Because the receptor tyrosine kinase c-Met plays a critical role in tumor growth, metastasis, tumor angiogenesis, and drug resistance, the c-Met axis represents an attractive therapeutic target. Herein, we report the first preclinical characterization of SCC244, a novel, potent, and highly selective inhibitor of c-Met kinase. SCC244 showed subnanomolar potency against c-Met kinase activity and high selectivity versus 312 other tested protein kinases, making it one of the most selective c-Met inhibitors described to date. Moreover, this inhibitor profoundly and specifically inhibits c-Met signal transduction and thereby suppresses the c-Met–dependent neoplastic phenotype of tumor and endothelial cells. In xenografts of human tumor cell lines or non–small cell lung cancer and hepatocellular carcinoma patient-derived tumor tissue driven by MET aberration, SCC244 administration exhibits robust antitumor activity at the well-tolerated doses. In addition, the in vivo antitumor activity of SCC244 involves the inhibition of c-Met downstream signaling via a mechanism of combined antiproliferation and antiangiogenic effects. The results of the current study provide a strong foundation for the clinical investigation of SCC244 in patients with tumors harboring c-Met pathway alterations. Mol Cancer Ther; 17(4); 751–62. ©2017 AACR.

c-Met is the prototypical member of a subfamily of receptor tyrosine kinases (RTK) that also includes RON. c-Met is structurally distinct from other RTKs and is the only known high-affinity receptor for hepatocyte growth factor (HGF; refs. 1, 2). Activation of the HGF/c-Met pathway provides a powerful signal for cell proliferation, survival, migration, invasion, angiogenesis, and morphogenic differentiation. The normal functions of the c-Met pathway are largely restricted to organ morphogenesis during development and to tissue damage repair and regeneration in adults (3–5).

c-Met and HGF are highly expressed in numerous cancers relative to the surrounding tissue, and their elevated expression is correlated with poor prognosis (3, 6–15). In addition to HGF stimulation, 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 a general and important feature of highly aggressive tumors (16–18). More importantly, in addition to its role as an oncogenic driver, c-Met has been increasingly implicated as a key mediator of acquired 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 undergoing preclinical and clinic assessment over the past decade, none of them have been approved for clinical use (14, 15, 24–28). Some of the c-Met pathway–targeted agents currently being evaluated in clinical trials are limited by their selectivity, potency, and safety profile, which may considerably hamper their ability to achieve optimal inhibition of the pathway at a better tolerated dose in cancer patients. More importantly, in the era of precision medicine, a highly selective c-Met inhibitor could be significantly beneficial 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, with properties that may overcome the aforementioned limitations. SCC244 is orally bioavailable, demonstrates greater than 2,400-fold selectivity for c-Met over a panel of 312 kinases, and has an IC50 in the subnanomolar range. This inhibitor potently and specifically inhibits c-Met–mediated signal transduction and the c-Met–dependent neoplastic phenotype of tumor and endothelial 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-tolerated doses and showed a superior safety margin. On the basis of this impressive preclinical activity, SCC244 received CFDA IND approval in January, 2017 and is currently in phase I trials. Herein, we present the first report of the preclinical antitumor activity of SCC244 against c-Met–dependent cancer cells in culture and tumor xenografts in mice.

Reagents

SCC244 [SCC-244, Glumetinib, Fig. 1A] was synthesized at Prof. Jingkang Shen's Laboratory at the Shanghai Institute of Materia Medica. The Protocols for the synthesis of SCC244 are presented in Supplementary Materials and Methods. SCC244 was dissolved in dimethyl sulfoxide at 10 mmol/L and subsequently serially diluted to specific concentrations.

Figure 1.

SCC244 is a potent and highly selective c-Met inhibitor, with subnanomolar biochemical and nanomolar cellular potency against c-Met. A, The chemical structure 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 were serum-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 to Western blot analysis.

Figure 1.

SCC244 is a potent and highly selective c-Met inhibitor, with subnanomolar biochemical and nanomolar cellular potency against c-Met. A, The chemical structure 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 were serum-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 to Western blot analysis.

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Crizotinib and INCB28060 (5) were purchased from Selleck Chemicals.

Kinase inhibition assay

Met Protein active (His tagged), TyrO3 Protein active (His tagged), Axl Protein active (His tagged), Ron Protein active (GST fusion), and Mer Protein active (GST fusion) were purchased from Eurofins. Met, Ron, Axl, TyrO3, and Mer kinases activity were assessed using both ELISA as reported previously (29) and radiometric protein kinase assays in Eurofins (UK). The kinase selectivity profile of SCC244 (1 μmol/L) was screened against a panel of other 308 recombinant kinases using radiometric protein kinase assays was also performed by Eurofins (UK) according to the manufacturer's specifications.

