1521-0103/350/1/36–45$25.00 http://dx.doi.org/10.1124/jpet.114.214817THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 350:36–45, July 2014Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

SOMG-833, a Novel Selective c-MET Inhibitor, Blocksc-MET–Dependent Neoplastic Effects and ExertsAntitumor Activity s

Hao-tian Zhang, Lu Wang, Jing Ai, Yi Chen, Chang-xi He, Yin-chun Ji, Min Huang,Jing-yu Yang, Ao Zhang, Jian Ding, and Mei-yu GengDepartment of Pharmacology, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China (H.-t.Z., J.-y.Y.);Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research (L.W., J.A., Y.C., C.-x.H., Y.-c.J., M.H., J.D., M.-y.G.)and CAS Key Laboratory of Receptor Research and Synthetic Organic & Medicinal Chemistry Laboratory (A.Z.), Shanghai Instituteof Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China

Received March 20, 2014; accepted April 14, 2014

ABSTRACTThe hepatocyte growth factor/c-MET signaling axis plays animportant role in tumor cell proliferation, metastasis, and tumorangiogenesis, and therefore presents as an attractive targetfor cancer therapy. Notably, most small-molecule c-MET in-hibitors currently undergoing clinical trials are multitargetinhibitors with the unwanted inhibition of additional kinases,often accounting for undesirable toxicity. Here, we discoveredSOMG-833 [3-(4-methylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl) quinoline] as a potent and selective small-molecule c-MET inhibitor, with an average IC50 of 0.93 nMagainst c-MET, over 10,000-fold more potent compared with19 tyrosine kinases, including c-MET family members andhighly homologous kinases. SOMG-833 strongly suppressedc-MET–mediated signaling transduction regardless of mecha-nistic complexity implicated in c-MET activation, includingMET gene amplification,MET gene fusion, and HGF-stimulatedc-MET activation. In a panel of 24 human cancer or genetically

engineered model cell lines, SOMG-833 potently inhibitedc-MET–driven cell proliferation, whereas cancer cells lackingc-MET activation were markedly less sensitive (at least 15-fold) to the treatment. SOMG-833 also suppressed c-MET–mediated migration, invasion, urokinase activity, and invasivegrowth phenotype. In addition, inhibition of primary humanumbilical vascular endothelial cell (HUVEC) proliferation anddownregulation of plasma proangiogenic factor interleukin-8secretion resulted from SOMG-833 treatment, suggestingits significant antiangiogenic properties. Together, theseresults led to the remarkable antitumor efficacy of SOMG-833 in vivo, as demonstrated in c-MET–dependent NIH-3T3/TPR-MET, U-87MG, and EBC-1 xenograft models.Collectively, our results suggested SOMG-833 as a promis-ing candidate for highly selective c-MET inhibition and apowerful tool to investigate the sole role of MET kinase incancer.

IntroductionThe receptor tyrosine kinase c-MET was first discovered

and identified as an oncogene in a fusion form known as TPR-MET (Cooper et al., 1984). Downstream activation of mitogen-activated protein kinase and phosphoinositide-3 kinase waswell established, signaling cascades responsible for the diverseprocess exerted by the hepatocyte growth factor (HGF)/c-METaxis, including proliferation, invasion,metastasis, and angiogenesis

(Birchmeier et al., 2003; Lemmon and Schlessinger, 2010;Trusolino et al., 2010).Several lines of evidence support the significant role of

HGF/c-MET in cancer development (Gherardi et al., 2012).Aberrant c-MET activation resulting from specific geneticlesions, transcriptional upregulation, or ligand-dependent auto-crine or paracrine mechanisms occurred in many types ofcancers (www.vai.org/met/) (Comoglio et al., 2008). Moreover, ithas been shown that the propagation of MET-dependentinvasive growth signals is a general and remarkable featureof highly aggressive tumors, which spawn “pioneer” cells thatmove out, infiltrate adjacent tissues, and establish metastaticlesions (Comoglio and Trusolino, 2002; Trusolino and Comoglio,2002; Boccaccio and Comoglio, 2006). This, together with theobservation that c-MET is expressed by endothelial cells, andthat HGF is a potent angiogenic factor, implies that inhibition

This work was supported by the National Program on Key Basic ResearchProject of China [Grant 2012CB910704]; the National Natural ScienceFoundation [Grants 91229205 and 81102461]; National S&T Major Projects[Grant 2012ZX09301001-007]; and China Marine Commonwealth ResearchProject [Grant 201005022-5].

H.-t.Z., L.W., and J.A. contributed equally to this work.dx.doi.org/10.1124/jpet.114.214817.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: AKT, protein kinase B; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; ELISA, enzyme-linkedimmunosorbent assay; ERK, extracellular signal-regulated kinase; HGF, hepatocyte growth factor; HUVEC, human umbilical vascular endothelialcells; IL-8, interleukin-8; MDCK, Madin2Darby canine kidney epithelial cell; MTT, thiazolyl blue tetrazolium bromide; OD, optical density; PI,propidium iodide; SOMG/SOMG-833, (3-(4-methylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl) quinoline); TPR, translocated promoterregion; TV, tumor volume; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor.

