preclinical evaluation of scc244 (glumetinib), a novel, potent, … · small molecule therapeutics...

Small Molecule Therapeutics

Preclinical Evaluation of SCC244 (Glumetinib),a Novel, Potent, and Highly Selective Inhibitor ofc-Met in MET-dependent Cancer ModelsJing Ai1, Yi Chen1, Xia Peng1, Yinchun Ji1, Yong Xi1, Yanyan Shen1, Xinying Yang1,Yi Su1, Yiming Sun1, Yinglei Gao1, Yuchi Ma2, Bing Xiong2, Jingkang Shen2,Jian Ding1, and Meiyu Geng1

Abstract

Because the receptor tyrosine kinase c-Met plays a critical rolein tumor growth, metastasis, tumor angiogenesis, and drugresistance, the c-Met axis represents an attractive therapeutictarget. Herein, we report the first preclinical characterization ofSCC244, a novel, potent, and highly selective inhibitor of c-Metkinase. SCC244 showed subnanomolar potency against c-Metkinase activity and high selectivity versus 312 other testedprotein kinases, making it one of the most selective c-Metinhibitors described to date. Moreover, this inhibitor pro-foundly and specifically inhibits c-Met signal transduction andthereby suppresses the c-Met–dependent neoplastic phenotype

of tumor and endothelial cells. In xenografts of human tumorcell lines or non–small cell lung cancer and hepatocellularcarcinoma patient-derived tumor tissue driven by MET aberra-tion, SCC244 administration exhibits robust antitumor activityat the well-tolerated doses. In addition, the in vivo antitumoractivity of SCC244 involves the inhibition of c-Met down-stream signaling via a mechanism of combined antiprolifera-tion and antiangiogenic effects. The results of the current studyprovide a strong foundation for the clinical investigation ofSCC244 in patients with tumors harboring c-Met pathwayalterations. Mol Cancer Ther; 17(4); 751–62. �2017 AACR.

Introductionc-Met is the prototypical member of a subfamily of receptor

tyrosine kinases (RTK) that also includes RON. c-Met is structur-ally distinct from other RTKs and is the only known high-affinityreceptor for hepatocyte growth factor (HGF; refs. 1, 2). Activationof the HGF/c-Met pathway provides a powerful signal for cellproliferation, survival, migration, invasion, angiogenesis, andmorphogenic differentiation. The normal functions of the c-Metpathway are largely restricted to organ morphogenesis duringdevelopment and to tissue damage repair and regeneration inadults (3–5).

c-Met and HGF are highly expressed in numerous cancersrelative to the surrounding tissue, and their elevated expressionis correlated with poor prognosis (3, 6–15). In addition to HGFstimulation, gene amplification, mutation, or rearrangement can

lead to aberrant c-Met activation. The propagation of the c-Met–dependent invasive growth process has been shown to be ageneral and important feature of highly aggressive tumors(16–18).More importantly, in addition to its role as anoncogenicdriver, c-Met has been increasingly implicated as a keymediator ofacquired or de novo resistance to approved therapies (19–23).Thus, c-Met is a promising therapeutic antitumor target.

Despite a remarkable number of c-Met inhibitors undergoingpreclinical and clinic assessment over the past decade, none ofthem have been approved for clinical use (14, 15, 24–28). Someof the c-Met pathway–targeted agents currently being evaluated inclinical trials are limited by their selectivity, potency, and safetyprofile, which may considerably hamper their ability to achieveoptimal inhibition of the pathway at a better tolerated dose incancer patients. More importantly, in the era of precision med-icine, a highly selective c-Met inhibitor could be significantlybeneficial for treating tumors driven by c-Met and serve as a"clean" component for combination strategies against c-Met–mediated drug resistance.

Recently, we identified a novel c-Met inhibitor, SCC244, withproperties that may overcome the aforementioned limitations.SCC244 is orally bioavailable, demonstrates greater than2,400-fold selectivity for c-Met over a panel of 312 kinases, andhas an IC50 in the subnanomolar range. This inhibitor potentlyand specifically inhibits c-Met–mediated signal transduction andthe c-Met–dependent neoplastic phenotype of tumor and endo-thelial cells in vitro. SCC244 showed robust antitumor activity in c-Met–dependent subcutaneous cell line–derived xenograft (CDX)and patient-derived xenograft (PDX) tumor models at well-tol-erated doses and showed a superior safetymargin. On the basis ofthis impressive preclinical activity, SCC244 received CFDA IND

1Division of Antitumor Pharmacology, State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,China. 2Department of Medicinal Chemistry, State Key Laboratory of DrugResearch, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,Shanghai, China.

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

Corresponding Authors: Jing Ai, Shanghai Institute of Materia Medica, ChineseAcademy of Sciences, Shanghai 201203, China. Phone: 8621-5080-6600, ext.2413; E-mail: [email protected]; Meiyu Geng, [email protected]; and Jian Ding,[email protected]

doi: 10.1158/1535-7163.MCT-17-0368

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

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Published OnlineFirst December 13, 2017; DOI: 10.1158/1535-7163.MCT-17-0368

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approval in January, 2017 and is currently in phase I trials. Herein,we present the first report of the preclinical antitumor activity ofSCC244 against c-Met–dependent cancer cells in culture andtumor xenografts in mice.

