug-sensitive forms of theMET receptorTo assess the biologic consequences of the inhibition

of MET-driven signaling pathways by EMD1214063,we first investigated its impact on cell-cycle distribu-tion in the cell lines expressing the various MET var-iants. As the results of Fig. 3 suggest, variants V1238I,M1268T, and H1112L exhibited the highest sensitivityto 30 nmol/L of EMD1214063 in terms of growth arrestas manifested by 57%, 47%, and 43% respective reduc-tion in S-phase cells. All these three cell lines displayeda concomitant accumulation of cells in G1, indicating aG1 arrest. Cells expressing the H1112Y and V1110Ivariants showed a 30% reduction of cells found in S-phase, whereas V1206L cells showed a very moderatereduction in S-phase and a similar increase in G1

content. On the other hand, no changes in cell-cycleprogression in terms of G1 and S-phase populationswere observed in Y1248H and L1213V cells treatedwith the EMD1214063, indicating a resistance of theseMET variants.

In addition, data from a viability assay (SupplementaryMaterials and Methods) show that cell-cycle arrest is alsoaccompaniedby increases in cell deathuponEMD1214063treatment for 48 hours, M1268T, and V1238I beingthe most sensitive variants with 9.6- and 7.4-fold incre-ase, respectively (Supplementary Fig. 1; SupplementaryTable S1).

EMD1214063 impacts cellularmorphology,motility,and anchorage-independent growth in drug-sensitive MET variants

Signaling pathways that are activated byMET not onlypromote proliferation but also cellular transformation,motility, migration and invasion, which are cruciallyimplicated in tumor progression. Therefore, we studiedthe effects of MET inhibition by EMD1214063 on biologicendpoints, in particular on cellular morphology, motility,and anchorage-independent growth.

The morphology of the cells was examined followingexposure to 50 nmol/L of EMD1214063 for 24 hours. Ascan be seen in Fig. 4A, of the eight lines that express theconstitutively active MET mutations, M1268T, H1112Y,and V1238I cells exhibited considerable morphologicchanges such as cell flattening and resumption of cell–cell contact upon exposure to EMD1214063. Similarchanges, although at lesser extent, were seen in cell linesexpressing the variants H1112L and V1110I. A minoreffect of EMD1214063 was seen with V1206L cells. Onthe other hand, the transformed morphology of cellsexpressing the MET mutants Y1248H and L1213V didnot show any alterations following EMD1214063 treat-ment. In that respect, the lack of MET downstream sig-naling inhibition by EMD1214063 seems to be very wellcorrelated with cell scattering and motility, as evaluatedby in vitrowound healing/scratch assay. Predictably, themotility of the five sensitive MET mutants was eithercompletely hindered as in the case of M1268T, H1112Y,H1112L, V1238I, or considerably diminished in the case ofV1110I (Fig. 4B). As expected, in the case of the threeresistant mutations (Y1248H, L1213V, and V1206L),EMD1214063 had almost no effect on the wound closureand, similar to the controls, the scratches were hardlyvisible due to effective cell invasion.

As MET RTK signaling is required for invasiveness ofMET-expressing tumor cells,wenext studied the ability ofEMD1214063 to reduce anchorage-independent growthofMETmutated variants. Taking into consideration the IC50

values of EMD1214063 to inhibit MET tyrosine phosphor-ylation (the IC50 values were ranging between 2 and 43nmol/L), we decided to use 15 nmol/L of EMD1214063to assess its effects on cellular growth in soft agar.Surprisingly, at this concentration, there was a consistentand remarkable reduction in colony formation in all

M1268T

pMET

MET

pAKT

pERK

pPLCγ

β-Actin

pMET

MET

pAKT

pERK

pPLCγ

β-Actin

Y1248H H1112Y L1213V

H1112L

0EMD1214063 (nmol/L) 10 25 75 0 10 25 75 0 10 25 75 0 10 25 75

0EMD1214063 (nmol/L) 10 25 75 0 10 25 75 0 10 25 75 0 10 25 75

V1110I V1206L V1238I

Figure 2. EMD1214063 treatment (16 hours) selectively inhibits METphosphorylation and phosphorylation of its downstream signalingmolecules in NIH3T3 mouse fibroblast cells stably expressing distinctMET-activating point mutations.






EMD1214063-sensitive MET-mutated variants (Fig. 5).However and again, no hindrance of this feature wasobserved in Y1248H and L1213V variants.