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of the HGF/c-MET signaling axis can potentially interfere withcancer onset and metastasis (You and McDonald, 2008). Inaddition, c-MET signaling is responsible for the resistance acqui-sition of approved therapies (Kentsis et al., 2012; Straussmanet al., 2012; Wilson et al., 2012). All of these emphasize HGF/c-MET as an attractive target for cancer therapy, and severaldifferent therapeutic approaches are being clinically tested(Comoglio et al., 2008).Notably, most c-MET inhibitors currently undergoing clini-

cal trials aremultitarget inhibitors. The unwanted inhibition ofadditional kinases often leads to undesirable toxicity (Broekmanet al., 2011). The broad toxicity profile of multitarget kinaseinhibitors also largely limited their chances in combinationregimens. In contrast, highly selective c-MET inhibitors couldlargely avoid off-target toxicities at therapeutic doses, and favortheir use in drug combinations.More importantly, in the new eraof personalized medicine, where cancer care relies on validatedbiomarkers to identify a patient subpopulation harboring thespecific molecular characteristics that is likely to benefit froma targeted therapy (Dietel and Sers, 2006; Hood and Friend,2011; Ma, 2012), there is a significant need for targeted drugswith high specificity. As such, highly selective c-MET in-hibitors represent the main direction for the development ofc-MET–targeted therapy.Here, we describe a novel, highly selective c-MET inhibitor,

SOMG-833 [3-(4-methylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl) quinolone], that showed strong potencyagainst c-MET kinase, suppressed c-MET phosphorylation,and the downstream signaling in c-MET–overactivated cancercell lines, as well as inhibited c-MET–dependent cellular eventsin tumor cells and primary endothelial cells. Moreover, SOMG-833exhibited significant antitumor activity in several c-MET–drivenxenograft models. All these findings promise SOMG-833 as apotential candidate for c-MET–driven human cancers.

Materials and MethodsCompounds

SOMG-833 was synthesized at Shanghai Institute ofMaterialMedica,Chinese Academy of Sciences (Shanghai, China) as we have reportedpreviously (Wang et al., 2011). This compound was fully characterizedand possessed a purity of 99%. The compound was prepared as a 10 mMstock solution in 100% dimethylsulfoxide and routinely stored at280°C.

Enzyme-Linked Immunosorbent Assay Kinase Assay andATP Competitive Assay

c-MET tyrosine kinase activity was evaluated by enzyme-linkedimmunosorbent assay (ELISA) as described before (He et al., 2014).Details of the procedures are described in Supplemental Materialsand Methods. For ATP competitive assay, various concentrations ofATP were diluted for the kinase reaction. The results were analyzedin Lineweaver-Burk plots.

Cell Culture

Human cancer cell lines SNU-1, SNU-5, AGS, DU-145, NCI-H661,NCI-H441, A549, H1581, Bx-PC3, HepG2, DBTRG, MCF-7, MDA-MB-231, HT-29, HCT-116, A375, and SK-MEL-28 were all purchasedfrom American Type Culture Collection (ATCC, Manassas, VA);human cancer cell lines MKN-45 and EBC-1 were purchased fromJCRB (Japanese Collection of Research Bioresources, Japan) andwere routinely maintained according to ATCC’s or JCRB’s recommen-dations. The BaF3 cell line was purchased from DSMZ (Braunschweig,

Germany). The Madin2Darby canine kidney epithelial cell line(MDCK) was kindly gifted from Prof. H. Eric Xu. SMMC-7721, BGC-803,U-87MG, and BEL-7404 cell lines were obtained from the Institute ofBiochemistry and Cell Biology (Chinese Academy of Sciences); GES-1,and SPC-A4 cell lines were obtained from the Shanghai Cancer Institute,Renji Hospital and Chest Hospital, Shanghai Jiaotong University Schoolof Medicine (Shanghai, China). Primary human umbilical vascularendothelial cells (HUVEC) were purchased from AllCells (Alameda, CA).Cells were cultured according to the suppliers’ instructions. The BaF3/TPR-MET cell line was a genetically generated BaF3 cell line that stablyexpressed a constitutively active oncogenic version of c-MET.

Western Blotting

EBC-1, MKN-45, and BaF3/TPR-MET cells were cultured underregular growth conditions to the exponential growth phase and treatedwith SOMG-833 for 2 hours. A549, NCI-H441, MDCK, and HUVECcells were serum starved for 24 hours and then incubated with thecompound for 2 hours, and 100 ng/ml HGF (PeproTech, Rocky Hill, NJ)was added for an additional 15 minutes. Cells were then lysed in 1 �SDS sample buffer and subsequently resolved by 10% SDS-PAGE andtransferred to nitrocellulose membranes. The membranes were firstprobed with phospho–c-MET, phospho–extracellular signal-regulatedkinase (ERK), ERK, phospho-AKT, AKT (all from Cell Signaling Tech-nology, Danvers, MA), c-MET (from Santa Cruz Biotechnology, SantaCruz, CA), or glyceraldehyde 3-phosphate dehydrogenase (KangChengBiotech, Shanghai, China) antibody and then with antirabbit orantimouse IgG horseradish peroxidase (Jackson ImmunoResearchLaboratories Inc., West Grove, PA). Immunoreactive proteins weredetected using ECL Plus or Femto (Thermo Fisher Scientific, Waltham,MA), and images were captured with ImageQuant LAS 4000 (GEHealthcare, Chalfont St. Giles, UK).