Materials and MethodsReagents

SCC244 [SCC-244, Glumetinib, Fig. 1A] was synthesized atProf. Jingkang Shen's Laboratory at the Shanghai Institute ofMateria Medica. The Protocols for the synthesis of SCC244 arepresented in SupplementaryMaterials andMethods. SCC244wasdissolved in dimethyl sulfoxide at 10 mmol/L and subsequentlyserially diluted to specific concentrations.

Crizotinib and INCB28060 (5) were purchased from SelleckChemicals.

Kinase inhibition assayMet Protein active (His tagged), TyrO3 Protein active (His

tagged), Axl Protein active (His tagged), Ron Protein active (GSTfusion), andMer Protein active (GST fusion)were purchased fromEurofins. Met, Ron, Axl, TyrO3, and Mer kinases activity wereassessed using both ELISA as reported previously (29) and radio-metric protein kinase assays in Eurofins (UK). The kinase selec-tivity profile of SCC244 (1 mmol/L) was screened against a panelof other 308 recombinant kinases using radiometric proteinkinase assays was also performed by Eurofins (UK) according tothe manufacturer's specifications.

For the ATP competition assay, various concentrations of ATPwere diluted for the kinase reaction.

CellsUnless otherwise mentioned, cells were purchased from ATCC.

MKN-45, MKN-1, MKN-74, MKN-28, and EBC-1 cells were pur-chased from Japanese Research Resources Bank. BaF3 and RT-112cells were purchased from Deutsche Sammlung von Mikroorga-nismen und Zellkulturen GmbH. SMMC-7721, MGC-803,U87MG, SPC-A1,NCI-H1299, NCI-H460, NCI-H292, MGC-823,786-O, and BEL-7404 cells were obtained from the Institute ofBiochemistry and Cell Biology, Chinese Academy of Sciences.Human gastric epithelial cell line SGC-7901 cells were obtainedfrom the Shanghai Cancer Institute, Renji Hospital, ShanghaiJiaotong University School of Medicine. SPC-A4 cells wereobtained from Shanghai Chest Hospital, Shanghai Jiaotong Uni-versity School of Medicine. HUVECs were obtained from Scien-Cell Research Laboratories. NCI-H3122 cells were obtained fromthe National Cancer Institute. ACHN cells were obtained fromBeNa Culture Collection. BaF3 cells were engineered to expresshuman wild-type TPR-Met that stably expressed a constitutivelyactive c-Met. All the cell lines used in this study were obtainedduring 2000 to 2015 and cultured according to themanufacturer'sinstructions. Cells were checked to confirmed to be mycoplasma-free, and the cells were passaged no more than 25–30 times afterthawing. Cell lines were characterized by Genesky BiopharmaTechnology using short tandem repeat markers (latest tested in2017).

Antibodies and growth factorsRecombinant human HGF was acquired from PeproTech Inc.

The antibodies specific to phospho-c-Met (Y1234/1235),phospho-AKT (S473), phospho-ERK1/2 (T202/Y204), AKT and

ERK1/2 were purchased from Cell Signaling Technology, theantibodies against phosphotyrosine (PY99), c-Met, and CD31were purchased from Santa Cruz Biotechnology, the antibodyagainst Ki67was purchased from Epitomics Inc, and the antibodyagainst GAPDH was purchased from Kangcheng Bio.

Western blotting analysisCells were cultured under regular growth conditions to the

exponential growth phase. Then, the cells were treated withcompounds for the indicated time and lysed in 1� SDS samplebuffer. If HGF treatmentwas required, cells were starved in serum-free medium for 24 hours and then treated with compounds plusrecombinant human HGF for the appropriate time. The celllysates were subsequently resolved on 10% SDS-PAGE and trans-ferred to nitrocellulose membranes. Proteins were probed with aspecific antibody then subsequently with a secondary horseradishperoxidase–conjugated antibody. Finally, immunoreactive pro-teins were detected using an enhanced chemiluminescence detec-tion reagent.

Cell proliferation assaysCells were seeded in 96-well plates at a low density in growth

media. The next day, appropriate controls or designated con-centrations of compounds were added to each well, and thecells were incubated for 72 hours. HUVECs (passage 3) wereseeded in 96-well plates in growth media overnight and trans-ferred to serum-free media for 24 hours. The following day,appropriate controls or designated concentrations of com-pounds were added to each well, and HGF was added todesignated wells at 100 ng/mL. The cells were incubated for48 hours. Finally, cell proliferation was determined using asulforhodamine B assay, a thiazolyl blue tetrazolium bromideassay or a cell counting kit (CCK-8) assay. IC50 values werecalculated by concentration-response curve fitting using a Soft-Max pro-based four-parameter method.

Cell-cycle analysisThe effects of compounds on cell-cycle progression and pop-

ulation distribution were determined by flow cytometry. Cellswere seeded at 2 � 105 cells in 6-well plates and treated withcompounds at the indicated concentration or with vehicle as acontrol. After 24 hours, the cells were collected, fixed, and stainedwith propidium iodide (10 mg/mL) for 30 minutes and thenanalyzed using a flow cytometer (FACSCalibur instrument; Bec-ton, Dickinson & Co.). Data were plotted using CellQuest soft-ware (Becton, Dickinson & Co).