EMD1214063 represses tumor growth in a xenograftanimal model

To test the efficiency of the MET inhibitor EMD1214063in vivo and based on our in vitro data, we established amouse xenograft tumormodel usingNIH3T3 cells expres-

singMET-mutated variantsH1112L andL1213V.Wehavechosen to use these mutants as we have detected morethan 120-fold difference for in vitro IC50 values betweenH1112L and L1213V (2.2 vs. 270.1 nmol/L, respectively).We injected 20,000 of tumor cells expressing Luc gene intothe right flankof aRag2 commongamma-nullmice and letthem to form palpable and visible tumors. Animals bear-ing H1112L or L1213V tumors were randomized for thetreatment with 50 mg/kg/d of EMD1214063 or vehicle

Figure 3. The specific MET TKIEMD124063 causes redistributionof the cell-cycle profile ininhibitor-sensitive cells, leadingoverall to a significant increasein G1 phase. The values representthe average in percentage of thetotal events of three independentexperiments (�SEM).

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only. Surprisingly, after only 5 days of treatment withEMD1214063, the H1112L tumors completely regressed(Fig. 6). On the other hand, tumor growth continued in thevehicle-treated mice with H1112L tumors as well as inmice bearing the L1213V tumors (both vehicle- andEMD1214063-treated). These results strongly support thedata obtained in in vitro studies and show the enormouscapacity of EMD1214063 to inhibit certain forms ofderegulated MET.

DiscussionPrimary and secondary acquired resistances to MET

small-molecule inhibitors result from preexisting andnewly appearing point mutations, respectively. Thisobservation follows the accumulating experience with

other TKIs that target molecular pathways such as thoserelated to EGFR and ABL signaling (27, 28). As alreadyascertained for Gleevec in chronicmyelogenous leukemiaand gastrointestinal stromal tumors, information relatedto presence of mutations and response of particular var-iants to given inhibitors is critical for treatment outcome.Since first described in 1997 (5), approximately 30 METmutated variants have been so far reported (29). None-theless, it is rather surprising to note that little data areavailable that systematically documents their responsive-ness towards selective and nonselectiveMET TKIs, whichare presently evaluated in clinical trials (21).

In the current study, we used cells expressing mutatedMET variants to characterize the inhibitory capacity ofEMD1214063, a novel selective and reversible ATP com-petitor that targets the MET receptor. We report thatEMD1214063 showed effective inhibition ofMET tyrosineautophosphorylation of five of the eight MET variantsanalyzed in this study. Sensitive MET mutations tar-geted with EMD1214063 resulted in a loss of cellularproliferation, change in cell-cycle redistribution, motilityimpairment, and reduced the ability of tumor cells to growindependently of an anchorage aswell as to sustain tumorgrowth in an in vivo xenograft model.

IC50 values for the studied mutants ranged between 2.2and 43 nmol/L for the H1112L and V1110I variants,respectively. In this regard, EMD1214063 displays anti-MET IC50 values, which are in the range of crizotinib, aMET small molecule that is currently in phase III clinicaltrials (30). These figures are also in agreement with IC50

values of EMD1214063 towards the wild-type form ofMET as recently reported (22). Intriguingly, and withrespect to inhibition of downstream MET-dependent sig-naling, we observed variations between the differentvariants. For example, while reduction in tyrosine phos-phorylation of PLCg is evident in cells expressing the

Figure 4. A, EMD1214063treatment induces selective anddifferential reversion oftransformed morphologies ofNIH3T3 cells stably expressingmutated MET receptor. B, METinhibition by EMD1214063causes selective inhibition ofspontaneous cell motility in ascratch assay. Experiments werecarried out independently atleast three times; representativepictures are shown.

Figure 5. EMD1214063 selectively affects anchorage-independentgrowth of eight distinct MET-mutated variants. The columnsrepresent the average number of colonies (�SEM) of six repetitionsas a percentage of the corresponding controls.






M1268T and V1238I variants, H1112Y and H1112L cellsdisplay only a moderate reduction in phosphorylation ofthis MET signaling effector. Similar observations for thisphenomenon, which remains still to be clarified, havebeen previously reported with the MET tool small-mol-ecule inhibitor SU11274 (26).

As EMD1214063 at a daily dose of 50 mg/kg has beenrecently shown to very effectively reduce in vivo tumorgrowth of humanKP-4 pancreatic tumor cells that expresswild-type MET (22), we have used this dosage in ourcurrentmousemodel. Interestingly, ourdataunderscore acompelling activity of EMD1214063 on the responsiveMET-mutated variant H1112L in vivo, which indicated

dramatic tumor shrinkage within only 5 days of overalltreatment, which are by far more pronounced comparingthe effects detectedwith this drug in the in vitro assays.Wetend to attribute this observation to potential effects thatthe inhibitormay exert onMET-dependent tumor–stromainteractions, which largely mediate tumor growth andprogression (31–33).

Of major importance is our observation that the autop-hosphorylation status of the MET-mutated variantsY1248H, L1213V, and V1206L displays a rather resis-tant-like response towards EMD1214063. In that respect,Y1248H and L1213V exhibit a similar resistant patterntowards EMD1214063 as to the MET tool compound

Figure 6. The specific MET inhibitorEMD1214063 leads to regressionof the MET-driven xenograftdrug-sensitive tumors bearingthe H1112L MET mutationbut not the drug-resistant METmutants L1213V. Tumor growthwas evaluated by bothluminescence and calipermeasurements.