Cell Proliferation/Survival Assays

Tumor cells were seeded in 96-well plates, 3000–8000/well, ingrowth media overnight and then exposed to designated concen-trations of SOMG-833 for 72 hours. A sulforhodamine B (Sigma-Aldrich, St. Louis, MO) or MTT (thiazolyl blue tetrazolium bromide;Sigma-Aldrich) assay was done to determine tumor cell proliferation.HUVEC cells (passage 3) were first serum starved in completemedium (AllCells) for 24 hours and treated by SOMG-833 for 72 hoursin media containing 3% bovine serum albumin and 100 ng/ml HGF.Appropriate controls were conducted [containing 100 ng/ml HGF(HGF1) or not (HGF2)]. A Cell Counting Kit-8 (Dojindo, Shanghai,China) assay was done to determine the viability of HUVEC cells.HGF-dependent proliferation inhibition % 5 [1 2 (ODtreatment 2ODHGF-/ODHGF1 2 ODHGF-)] � 100%. IC50 values were calculatedby concentration-response curve fitting a four-parameter method.

Cell Cycle and Apoptosis Assay

1 � 105 EBC-1 or MKN-45 cells were seeded in six-well plates(Corning, NY) overnight; the following day, cells were treated withdifferent concentrations of SOMG-833 for 24 hours. After treatment,the cells were trypsinized, fixed in 70% ethanol, incubated in 20 ng/mlRNase and 10 ng/ml propidium iodide, and analyzed using a flowcytometer (FACS Calibur; BD, Franklin Lakes, NJ). The data wereanalyzed using softwareModifit LT. Cell apoptosis was determined byan Annexin V-fluorescein isothiocyanate/PI Apoptosis Detection kit(Vazyme, Piscataway, NJ).

Cell Migration and Invasion

For migration assays, NCI-H441 cells suspended in serum-freeDulbecco’s modified Eagle’s medium (DMEM) at a density of 1.5� 105

cells/ml were seeded (0.1 ml) in the plate inserts of the transwellchamber (pore size, 8 mm; Corning Life Sciences, Lowell, MA), andserum-free DMEM (0.6 ml) containing 100 ng/ml HGF (only added tothe lower well) or not with designated concentrations of SOMG-833

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was added. NCI-H441 cells were subsequently cultured for 24 hours.Cells that migrated to the lower wells were then fixed by 90% ethanol,stained by 0.1% crystal violet, and photographed. The crystals stainedon the lower side of the well were dissolved by 100 ml of 10% aceticacid, and the absorbance of the resulting solution was measured at600 nmusing amultiwell spectrophotometer (SpetraMAX190;MolecularDevices, Sunnyvale, CA). The group only stimulated byHGFwas designedas a positive control (HGF1, 100% migrated/invasive). The relativemigration/invasion was calculated as (ODtreated/ODHGF1) � 100%.

For the invasion assay, NCI-H441 cells were cultured in the topchambers containing Matrigel-coated membrane inserts (BD Bio-sciences, San Jose, CA). The ensuing procedure was identical to themigration assay.

Cell Scattering Assay

MDCK cells (1.5� 103 cells per well) were plated into 96-well platesand grown overnight. Increasing concentrations of SOMG-833 and100 ng/ml HGF were added to the appropriate wells and incubated at37°C, 5% CO2 for 24 hours. The cells were fixed with 4% para-formaldehyde, stained by 0.5% violet purple, and photographed undera microscope.

Cell Branching Morphogenesis

Cells at a density of 20,000 cells/ml in serum-free DMEM weremixed with collagen I solution (BD Biosciences) at a proportion of 4:6(the pH was adjusted to alkaline) and then plated at 0.1 ml/well ofa 96-well culture plate and incubated for 45 minutes at 37°C, 5% CO2

to allow collagen gelling. HGF (100 ng/ml) with or without SOMG-833at various concentrations dissolved in 100 ml of DMEM was added toeach well. The mediumwas replaced with fresh growthmedium every2 days. Pictures were taken under a microscope after 4 days.

Urokinase Plasminogen Activator Activity Detection Assays

Urokinase plasminogen activator (uPA) activity detection wascarried out according to protocols reported previously (Webb et al.,2000). Detailed procedures can be found in Supplemental Materialsand Methods.

In Vivo Studies

Animals. Female nu/nu mice (4–6 weeks old) were maintainedunder clean room conditions and housed on particulate air–filteredventilated racks. Animal experiments were performed according toinstitutional ethical guidelines of animal care.

Subcutaneous Xenograft Models in Athymic Mice. Tumor cellsat a density of 5� 106 in 200 ml were first implanted subcutaneously intothe right flank of each nudemouse and then allowed to grow to 700–800mm3, defined as a well-developed tumor. After that, the well-developedtumors were cut into 1-mm3 fragments and transplanted subcutane-ously into the right flank of nude mice using a trocar. When the tumorvolume reached 100–150 mm3, the mice were randomly assigned tocontrol and treatment groups (n 5 5 per group).

Efficacy Studies. Control groups were given normal saline alone,and treatment groups received SOMG-833 via intraperitoneal in-jection once daily. The tumor volume (TV) was calculated as follows:TV5 [length (mm)�width2 (mm2)]/2. RTV (relative tumor volume)5TVDay N/TVDay 0 � 100%. Percent inhibition values were measured onthe final day of the study for drug-treated compared with vehicle-treatedmice and were calculated as follows: (1 – [(treated final day2 treated day 0) /(control final day – control day0)]) � 100%.

Signal Transduction Studies. At designated times (3 days) afterSOMG-833 administration, mice were humanely euthanized and tumorswere resected. Tumors were snap-frozen in liquid nitrogen, proteinlysates were generated, and protein concentrations were determinedusing a bovine serum albumin assay (Thermo Fisher Scientific). Thetotal tissue protein lysates were then conducted by Western blot for

detecting the phospho–c-MET, phospho-ERK, and phospho-AKT (CellTechnology Signaling).