Tumor cell migration and Matrigel invasion assaysFor the migration assay, NCI-H441 cells suspended in serum-

free medium (1.5 � 105 cells per well) were seeded in 24-wellTranswell plates (pore size, 8 mm; Corning). The bottom cham-berswere filledwith serum-freemedium supplementedwithHGF(100 ng/mL), and appropriate controls or designated concentra-tions of compounds were added to both sides of the membrane.The cultures were maintained for 24 hours, and then the non-motile cells at the top of the filter were removed using a cottonswab. The migrating cells were fixed in paraformaldehyde (4%)and stained with crystal violet (0.1%) for 15 minutes at roomtemperature. For the invasion assay,NCI-H441 cellswere culturedin the top chambers containing Matrigel-coated membraneinserts. The ensuing procedure was identical to that of the

Ai et al.

Mol Cancer Ther; 17(4) April 2018 Molecular Cancer Therapeutics752



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migration assay. The dye that was taken up by the cells bound tothe membrane was released by the addition of 100 mL 10% aceticacid, and the absorbance of the resulting solutionwasmeasured at

595 nm using a multiwell spectrophotometer (SpetraMAX 190,from Molecular Devices). The assay was performed in triplicate.Images were obtained using an Olympus BX51 microscope.

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Figure 1.

SCC244 is a potent and highly selective c-Met inhibitor, with subnanomolar biochemical and nanomolar cellular potency against c-Met. A, The chemicalstructure of SCC244. B, The inhibition curve of SCC244 against c-Met kinase activity. C, The ATP-competitive inhibition of c-Met kinase activity by SCC244. D,Kinase-selectivity profile of SCC244 in 313 protein kinases. E, SCC244 suppresses c-Met phosphorylation and downstream signaling in various cells. EBC-1, MKN-45,and BaF3/TPR-Met cells treated with increasing concentrations of SCC244 for 2 hours were lysed and subjected to Western blot analysis. U87MG cells wereserum-deprived for 24 hours prior to 2-hour treatment with SCC244 and then stimulated with HGF for 15 minutes. Next, the cells were lysed and subjected toWesternblot analysis.

Preclinical Characterization of c-Met Inhibitor SCC244

www.aacrjournals.org Mol Cancer Ther; 17(4) April 2018 753




Madin-Darby canine kidney cell scattering assayMadin-Darby canine kidney cells (MDCK) were plated in

96-well plates and grown overnight. Increasing concentrationsof compounds and HGF (100 ng/mL) were added to the appro-priate wells, and the plates were incubated at 37�C and 5% CO2for 24 hours. The cells were fixed with 4% paraformaldehyde for15 minutes at room temperature and then stained with 0.2%crystal violet. The assay was performed in triplicate. Images wereobtained using an Olympus IX51 microscope.

Cell branching morphogenesisMDCK cells at a density of 2 � 104 cells/mL in DMEM were

mixed with an equal volume of collagen I solution and plated at0.1 mL/well in a 96-well culture plate. After incubation for 1–2hours at 37�C and 5% CO2 to allow the collagen to gel, HGF(100 ng/mL) with or without compounds at various concentra-tions dissolved in 100 mL of growth medium was added to eachwell. The medium was replaced with fresh growth medium every2 days. After 5 days, imageswere obtained using anOlympus IX51microscope.

Animal studiesCDX model. Female nude mice (4–6 weeks old) were housed andmaintained under specific pathogen-free conditions. The tumorcells at a density of 5�106 in 200mLwere injected subcutaneously(s.c.) into the rightflankof nudemice and then allowed to grow to700–800 mm3, which was defined as a well-developed tumor.Subsequently, the well-developed tumors were cut into 1-mm3

fragments and transplanted subcutaneously into the right flankof nude mice using a trocar. When the tumor volume reached100–150 mm3, the mice were randomly assigned into a vehiclecontrol group (n ¼ 12) and treatment groups (n ¼ 6 per group).The control group was given only vehicle, and the treatmentgroups received SCC244 or INCB28060 at the indicated dosesvia oral injection once daily for 2–3weeks. The sizes of the tumorswere measured twice per week using a microcaliper.

PDX model. Animal studies using non–small cell lung cancer(NSCLC) and hepatocellular carcinoma(HCC) PDXmodels wereconducted by WuXi AppTec (Shanghai, China) (LI-03-0022,LI-03-0117, LI-03-0240, LI-03-0317, and LU-01-0439; n ¼ 5 pergroup for LI-03-0022, LI-03-0117, LI-03-0240, LI-03-0317, n¼ 8per group for LU-01-0439) and Crown Bioscience (LI0612,LU1902, LU2071 and LU2503; n ¼ 8 per group). The mice wasgiven only vehicle or SCC244 (10 mg/kg) via oral injection oncedaily for 18–21 days. The sizes of the tumors weremeasured twiceper week using a microcaliper.