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SU11274 (13). The current data confirm therefore thepotential importance of a histidine to tyrosine substitutionof residue 1248, which is located in the activation loop oftheMETkinase for resistance toMETTKIs. Indeed, and infurther support to the current and previous observations(13), a recent study by Qi and colleagues showed therelevance of the Y1248H mutation as a major resistancemechanism both in in vitro and in vivomodels for the toolMET TKI PHA665752 as well as to crizotinib (30). One ofthe potential strategies to overcome the resistanceimposed by the Y1248H variant would be to use METinhibitors that interact with the receptor in a differentmode of binding. In 2008, Bellon and colleagues providedfor the first time evidence that AM7, a class II MET TKIthat binds the MET kinase linker and extends into a newhydrophobic binding site behind the C-helix, is able toinhibit Y1248H with an IC50 of 10 nmol/L (34). In a morerecent study, Yakes and colleagues provided evidencethat the nonselective class II inhibitor, cabozantinib,which apart ofMET targets alsoVEGFR2, RET, KIT, FLT3,and TIE2, is also able to inhibit the Y1248H variants withan IC50 of 3.8 nmol/L (35). In contrary to these potentialY1248H-targeting compounds, it is important to note thatcurrently no MET TKI has been reported to block thekinase activity of the L1213V variant. In that respect, it isworthwhile emphasizing, that although L1213V is prima-ry mutation with low penetrance in human cancers (36),its significance could be as a secondarymutation thatmayaccount for tumor relapse.Since originally described,mutations of theMET recep-

tor have been associated with a complex biologic pattern.Bardelli and colleagues were the first to suggest thatmutations in MET signaling lead to a substrate shift, thatresults in the activation of downstream effectors that arenot activated by the wild-type receptor (9). This is furthersupported by the observation that MET mutants cancouple with different signaling pathways, leading todiverse biologic consequences (10, 37–40). These observa-tions also support the findings by Graveel and colleagueswho showed that knock-inmice expressingMET-mutatedvariants developed tumors of different histotypes (41).The specific complexity of MET mutations and down-stream signaling seem to be unique to this RTK system.Moreover, their importance is further manifested with

respect to their emerging significance in targeted therapyfor MET-driven and MET-positive tumors.Finally, it is probably worthwhile mentioning that

unlike BCR-ABL, KIT and EGFR, for which mutations

that confer resistance to targeted therapies were firstidentified in the clinic due to lack of tumor response orrelapse,MET inhibitors still have not enter routine clinicalpractice. Yet, the high number of already identified METvariants along with experience made with the aforemen-tioned clinical targets strongly suggest for an early screen-ing and characterization of responses of clinically-rele-vant MET TKIs for particular mutations, similar to thecurrent study with EMD1214063.

Finally, to better elucidate the molecular basis for thecurrently reported observations, structural studies thatuse crystallographic approaches will have to be carriedout using these MET mutants in combination withEMD1214063.

Disclosure of Potential Conflicts of InterestA. Blaukat has ownership interest (including patents) in Merck KGaA.

F. Bladt is employed as a Director of Biomarker Discovery (other thanprimary affiliation; e.g., consulting) in Merck Serono and has ownershipinterest (including patents) in EMD1214063 patents. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Medov�a, B. Pochon, D.M. Aebersold,A. Blaukat, Y. ZimmerDevelopment ofmethodology:M.Medov�a, B. Pochon, B. Streit,W. Blank-Liss, D. Stroka, Y. ZimmerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Medov�a, B. Pochon, B. Streit, P. Francica,D. Stroka, A. KeoghAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):M. Medov�a, B. Pochon, B. Streit, D. Stroka,Y. ZimmerWriting, review, and/or revision of the manuscript: M. Medov�a,D. Stroka, A. Blaukat, F. Bladt, Y. ZimmerAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):B. Streit, D.M.Aebersold, Y.ZimmerStudy supervision: Y. Zimmer

AcknowledgmentsThe authors thank Mat�u�s Medo (University of Fribourg, Fribourg,

Switzerland) for his contribution to data analysis. This study is dedicatedto the memory of our dear colleague and friend Benoı̂t Pochon.

Grant SupportThe current study was funded by a Swiss National Science Foundation

grant 31003A-125394 (Y. Zimmer) and by Stiftung zur Krebsbek€ampfung(grant no.265; Y. Zimmer).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 27, 2013; revised August 20, 2013; acceptedSeptember 5, 2013; published OnlineFirst September 23, 2013.

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Medov�a et al.





Published OnlineFirst September 23, 2013.Mol Cancer Ther Michaela Medová, Benoît Pochon, Bruno Streit, et al. Activity against Selected MET-Mutated VariantsGrowth Factor Receptor EMD1214063 Displays Inhibitory The Novel ATP-Competitive Inhibitor of the MET Hepatocyte

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