Cytokine Secretion Detection

The serum from EBC-1 xenograft mice was collected from vehicle andSOMG-833–treated groups on the final day (day 14) of the experiment.Cytokine secretion was detected using ELISA assays (70-E-EK1081;MultiSciences Biotech, Hangzhou, China) and a Bio-Plex Pro HumanCytokine 27-Plex Assay (M50-00031YV; Bio-Rad, Hercules, CA).

Statistics

Data were presented as the mean6 S.D. (in vitro) or mean6 S.E.M.(in vivo). The two-tailed Student’s t test was performed to analyzestatistical differences between groups, and P # 0.05, P # 0.01, P #

0.001 were considered significant.

ResultsSOMG-833 Is an ATP-Competitive Inhibitor of c-MET

Kinase with High Selectivity. SOMG-833 was initiallyidentified as a potent small-molecule inhibitor of c-MET withan IC50 of 0.93 6 0.15 nM in a biochemical enzymatic assay(Fig. 1A) (Wang et al., 2011). We were prompted to investigatewhether this potency was specifically against c-MET. A panelof 20 human kinases was profiled, including c-MET familymember RON (macrophage-stimulating protein receptor) andhighly homologous kinase AXL (tyrosine-protein kinase receptorUFO). In contrast to its high potency against c-MET, SOMG-833barely inhibited other 19 tested kinases at concentrations upto 10 mM (Table 1), indicating that SOMG-833 was a selectivec-MET inhibitor.Most kinase inhibitors discovered to date are ATP competi-

tive. To examine whether SOMG-833 functioned in this manner,we evaluated the inhibitory potency of SOMG-833 on c-METactivity using an ATP competitiveness assay. With the in-creasing concentration of ATP, the inhibitory activity of SOMG-833 upon c-MET kinase was decreased. In a Lineweaver-Burkplot, the different concentration curves of SOMG-833 intersectedat a specific point (known as 1/Vmax) at the y-axis with a broadconcentration from 0.32 to 200 nM (Fig. 1B), showing thatSOMG-833 was an ATP-competitive inhibitor. Together, thesedata suggested that SOMG-833 is a potent and selective c-METinhibitor that blocks c-METkinase activity in anATP-competitivemanner.SOMG-833 Inhibits c-MET Phosphorylation and Blocks

Downstream Signals. To confirm cellular effectiveness ofSOMG-833 targeting c-MET kinase, four cell lines with differentmechanisms of c-MET activation were chosen, i.e., humannon–small-cell lung cancer cell line EBC-1 and gastric tumorcell line MKN-45 with MET gene amplification, a geneticallyengineered cell line BaF3/TPR-MET stably expressing a consti-tutively active oncogenic version TPR-MET, and non–small–celllung cancer cell line A549 responsive to HGF stimulation. Thesecells were treated with various concentrations of SOMG-833,and c-MET signaling was examined using Western blot assays(Fig. 2A). The results showed that SOMG-833 inhibited c-METphosphorylation in a dose-dependent manner, with a completeabolishment at 1 mM in all tested cells. Similar results wereobserved in EBC-1 andMKN-45 cells using immunofluorescenceassay (Fig. 2B). ERK1/2 and AKT are the key downstreammolecules of c-METandplay important roles in c-MET functioning.In linewith suppressed c-METphosphorylation, phospho-ERKand

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phospho-AKT were significantly inhibited by SOMG-833 ina dose-dependent manner in all four tested cell lines (Fig. 2A).These data showed that SOMG-833 effectively suppressed c-METsignaling in cancer cells, regardless of mechanistic complexity inc-MET activation across different cellular contexts.SOMG Selectively Inhibits c-MET–Dependent Tumor

Cell Proliferation. Sustained c-MET signaling elevationcould trigger uncontrolled cell proliferation, one of the hall-marks of cancer. We then evaluated the effect of SOMG-833 onc-MET–dependent cell proliferation. SOMG-833 significantlyinhibited the proliferation of EBC-1, MKN-45, SNU-5, and BaF3/TPR-MET cell lines, whose growthwas driven by activated c-METsignaling arising fromMET gene amplification orTPR-MET genefusion, with a mean IC50 value of 0.160–0.457 mM (Fig. 3A;Supplemental Table 1). The inhibitory effect of SOMG-833 onEBC-1 and MKN-45 cell proliferation was further confirmed ina colony formation assay (Fig. 3B). By expanding to a panel ofcancer cell lines originating from different tissues with MET lowexpression or activation, SOMG-833 showed at least over 15-foldless potency (Fig. 3A). These data demonstrated that SOMG-833specifically inhibited c-MET–dependent cancer cell growth.SOMG-833 Inhibits c-MET–Dependent Cell Prolifer-

ation through Arresting Cells at the G1/S Phase. c-METinhibition is known to block cell proliferation via cell cycle