Tumor volume ¼ (length� width2)/2. The tumor volume wasshown on indicated days as the median Tumor volume � SEindicated for groups of mice. Tumor growth inhibition (TGI) (%)valuesweremeasuredon thefinal day of study for thedrug-treatedmice compared with vehicle-treated mice and were calculated as100 � {1 � [(VTreated Final day � VTreated Day 0)/(VControl Final day �VControl Day 0)]. Response in individual mouse of PDX model wasalso evaluated on the basis of the Response Evaluation Criteria inSolid Tumors (RECIST guideline, version 1.1; refs. 30, 31).

Study approval. Animal studies using CDXmodels were approvedby the Institutional Animal Care and Use Committee at ShanghaiInstitute of Materia Medica (approval no. 2014-03-DJ-13,

no. 2015-04-DJ-17). Animal studies using PDX models wereapproved by the Institutional Animal Care and Use Committee(IACUC) at WuXi AppTec (approval no. R20150728-Mouse andRat, no. N20160615-Mouse and Rat-B) and at Crown Bioscience(approval No. AN-1507-011-341), respectively.

IHCTumor specimens were fixed in 4% paraformaldehyde for 24

hours. The tumor samples were subsequently paraffin-embeddedand sliced onto microscope slides. After dewaxing and blockingendogenous peroxidase activity with 3% H2O2, the sections wereincubated with 1.5% normal goat serum and then incubatedovernight at 4�C with anti-Ki67 or anti-CD31 antibody. Then,the sections were incubated with biotin-conjugated anti-rabbitIgG for 2 hours at 37�C and then with avidin–biotin–peroxidasecomplex (ABC) for 1 hour using a Vectastain ABC kit (VectorLaboratories). Staining was detected using the DAB (3,30-diami-nobenzidine tetrahydrochloride) Liquid System (ZSGB-Bio) and,in each section, was imaged in 5 different fields. The density ofmicrovessel was assessed by IHC analysis with antibodies to theendothelial marker CD31 and determined according to themeth-od reported previously (32, 33). Briefly, the immunostainedsections were initially screened at low magnifications (40�) toidentify hotspots, which are the areas of highest neovasculariza-tion. Any yellow brown–stained endothelial cell or endothelialcell cluster that was clearly separate from adjacent microvessels,tumor cells, and other connective tissue elements was considereda single, countable microvessel. Within the hotspot area, thestained microvessels were counted in a blinded manner in 12fields (4 fields per section, 3 sections per tissue) of the tissue usinga light microscope at 200�magnification. The number of vesselsin each field was averaged.

Human IL8 immunoassaySerum levels of human IL8 were detected using a Human IL8

Quantikine ELISA Kit (R&D Systems, Inc.) according to themanufacturer's instructions.

Pharmacodynamic studiesTo assess the pharmacodynamics of SCC244 in tumors, mice

bearing established xenograft tumors were treated with a singledose of the compound at 10 or 2.5 mg/kg, and tumors wereharvested at several time points. At a designated time followingadministration, mice were humanely euthanized, and theirtumors were resected. The tumors were snap-frozen in liquidnitrogen and then homogenized in 500 mL of protein extractionsolution (radioimmunoprecipitation assay, RIPA). The tumorextracts were then subjected to Western blot analysis. The indi-vidual bands of phospho-c-Met, phospho-AKT, and phospho-ERKwere scanned and quantified usingGel Pro Analyzer software(Media Cybernetics, Inc.). The relative tyrosine phosphorylationof each sample at the indicated time points was then calculated,with the average value of vehicle-treated sample used as 100%.

Statistical analysisData from in vitro assays are presented as the mean � SD,

whereas data from in vivo efficacy evaluations are presented as themean � SE. Significance was determined by Student t test, anddifferences were considered statistically significant at �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Ai et al.





ResultsSCC244 is ahighly selective c-Met inhibitorwith subnanomolarbiochemical potency

Through medicinal chemistry studies, we have discovered aseries of small-molecule inhibitors of c-Met kinase exemplified bySCC244 (Fig. 1A; ref. 34). SCC244 exhibited high potency (IC50¼0.42 � 0.02 nmol/L) against purified c-Met kinase activity usingELISA kinase assay. The potency was comparable with that ofINCB28060, the most advanced selective c-Met inhibitor, andmuchhigher than that of crizotinib (Fig. 1B; Supplementary TableS1). According to additional kinetic studies, SCC244 is an ATP-competitive c-Met inhibitor (Fig. 1C). Furthermore, to explorewhether this potency was specific for c-Met, SCC244 was evalu-ated against a panel of 312 other kinases. And we found SCC244has greater than 2,400-fold selectivity for c-Met over those 312kinases evaluated, including the c-Met family member RON andhighly homologous kinases Axl, Mer, and TyrO3 (Fig. 1D; Sup-plementary Table S2). These data indicated that SCC244 was ahighly specific c-Met kinase inhibitor.