arrest (Bertotti et al., 2009). To confirm whether theantiproliferative activity of SOMG-833 was associated withblockage of c-MET signaling, EBC-1 and MKN-45 cells weretreated with various concentrations of SOMG-833 for 24 hoursand cell-cycle distribution was analyzed. SOMG-833 induceda G1/S phase arrest in the EBC-1 cells, with 82.05% of the cellpopulation in the G1 phase in the presence of 1 mM SOMG-833(versus 55.78% in the control group) (Fig. 3, C and D). Similarresults were recapitulated in MKN-45 cells (Fig. 3, E and F).Consistently, the cyclin-dependent kinase inhibitors p27 andp21 were significantly increased, whereas the expression ofG1/S modulators CylinD1 and CyclinE1 were downregulatedby SOMG-833 (Fig. 3G). Meanwhile, no obvious sub-G1 cellpopulation was observed upon SOMG-833 treatment (Fig. 3, CandE). Treatmentwith SOMG-833 for up to 48 hours displayedno apparent apoptotic cells as detected by annexin V/PI dualstaining (Fig. 3H), suggesting the G1/S phase arrest contrib-utedmost to the proliferation inhibition induced by SOMG-833.SOMG-833 Potentially Inhibits c-MET–Mediated Me-

tastasis. HGF/c-MET axis activation promoted cell invasionandmigration to allow cancer metastasis (Jeffers et al., 1996b).We then tested the effects of SOMG-833 on these processes usingtranswell-based migration and invasion assays. SOMG-833 in-hibited HGF-induced NCI-H441 cell migration (Fig. 4, A and B).Similar results were observed in a wound-healing assay usingMDCK cells (Supplemental Fig. 1). Further, SOMG-833 stronglysuppressed HGF-induced NCI-H441 cell invasion (Fig. 4, C andD) under a conditionwhere no significant viability inhibitionwasobserved (Fig. 4I).Cell invasion and metastasis requires degradation of sur-

rounding ECM. HGF/scattering factor–induced uPA plays acentral role in catalyzing ECM/BM (extracellular matrix/basementmembrane) degradation, mainly through cleavage of plasmin-ogen into the broader specificity protease plasmin (Jefferset al., 1996a). The MDCK cell line was a widely used model toevaluate uPA-plasmin network expression upon HGF stimu-lation (Webb et al., 2000). We found that SOMG-833 inhibitedthe activity of plasmin cleaved by uPA upon HGF stimulationin a dose-dependent manner (Fig. 4G).Upon HGF stimulation, c-MET induces several biologic re-

sponses that collectively give rise to a process known as in-vasive growth, which is pivotal in driving cancer cell invasionand metastasis (Boccaccio and Comoglio, 2006). Thus, we nextexamined whether SOMG-833 inhibited c-MET–associated in-vasive growth. In vitro, this morphogenetic program wasrecapitulated by stimulating cultured MDCK epithelial cells

TABLE 1Kinase-selectivity profile of SOMG-833IC50 values are shown as the mean 6 S.D. or estimated values from three separateexperiments.

Tyrosine Kinase IC50 Tyrosine Kinase IC50

nM nM

c-MET 0.93 6 0.15 RET .10,000RON .10,000 EGFR .10,000Axl .10,000 ErbB2 .10,000Tyro-3 .10,000 ErbB4 .10,000ALK .10,000 c-Src .10,000Flt-1 .10,000 ABL .10,000KDR .10,000 EPH-A2 .10,000c-Kit .10,000 EPH-B2 .10,000PDGFRa .10,000 IGF1R .10,000PDGFRb .10,000 FGFR1 .10,000

ABL, Abelson murine leukemia viral oncogene homolog 1; Axl, tyrosine-proteinkinase receptor UFO; c-Kit, tyrosine-protein kinase kit; c-Src, tyrosine-protein kinaseCSK; EGFR, epidermal growth factor receptor; EPH, Ephrin-type receptor; ErbB2(HER2), human epidermal growth factor receptor 2; ErbB3, human epidermal growthfactor receptor 3; FGFR1, fibroblast growth factor receptor 1; Flt-1, vascular endothelialgrowth factor receptor 1; IGF1R1, insulin-like growth factor 1; VEGFR2, vascularendothelial growth factor receptor 2; PDGFR, platelet-derived growth factor receptor;RET, rearranged during transfection tyrosine kinase; RON, macrophage-stimulatingprotein receptor; Tyro-3, tyrosine-protein kinase receptor.

Fig. 1. (A) Chemical structure of SOMG-833. (B)Lineweaver-Burk double-reciprocal plots depicted theATP-competitive nature of SOMG-833. Velocity versusATP (5, 25, and 625 mM) at varied concentrations ofSOMG-833 (0, 0.32, 1.6, 8, and 200 nM) are shown. Thedata were representative of three independent experiments.

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with HGF in suspension in a three-dimensional extracellularmatrix (collagen) to form multicellular-branched structures,named morphogenesis (Montesano et al., 1991; Jeffers et al.,1996b). In addition, induction of epithelial cell scattering isa unique feature of HGF and is fundamental for HGF/c-METsignaling–elicited invasive growth (Birchmeier et al., 2003).Wetherefore chose these two representativemodels, cell scatteringand morphogenesis, to evaluate the impact of SOMG-833 onc-MET–mediated invasive growth. MDCK cells were stimulatedwith 100 ng/ml HGF in the presence of various concentrationsof SOMG-833. At a concentration of 3 mM, SOMG-833 showedstrong inhibitory effects on cell scattering (Fig. 4E) and morpho-genesis (Fig. 4F), indicating SOMG-833 inhibited HGF-inducedc-MET–mediated invasive growth.In accordance with these effects, SOMG effectively blocked

phosphoinositide 3-kinase–AKT andMAPKs (mitogen-activatedprotein kinases) pathways (Fig. 4H), which mediated c-MET–dependent survival, invasion, and morphogenesis (Zhang andVande Woude, 2003). Together, SOMG-833 showed its potencyagainst c-MET–dependent migration and invasion, thus lower-ing the risk of tumor metastasis.SOMG-833 Inhibits c-MET–Dependent Proliferation

of HUVEC. In addition to its crucial role in cancer cells, HGF/c-MET signaling is a potent inducer of endothelial cell growthand promoted angiogenesis (Grant et al., 1993; Abounaderand Laterra, 2005; Ren et al., 2005; You andMcDonald, 2008).Hence, we also assessed the antiangiogenesis potential of

SOMG-833. As shown in Fig. 4J, SOMG-833 dose-dependentlyinhibited HGF-stimulated growth of primary HUVEC with anaverage IC50 of 0.1 mM. Consistently, HGF-dependent c-METphosphorylation and its downstream signaling were potentlyinhibited in HUVEC cells by SOMG-833 (Fig. 4K).SOMG-833 Shows Strong Antitumor Activity In Vivo.

To assess the in vivo antitumor efficacy of SOMG-833, threerepresentative tumor xenograft models driven by dysregulatedc-METwere chosen: aNIH-3T3/TPR-Metmodel, in which tumorgrowth was driven by constitutive active MET fusion indepen-dent of HGF stimulation; a U-87MG human glioblastoma modelwith HGF and c-MET comprising an autocrine loop; and anEBC-1 xenograft model specifically driven byMET amplification.In the NIH-3T3 model, upon 14-day SOMG-833 adminis-

tration, tumor growth inhibition was observed in the SOMG-833–treated group, with inhibitory rates of 79.0% (P , 0.01)and 51.0% (P, 0.05) at doses of 80 and 40 mg/kg, respectively(Fig. 5A). In the U-87MG glioblastoma model, SOMG-833showed a similar dose-dependent inhibition of tumor growthwith inhibitory rates of 73.0% (P, 0.01) and 48.0% (P, 0.05),respectively (Fig. 5B). In the EBC-1 xenograft model, SOMG-833 strongly inhibited tumor growth inhibition at doses of40 mg/kg (56.0%, P , 0.05) and 80 mg/kg (97.9%, P , 0.001)(Fig. 5C). By examining EBC-1 tumor tissue samples collectedat different time points after SOMG-833 treatment on day 3, weobserved marked inhibition of intratumoral phospho–c-METand its downstream key effectors phospho-AKT and phospho-ERK

Fig. 2. SOMG-833 inhibited c-MET phosphorylation and downstream signaling in various cells. (A) SOMG-833 effectively inhibited the phosphorylationof c-MET and the c-MET pathway downstream effectors ERK1/2 and AKT in EBC-1, MKN-45, BaF3/TPR-MET, and HGF-stimulated A549 cells. EBC-1,MKN-45, and BaF3/TPR-MET cells treated for 2 hours with SOMG-833 at the indicated concentrations were lysed and subjected to Western blotanalysis. A549 cells treated with SOMG-833 for 2 hours following 100 ng/ml HGF stimulation for 15 minutes were then lysed and subjected to Westernblot analysis. (B) Inhibitory effects of SOMG-833 on c-MET phosphorylation in EBC-1 and MKN-45 cells. Cells treated with 0.3 mM SOMG-833 for2 hours were subjected to immunofluorescence straining using specific phospho–c-MET antibody (scale bars, 200 mM). Representative data are shownfrom three independent experiments.

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levels in the SOMG-833–treated group (Fig. 5F), suggesting theinhibition of tumor growth by the administration of SOMG-833was associated with the blockage of c-MET signaling.c-MET–driven cancer malignancy is mediated by its impact

on both tumor cells and endothelial cells. The intratumoral

mitotic index (Ki67) was assessed using immunohistochemicalanalysis. A significant decrease in Ki67 expression level wasobserved at 80 mg/kg per day of SOMG-833 in the EBC-1xenograft models (Fig. 5D), indicating the potent inhibition ofmitogenesis in vivo.Meanwhile, the contribution of antiangiogenic

Fig. 3. SOMG-833 inhibited c-MET–dependent cell proliferation throughG1/S cell cycle arrest. (A) Cell linesseeded in 96 wells were incubated witha range of concentrations of SOMG-833for 72 hours, and then cell proliferationwas determined using MTT assays orthe sulforhodamine B assays. The IC50values of SOMG-833 are plotted as themean 6 S.D. (micromolar) or estimatedvalues from three independent experi-ments. (B) Proliferation inhibition onEBC-1 and MKN-45 cells treated bySOMG-833 (0.3 mM) was determinedusing clone formation assay. (C–F) In-duction of G1 phase arrest by SOMG-833. Cells were treated with increasingconcentrations of SOMG-833 or vehiclefor 24 hours. The DNA content was mea-sured by fluorescence-activated cell sort-ing (FACS) analysis (C and E). Thepercentage of EBC-1 (D) and MKN-45 (F)cells in different cell cycle phases deter-mined by FACS and analyzed with Mod-ifitLT V3.0 was plotted. The data areshown as the mean 6 S.D. and represen-tative images are shown. (G) EBC-1 andMKN-45 cells were treated with SOMG-833 for 24 hours, and indicated G0/G1-S–phase regulated proteins were analyzedby immunoblot. (H) Apoptosis detection ofMKN-45 cells treated by 0.3 mM SOMG-833 for 48 hours using annexin V and PIdouble staining. The percentage of earlyapoptotic cells (bottom-right corner) andlate apoptotic cells (top-right corner) areshown as indicated. Representative dataare shown from three independent experi-ments. CDK2, cyclin-dependent kinase 2.