SCC244 blocks c-Met activation and downstream signaling incancer cells

To investigate whether SCC244 could inhibit c-Met activity incancer cells, EBC-1 andMKN-45 cells harboring an amplifiedMETgene and U87MG cells responsive to HGF stimulation were used.SCC244 was found to strongly inhibit c-Met phosphorylation inall tested cell lines (Fig. 1E), as well as AKT and ERK phosphor-ylation, which are important molecules in the PI3K and Rassignaling axis required for sustaining oncogene addiction (35).Similar results were also observed in a model cell line BaF3/TPR-Met, which contains the MET chromosomal rearrangement(TPR-MET) oncogenic form of c-Met, treated with increasingconcentrations of SCC244 (Fig. 1E). These data indicated thatSCC244 significantly inhibits c-Met activation and signaling,regardless of the mechanistic complexity of c-Met activationacross different cellular contexts.

SCC244 elicits selective and profound effects against c-Met–driven cancer cell proliferation

Because increased c-Met activity has been shown to inducetumor cell proliferation, the antiproliferative effects of SCC244were evaluated against a panel of human cancer cell lines withdistinct genotypes and normal cell lines. SCC244 was prefer-entially efficacious against EBC-1, MKN-45, SNU-5, cancer celllines (Fig. 2A; Supplementary Table S3). The growth of thesecells lines is driven by MET amplification, and they have beenreported to be extremely sensitive to c-Met inhibition (36, 37).SCC244 also significantly inhibited the proliferation of BaF3/TPR-Met cells (Fig. 2A, Supplementary Table S3), which alsofeature c-Met–addicted cell growth. Moreover, the antiproli-ferative activity of SCC244 is comparable with that ofINCB28060 and much potent than that of crizotinib. In con-trast, SCC244 exhibited almost 10,000-fold less potency inother tested cells with low expression or activation of c-Met(Fig. 2A; Supplementary Table S3). c-Met inhibition is knownto exert antiproliferative effects via arresting cells in the G1–Sphase (35, 38); therefore, SCC244 treatment consistentlyinducing G1–S cell-cycle arrest (Fig. 2B and C) further con-firmed that the potent antiproliferative activity of the com-pound was because it specifically targeted c-Met signaling.

Collectively, the data suggested that SCC244 selectively inhib-ited c-Met–driven cell proliferation with high potency.

SCC244 inhibited c-Met–dependent neoplastic phenotypes ofmetastasis and angiogenesis

c-Met is implicated in a variety of tumor cell and endothelialcell functions. Thus, SCC244 was evaluated in a series of cell-based functional assays. Because c-Met activation is important forpromoting cancer cell migration and invasion, both contributorsto tumor metastasis, we evaluated the effect of SCC244 in thisregard. SCC244 strongly suppressed HGF-inducedNCI-H441 cellmotility and invasion in a dose-dependent manner and wassufficient to block the movement of most cells at a dose of10 nmol/L (Fig. 3A–D).

Activation of c-Met drives a complex genetic program termedinvasive growth, which is pivotal in driving cancer cell invasionand metastasis (18, 39). In vitro, this morphogenetic program isreplicated by stimulating cultured MDCK epithelial cells withHGF in a 3D multicellular-branched morphogenesis model and2D scattering assay. We therefore chose cell scattering and mor-phogenesis as two representativemodels to evaluate the impact ofSCC244 on c-Met–mediated invasive growth. As expected,MDCKcells appeared to dissociate and form multicellular-branchedstructures upon HGF stimulation. Moreover, SCC244 showedstrong inhibitory effects on cell scattering (Fig. 3E) and morpho-genesis (Fig. 3F), indicating that SCC244 inhibited HGF-inducedc-Met–mediated invasive growth.

HGF/c-Met signaling is a potent direct inducer of endothelialcell proliferation and thereby promotes angiogenesis, which isalso an important process in tumor formation and metastasis(4, 40). Upon assessing the effect of SCC244 on primary endo-thelial cell proliferation, we found that the compound inhibitedHGF-stimulated c-Met signaling–activation and cell proliferation(Fig. 3G, IC50 ¼ 8.8 � 0.4 nmol/L).

The strong inhibition of c-Met–dependent phenotypes suggeststhat the pharmacologic activity of SCC244 observed in thesefunctional assays is mediated by the inhibition of c-Met.

SCC244 significantly inhibited c-Met–driven tumor growth incancer CDX models

Encouraged by the potency of SCC244 in reversing c-Met–dependentneoplasticphenotypes in vitro,weproceeded toevaluateits antitumor efficacy in vivo. CDX models representative of cancerindications in which the dysregulation of c-Met is implicated wereused, including the gastric carcinomaMKN-45 and SNU-5modelsand lung cancer EBC-1 model. In the MKN-45 model, SCC244significantly inhibited tumor growth with inhibitory rates of99.3%, 88.6%, and 63.6% at doses of 10, 5, and 2.5 mg/kg,respectively. In addition, tumor stasis was observed following a21-day treatment with 5 and 10 mg/kg SCC244 (Fig. 4A). Similarresults were obtained in the SNU-5 model treated with SCC244,and tumor regression was observed in the high dose group(Fig. 4A). In the EBC-1 study, all mice receiving SCC244 exhibiteda greater than 66.0% decrease in tumor mass, and in both the10 and 5 mg/kg treatment groups, 1 of 6 mice exhibited noevidence of a tumor (Fig. 4A). Moreover, in all the tested models,the efficacy of SCC244 at 10 mg/kg is comparable with that ofINCB28060 at 15 mg/kg and crizotinib at 50 mg/kg (Fig. 4A;Supplementary Table S4). In addition, SCC244 was tolerated well,with no significant body weight loss in all treated groups, even atthe highest dose of 50 mg/kg (Supplementary Fig. S1A–S1D).






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SCC244 specifically and potently inhibits proliferation of c-Met–addicted human cancer cells. A, The antiproliferative activity of SCC244 against a panel oftumor cell lines and normal cells originating from different tissue typeswas determined by a sulforhodamine B (SRB) assay, an MTT assay, or a CCK-8 assay. The IC50values were plotted as the mean � SD (nmol/L) or were estimated values from three separate experiments. B and C, SCC244 induced G1–S phase cell-cyclearrest in c-Met–addicted human cancer cells. EBC-1 andMKN-45 cellswere treatedwith the indicated concentrations of SCC244 for 24 hours. The percentages of cellsin different cell-cycle phases determined by FACS and analyzedwithModifit LTwere plotted. The data shown are themean� SD from two independent experiments.

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SCC244 inhibited c-Met–dependent neoplasticphenotypes of metastasis and angiogenesis. A,SCC244 suppressed HGF-induced NCI-H441 cellmigration. Representative images are shown (scalebars, 1 mm). B, SCC244 suppressed HGF-inducedNCI-H441 cell invasion. Representative images areshown (scale bars, 1 mm). C and D, The relativemigration (C) and invasion (D) were plotted. The datashown are the mean � SD from three independentexperiments, assuming 100%migration or invasion ofcells stimulated with HGF. �� , P < 0.001 versus HGF-stimulating group, determined using t test. E, SCC244inhibited HGF-induced scattering of MDCK cells. Cellswere grown as small colonies at low density andtreated with HGF (50 ng/mL) in the presence ofincreasing concentrations of SCC244 for 18 to 24hours. Representative images from three separateexperiments are shown (scale bars, 50 mm). F,SCC244 significantly inhibited HGF-stimulatedinvasive cell growth. The MDCK branchingmorphogenesis in collagen induced by HGF wasinhibited by SCC244. Images were obtained 5 daysafter treatment. Representative images from threeseparate experiments are shown (scale bars, 20 mm).G, SCC244 inhibited HGF-stimulated primary HUVECc-Met signaling and proliferation. Prestarved primaryHUVECs treated with SCC244 for 2 hours and thenstimulated with 100 ng/mL HGF for 15 minutes werelysed and subjected to Western blot analysis.Prestarved primary HUVECs were treated with HGF(100 ng/mL) and increasing concentrations ofSCC244 for 48 hours. Cell viability was measured byCCK-8 assay. Representative data are shown fromthree independent experiments.






SCC244was also evaluated for its effect on tumormitotic index(Ki67) using IHCmethods. A significant dose-dependent decreasein Ki67 levels was observed upon SCC244 treatment in the EBC-1and SNU-5 models (Fig. 4B). The in vivo antiangiogenic effect ofSCC244was assessed for intratumoralmodulation ofmicrovesseldensity (MVD) by immunostaining for platelet endothelial celladhesion molecule 1 (CD31). A significant reduction of CD31-positive endothelial cells was observed upon SCC244 treatment(Supplementary Fig. S1E and S1F). The plasma level of proangio-genic factor IL8, the release of which was stimulated by c-Met/HGF activation, was alsomeasured. SCC244 significantly reducedhuman IL8plasma levels (Fig. 4C). These results indicated that the

antitumor activity of SCC244 is mediated by direct effects ontumor cell growth and by antiangiogenic mechanisms.

To evaluate in vivo c-Met inhibition by SCC244, EBC-1 tumorswere harvested at several time points after administering a singledose of 2.5 or 10 mg/kg SCC244, and the c-Met–signalingphosphorylation in tumors was examined. Almost completeinhibition of c-Met, ERK, and Akt phosphorylation for 8 hoursis observed at the dose of 10mg/kg, which is consistent with near-complete tumor growth inhibition/cytostasis (104.4% TGI). Thedose with suboptimal efficacy (2.5 mg/kg, 66.0% TGI) led topotent inhibition of c-Met signaling for only a portion of theexperiment, with almost full recovery by 8 hours (Fig. 4D). These

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SCC244 significantly inhibited c-Met–driven tumorgrowth in cancer CDXmodels.A, SCC244 inhibits tumor growth inMKN-45, SNU-5, andEBC-1 xenografts. SCC244was administered orally once daily after the tumor volume reached 100 to 200 mm3. Tumor volumes were measured twice a week and are shown as themean � SE. Significant difference from the vehicle group was determined using a t test. �� , P < 0.01; �� , P < 0.001. B, An IHC evaluation of Ki67 expression wasdetermined in the EBC-1 and SNU-5 xenograft models 2 hours after the final administration of SCC244 (scale bars, 100 mm). C, SCC244 reduced secretion ofhuman IL8 in the EBC-1 and SNU-5 xenograft models. Serum levels of human IL8 were determined by ELISA of EBC-1 and SNU-5 xenografts on day 21. �� , P < 0.001versus vehicle group, as determined using a t test. D, Mice bearing EBC-1 subcutaneous tumor xenografts were orally administered 2.5 or 10 mg/kg SCC244only once. Tumors were harvested at several time points, p-c-Met, p-AKT, and p-ERK in tumor samples were detected by immunoblotting, and relativephosphorylation was calculated as described in Materials and Methods and are presented as the mean � SD.