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Fig. 4. SOMG-833 potently inhibited HGF-induced c-MET–dependent cell metastasis and angiogenesis. (A and C) The migratory ability (A) andinvasive ability (C) of NCI-H441 cells induced by 100 ng/ml HGFwas impaired by SOMG-833. Representative pictures are shown (scale bars, 200 mM). (Band D) The relative quantitative determination of migrated and invasive cells were plotted. The data shown are the mean6 S.D. from three independentexperiments, assuming 100%migration or invasion of cells stimulated with HGF. (E) Inhibition of HGF-dependent MDCK cell scattering by SOMG-833at indicated concentrations for 24 hours. Partial enlarged view of cluster of cells is shown (scale bars, 200 mM). (F) SOMG-833 inhibited the MDCKbranching morphogenesis on collagen stimulated by HGF. The “2” and “+” represent the HGF (100 ng/ml)-untreated and -treated groups. Images werephotographed 4 days after treatment. Scale bars, 100 mM. (G) SOMG-833 inhibited HGF-induced uPA activation in MDCK cells. Cells stimulatedor untreated with HGF are shown as HGF+ or HGF-. Data are presented as the mean 6 S.D. (H) SOMG-833 inhibited c-MET phosphorylation and

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efficacy was assessed. SOMG-833 was evaluated for modulationof microvessel density assessed by immunostaining for CD31(platelet endothelial cell adhesion molecule 1). At the 80 mg/kgper day dose, the SOMG-833–treated sample showed a sig-nificant reduction of CD31-positive microvessels (Fig. 5D).Moreover, we found that the levels of proangiogenic factorinterleukin-8 (IL-8), which is regulated by c-MET/HGF (Yoshidaet al., 1997; Li et al., 2003; Zhang et al., 2003), in the SOMG-833(80 mg/kg)–treated group, were downregulated compared withthe vehicle group using ELISA assay (Fig. 5E) and Bio-200System (Bio-Rad Laboratories) (Supplemental Fig. 2). Theseresults indicated that the antitumor activity of SOMG-833 ismediated by direct effects on tumor cell growth as well as anti-angiogenic mechanisms. Together, SOMG-833 showed robustantitumor efficacy which was correlated with the inhibitionof c-MET–mediated signaling in c-MET–dependent tumormodels.

DiscussionAberrant c-MET activation has been frequently found in

many human solid tumors and hematologic malignancies.Overactivation of c-MET is known to initiate tumorigenesisand promote metastasis, as well as cause therapeutic re-sistance (Engelman et al., 2007), underscoring the importanceof developing therapeutic strategies capable of interruptingc-MET signaling (Gherardi et al., 2012). In fact, recent clinicaltrials of c-MET pathway-targeted agents have yielded convinc-ing evidence for the benefit of targeting c-MET in cancer therapy,including monotherapy and combined therapy (Sequist et al.,2011; Bendell et al., 2013). Previously, we conducted a screen todiscover specific c-MET inhibitors, and SOMG-833 was selectedfor further characterization of c-MET–targeting antitumor efficacy(Wang et al., 2011).An important feature of SOMG-833 is its selectivity against

c-MET. SOMG-833 presented an IC50 for c-MET in thenanomolar range and showed at least more than 1000-foldselectivity over a panel of 19 human kinases, including c-METfamily member Ron (Wang et al., 2003). Consistently, cancercells with low c-MET activity were markedly less sensitive (atleast 15-fold) to SOMG-833 than c-MET–addicted cells. It isworth noting that most of the reported c-MET kinase inhibitorsbeing clinically evaluated are multitarget inhibitors, oftenresulting in unwanted broad nonspecific toxicity (Broekmanet al., 2011). Highly selective for c-MET kinase inhibition,SOMG-833 could specifically achieve the therapeutic potential ofc-MET inhibition in patients harboring c-MET aberrations, andits use in biomarker-directed drug combinations in personal-ized medicines becomes possible. In addition, the feature ofhigh selectivity makes SOMG-833 suitable for use as a toolinhibitor in preclinical models to dissect the role of c-METkinase activity in cancer progression.

Activation of c-MET drives a complex morphogenetic pro-gram termed invasive growth (Boccaccio and Comoglio, 2006).Under normal conditions, invasive growth is based upon afinely tuned interplay between related phenomena, includingcell proliferation, motility, ECM degradation, and survival. Intransformed tissues, aberrant implementation of this interplayis responsible for cancer progression and metastasis (Comoglioand Trusolino, 2002). In our study, using a series of cell models,we were able to dissect the key biologic steps of invasivegrowth, including cell proliferation, scattering, migration, andinvasion, and found potency of SOMG-833 against theseindividual steps. Moreover, SOMG-833 reversed the compre-hensive three-dimensional branching morphogenesis pheno-type stimulated by HGF, further confirming the strong inhibitoryeffect of SOMG-833 on c-MET–mediated invasive growth. All ofthese indicated a potential role of SOMG-833 against tumorprogression and metastasis.HGF and its receptor c-MET have been implicated in the