Ai et al.





findings suggest that the extent and duration of c-Met targetinginhibition is due to the antitumor efficacy of SCC244.

SCC244 showed significant antitumor efficiency in NSCLC andHCC tumor PDX models with MET aberration

CDXs have proven utility as models for pharmacologicstudies and as models for efficacy in cases of oncogene addic-tion. However, the effectiveness of CDXs is limited by the lackof molecular and cellular heterogeneity. PDX models offerpromise as improved disease models through their increaseddiversity of molecular lesions and preservation of 3D tumor–stromal cell components and interactions. Thus, we studiedthe antitumor efficacy of SCC244 by interrogating a panel of 4human NSCLC tumor models and 5 HCC tumor modelsknown to harbor MET-driven mutation [gene amplificationor/and Exon 14 skipping mutation (MET ex14)] or c-Metoverexpression (Table 1). We found that SCC244 showedrobust antitumor efficiency in all these 9 models, with thetumor growth–inhibitory rate from 87.7% to 115.8% at theoptimal dose of 10 mg/kg (Fig. 5; Table 1). Tumor shrink wasobserved in some tumor-bearing mice even during the first 7days. Moreover, an undetectable tumor (complete response)was observed in some tumor-bearing mice of the LU2503 andLI0612 models (Fig. 5B and E; Table 1). In addition, twoNSCLC PDX model (LU2071, LU-01-0439) has MET copynumber less than 5, which cannot be considered a clinicallyrelevant gene amplification. But it cannot be ignored that theyhave conspicuous intratumoral c-Met expression, which levelwas comparable with that of the MET-amplified LU1902LU2503 model (Supplementary Fig. S2). Moreover, thesetwo c-Met–overexpressing NSCLC PDX models respondedwell to SCC244 treatment (Fig. 5C and D). Our result isconsistent with the clinical finding that patients with c-Metoverexpression not caused by amplification also respond toc-Met inhibitor (41, 42).

These data further confirmed the high potency of SCC244against c-Met–driven tumor growth.

DiscussionGenotype-based targeted therapies for molecularly selected

populations hold promise for improving the therapeutic out-comes of cancer patients. Mutant MET, a clinically relevantmolecular oncogenic driver, is found in a diverse spectrum ofmalignancies, including NSCLC, gastric cancer, liver cancer, and

esophageal cancer (4, 12, 14, 15, 23, 43–49). Aberrant c-Metactivation is closely associated with tumor formation andmetastasis, as well as resistance to approved therapies(19–21). Therefore, inhibiting c-Met signaling could have sig-nificant potential for the treatment of human cancers driven byc-Met overactivation.

In this study, we reported SCC244, a c-Met inhibitor withsuperior efficacy. SCC244 has subnanomolar biochemical andnanomolar cellular potency against c-Met kinase and c-Met–dependent neoplastic phenotypes, including cancer cell prolifer-ation, migration, invasion, and invasive growth. In 3 c-Met–driven CDX models, SCC244 significantly inhibited tumorgrowth, causing tumor stasis or regression. The efficacy of10 mg/kg SCC244 is comparable with that of 15 mg/kgINCB28060,which is themost advanced c-Met selective inhibitor.PDX models are more informative than standard CDX modelsbecause theymaintain tumor heterogeneity and preserve. We alsoinvestigated the in vivo efficacy of SCC244 in 9 NSCLC and HCCcancer PDX models harboring MET aberration. SCC244 showedsignificant antitumor efficiency in all these models at the optimaldose of 10 mg/kg. SCC244 induced rapid tumor shrink in 2 CDXand 7 PDX models. In these models, tumors rapidly decreased insize during the first 7 days.

Furthermore, most c-Met inhibitors currently undergoing clin-ical trials are multitarget inhibitors, which may cause unwantedoff-target toxicity. In the new era of personalizedmedicine, highlyselective c-Met inhibitors could largely avoid toxicity arising fromtargeting extra molecules and thus could provide a better treat-ment option for the subpopulation of c-Met–driven cancers andserve as a "clean" component for rational combination. Anotheradvantageous characteristic of SCC244 is its high selectivity forc-Met, SCC244 showed at least a 2,400-fold selectivity for c-Metover a panel of 312 kinases, establishing it as one of the mostselective c-Met inhibitors described to date. Consistent with thisobservation, cancer cells with low c-Met activation or expressionweremarkedly less sensitive to SCC244 than c-Met–addicted cells(almost 10,000-fold less potency). Lacking the troublesome issueof off-target kinase inhibition, SCC244 may have a favorablesafety profile. Indeed, SCC244 demonstrated a broad therapeuticwindow inpreclinical studies and induced tumor stasis and shrinkat oral doses of 5 or 10 mg/kg with no body weight loss, even at adose of 50 mg/kg. The preclinical safety profile of SCC244 wasfurther characterized through repeated-dose studies up to 1month in duration in rats and dogs. The SD rats were given oncedaily oral administrationwith SCC244 at 10, 30, or 100mg/kg for

Table 1. Antitumor effect of SCC244 in PDX models with MET aberration used in our study

Number Tumor type Models MET status Copy numbera TGI (%) Treatment responseb

1 NSCLC LU1902 Amp 24.3 94.3 8 (5 SD, 3 PD)2 NSCLC LU2503 Amp, MET ex14 15.4 107.3 8 (4 CR, 4 PR)3 NSCLC LU2071 Overexpression 3.4 112.3 8 (1 PR, 6 SD, 1 PD)4 NSCLC LU-01-0439 Overexpression 3.3 115.8 8 (8 PR)5 HCC LI0612 Amp 28.1 112.1 8 (1 CR, 3 PR, 4 SD)6 HCC LI-03-0022 Amp 5.3 87.7 5 (1 PR, 4 PD)7 HCC LI-03-0117 Amp 5.1 104.2 5 (5 PR)8 HCC LI-03-0240 Amp 5.6 89.2 5 (1 SD, 4 PD)9 HCC LI-03-0317 Amp 5.5 112.9 5 (2 PR, 3 SD)

Abbreviations: Amp, amplification; CR, complete response; HCC, hepatocellular carcinoma; NSCLC, non–small cell lung cancer; PD, progressive disease; PR, partialresponse; SD, stable disease; TGI, tumor growth inhibition.aMET copy number of individual PDX model was obtained using whole exome sequencing provided by Crownbio and SNP6.0 chip by WuXi AppTec, respectively.bResponse in individual mouse of PDX model was also evaluated on the basis of the RECIST guideline, version 1.1






28 days. A No-Observed-Effect-Level (NOEL) of 10 mg/kg/daywas established and the MTD was more than 100 mg/kg/day.Only slight increases in WBC, mainly lymphocytes and neutro-phils, were noted by the end of the dosing phase at the dose of 30and 100 mg/kg/day while recovered by the end of the 28-daydosing-free phase. For repeated-dose study in dog, the Beagledogs were given once daily oral administration with SCC244 at 5,20, or 60 mg/kg for 28 days. The NOAEL was considered to be5 mg/kg/day and the MTD was 60 mg/kg/day. The mainfindings noted by the end of the dosing phase at dose of 20 and60 mg/kg/day included changes in ECG parameters, gastrointes-tinal adverse effects, lymphoid atrophy in thymus as well asincreased myeloid cells and megakaryocytes in the bone marrow

while they were not present by the end of the 28-day recoveryphase. SCC244 achieved a greater than 40-fold therapeutic indexwhen comparing its efficacious dose (tumor stasis) with its MTDin both rats and dogs. Because of its superior potency andfavorable preclinical safety window, SCC244 could maximize thetherapeutic potential of c-Met inhibition alone in human cancersaddicted to c-Met aberrations.

In conclusion, our data demonstrated that SCC244 is apotent and highly selective c-Met kinase inhibitor, able toinduce tumor regression in MET-driven CDX and PDX tumormodels with a favorable preclinical safety profile. Overall, theseresults support its clinical development in patients with MET-addicted tumors.

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SCC244 induces tumor regression in NSCLC and HCC PDX models with MET aberration. Nude mice bearing NSCLC PDXs LU1902 (A), LU2503 (B), LU2071 (C), andLU-01-0439 (D), HCC PDXs LI0612 (E), LI-03-0022 (F), LI-03-0117 (G), LI-03-0240 (H), and LI-03-0317 (I) were administered with vehicle control or SCC244(10 mg/kg) once daily for 18 or 21 consecutive days. Tumor volumes were measured twice a week and are shown as the mean � SE.

Ai et al.





Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Ai, J. Ding, M. GengDevelopment of methodology: X. YangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Chen, X. Peng, Y. Ji, Y. Xi, Y. Shen, Y. Su, Y.-M. Sun,Y. GaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Ai, Y. Chen, X. Peng, M. GengWriting, review, and/or revision of the manuscript: J. Ai, X. Peng, M. GengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Yang, Y. MaStudy supervision: J. Ai, J. Ding, M. GengOther (compound design and synthesis): B. Xiong, J. Shen

AcknowledgmentsThis research was supported by grants from the "Personalized Medicines –

Molecular Signature-based Drug Discovery and Development," Strategic Prior-ity Research Program of the Chinese Academy of Sciences (no. XDA12020000,for M. Geng; XDA12020103, for J. Ai), and the Natural Science Foundationof China (no. 81473243 and 81773762, for J. Ai). We thank Dr. H. Eric Xu(Shanghai Institute of Materia Medica, Chinese Academy of Sciences) forkindly providing MDCK cells.

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 April 26, 2017; revised July 27, 2017; accepted November 30, 2017;published OnlineFirst December 13, 2017.

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2018;17:751-762. Published OnlineFirst December 13, 2017.Mol Cancer Ther Jing Ai, Yi Chen, Xia Peng, et al. ModelsHighly Selective Inhibitor of c-Met in MET-dependent Cancer Preclinical Evaluation of SCC244 (Glumetinib), a Novel, Potent, and

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