regulation of tumor angiogenesis through multiple mecha-nisms (Zhang et al., 2003). In the present study, SOMG-833showed the ability to inhibit HGF-stimulated c-MET–mediatedendothelial cell survival, and to reduce microvessel density inthe EBC-1 model. In addition to their reported direct role inregulating endothelial cell function, c-MET and HGF are alsoimplicated in the regulation of secretion of angiogenic factorsby epithelial and tumor cells (Zhang et al., 2003; Knowleset al., 2009; Hill et al., 2012). Zou et al. (2007) have reportedthat PF-2341066 (Crizotinib; Pfizer, New York, NY) coulddecrease IL-8 and VEGF in c-MET–dependent gastricGTL-16 and glioblastoma U-87MG models, and Torti et al.have found that reduced secretion of IL-8 may serve as anindicative biomarker responding to c-MET inhibition bypharmacological inhibitors in c-MET–dependent gastricxenografts (Torti et al., 2012). Consistently, we foundSOMG-833 could downregulate serum IL-8 level in EBC-1xenograft, indicating that antiangiogenic activity observedwith SOMG-833 may be mediated by direct and indirectmechanisms. However, the serum VEGF in the vehicle groupwas very low in the EBC-1 xenograft (,20 pg/ml) and almost nochanges were observed upon SOMG-833 treatment (data notshown), suggesting that the regulation of angiogenic factor typesby the HGF/c-MET axis are not common to all tumor types.In conclusion, we assessed the antitumor efficacy of SOMG-

833, a novel selective c-MET inhibitor. SOMG-833 showedstrong potency against c-MET kinase, and inhibited c-METphosphorylation and the downstream signaling across differ-ent oncogenic forms in c-MET overactivated cancer cells.In turn, SOMG-833 inhibited c-MET–driven cellular pheno-type in tumor cells and primary endothelial cells. Further-more, SOMG-833 treatment resulted in significant antitumoractivity in several c-MET–driven xenografts. In addition,intratumoral inhibition of c-MET phosphorylation, proliferation

downstream signaling in HGF-stimulated NCI-H441 and MDCK cells. NCI-H441 and MDCK cells treated with SOMG-833 for 2 hours following 100 ng/mlHGF stimulation for 15 minutes were then lysed and subjected to Western blot analysis. (I) The effects of SOMG-833 on NCI-H441 cell viability. Cellviability of NCI-H441 treatedwith SOMG-833 for 24 hours at indicated concentrations was determined byMTT assay. The data shown are themean6 S.D.(J) SOMG-833 inhibited HGF-dependent cell proliferation of primary HUVEC cells. Primary HUVEC cells were treated by SOMG-833 (0.1, 0.3, and 1 mM)for 72 hours with HGF (100 ng/ml). Cell viability was measured by Cell Counting Kit-8 assay. (K) SOMG-833 inhibited c-MET phosphorylation anddownstream signaling in HGF-stimulated primary HUVEC cells. Primary HUVEC cells treated with SOMG-833 for 2 hours following 100 ng/ml HGFstimulation for 15 minutes were then lysed and subjected to Western blot analysis. Representative data are shown from three independent experiments.

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index (Ki67), and IL-8 level suggested that the concurrent anti-proliferative and antiangiogenic activity of SOMG-833 accountedfor its anticancer efficacy in vivo.

Acknowledgments

The authors thank Prof. H. Eric Xu (Shanghai Institute of MateriaMedica) for providing the MDCK cells.

Authorship Contributions

Participated in research design: Geng, Yang, Ai, Ding.Conducted experiments: H.-t. Zhang, Wang, Chen, He, Ji.Contributed new reagents or analytic tools: A. Zhang.Performed data analysis: H.-t. Zhang, Wang, Chen, Ai, Geng.Wrote or contributed to the writing of the manuscript: H.-t. Zhang,

Ai, Huang, Geng.

Fig. 5. Antitumor efficacy of SOMG-833 in vivo (A–C). Tumor growth inhibition upon SOMG-833 treatment in NIH-3T3/TPR-MET (A), U-87MG (B), and EBC-1(C) xenografts. SOMG-833was administered intraperitoneally once daily for 14–21 days after the tumor volume reached 100–150mm3. Results are expressed asthe mean6 S.E.M. (n = 5 per group). The percentage of tumor volume inhibition values (Inh.) was measured on the final day of the study for the drug-treatedmice compared with the control mice. *P , 0.05, **P , 0.01, and ***P , 0.001 versus vehicle group, using Student’s t test. (D) An immunohistochemicalevaluation of Ki67, phospho–c-MET, and CD31 expression at 80 mg/kg SOMG-833 was determined for the EBC-1 xenografts on the day 14. Representativeimages are shown (scale bar, 1 mm). Partially enlarged views are demonstrated in the upper corners. (E) Serum levels of human IL-8 at 80 mg/kg SOMG-833were determined by ELISA assay of EBC-1 xenografts on day 14. The data shown are the mean 6 S.D. (F) Inhibition of c-MET signaling transduction uponSOMG-833 treatment of EBC-1 xenografts.Mice were humanely euthanized on study day 3 at 0.5 and 2 hours postadministration of SOMG-833 and the tumorswere resected. Protein extracts from tumor tissues were analyzed for phospho–c-MET levels and downstream effectors phospho-ERK and phospho-AKT.

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Address correspondence to: Jing-yu Yang, Department of Pharmacology,Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic ofChina. E-mail: yangjingyu2007@gmail.com and Mei-yu Geng, Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, ShanghaiInstitute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,People’s Republic of China. E-mail: mygeng@simm.ac.cn

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