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

Cells

Unless otherwise mentioned, cells were purchased from ATCC. MKN-45, MKN-1, MKN-74, MKN-28, and EBC-1 cells were purchased from Japanese Research Resources Bank. BaF3 and RT-112 cells were purchased from Deutsche Sammlung von Mikroorganismen 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 of Biochemistry and Cell Biology, Chinese Academy of Sciences. Human gastric epithelial cell line SGC-7901 cells were obtained from the Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine. SPC-A4 cells were obtained from Shanghai Chest Hospital, Shanghai Jiaotong University School of Medicine. HUVECs were obtained from ScienCell Research Laboratories. NCI-H3122 cells were obtained from the National Cancer Institute. ACHN cells were obtained from BeNa Culture Collection. BaF3 cells were engineered to express human wild-type TPR-Met that stably expressed a constitutively active c-Met. All the cell lines used in this study were obtained during 2000 to 2015 and cultured according to the manufacturer's instructions. Cells were checked to confirmed to be mycoplasma-free, and the cells were passaged no more than 25–30 times after thawing. Cell lines were characterized by Genesky Biopharma Technology using short tandem repeat markers (latest tested in 2017).

Antibodies and growth factors

Recombinant 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, the antibodies against phosphotyrosine (PY99), c-Met, and CD31 were purchased from Santa Cruz Biotechnology, the antibody against Ki67 was purchased from Epitomics Inc, and the antibody against GAPDH was purchased from Kangcheng Bio.

Western blotting analysis

Cells were cultured under regular growth conditions to the exponential growth phase. Then, the cells were treated with compounds for the indicated time and lysed in 1× SDS sample buffer. If HGF treatment was required, cells were starved in serum-free medium for 24 hours and then treated with compounds plus recombinant human HGF for the appropriate time. The cell lysates were subsequently resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes. Proteins were probed with a specific antibody then subsequently with a secondary horseradish peroxidase–conjugated antibody. Finally, immunoreactive proteins were detected using an enhanced chemiluminescence detection reagent.

Cell proliferation assays

Cells were seeded in 96-well plates at a low density in growth media. The next day, appropriate controls or designated concentrations of compounds were added to each well, and the cells were incubated for 72 hours. HUVECs (passage 3) were seeded in 96-well plates in growth media overnight and transferred to serum-free media for 24 hours. The following day, appropriate controls or designated concentrations of compounds were added to each well, and HGF was added to designated wells at 100 ng/mL. The cells were incubated for 48 hours. Finally, cell proliferation was determined using a sulforhodamine B assay, a thiazolyl blue tetrazolium bromide assay or a cell counting kit (CCK-8) assay. IC50 values were calculated by concentration-response curve fitting using a SoftMax pro-based four-parameter method.

Cell-cycle analysis

The effects of compounds on cell-cycle progression and population distribution were determined by flow cytometry. Cells were seeded at 2 × 105 cells in 6-well plates and treated with compounds at the indicated concentration or with vehicle as a control. After 24 hours, the cells were collected, fixed, and stained with propidium iodide (10 μg/mL) for 30 minutes and then analyzed using a flow cytometer (FACSCalibur instrument; Becton, Dickinson & Co.). Data were plotted using CellQuest software (Becton, Dickinson & Co).

Tumor cell migration and Matrigel invasion assays

For the migration assay, NCI-H441 cells suspended in serum-free medium (1.5 × 105 cells per well) were seeded in 24-well Transwell plates (pore size, 8 μm; Corning). The bottom chambers were filled with serum-free medium supplemented with HGF (100 ng/mL), and appropriate controls or designated concentrations of compounds were added to both sides of the membrane. The cultures were maintained for 24 hours, and then the nonmotile cells at the top of the filter were removed using a cotton swab. The migrating cells were fixed in paraformaldehyde (4%) and stained with crystal violet (0.1%) for 15 minutes at room temperature. For the invasion assay, NCI-H441 cells were cultured in the top chambers containing Matrigel-coated membrane inserts. The ensuing procedure was identical to that of the migration assay. The dye that was taken up by the cells bound to the membrane was released by the addition of 100 μL 10% acetic acid, and the absorbance of the resulting solution was measured 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.

Madin-Darby canine kidney cell scattering assay

Madin-Darby canine kidney cells (MDCK) were plated in 96-well plates and grown overnight. Increasing concentrations of compounds and HGF (100 ng/mL) were added to the appropriate wells, and the plates were incubated at 37°C and 5% CO2 for 24 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then stained with 0.2% crystal violet. The assay was performed in triplicate. Images were obtained using an Olympus IX51 microscope.

Cell branching morphogenesis

MDCK cells at a density of 2 × 104 cells/mL in DMEM were mixed with an equal volume of collagen I solution and plated at 0.1 mL/well in a 96-well culture plate. After incubation for 1–2 hours at 37°C and 5% CO2 to allow the collagen to gel, HGF (100 ng/mL) with or without compounds at various concentrations dissolved in 100 μL of growth medium was added to each well. The medium was replaced with fresh growth medium every 2 days. After 5 days, images were obtained using an Olympus IX51 microscope.

Animal studies

CDX model.

Female nude mice (4–6 weeks old) were housed and maintained under specific pathogen-free conditions. The tumor cells at a density of 5 × 106 in 200 μL were injected subcutaneously (s.c.) into the right flank of nude mice and then allowed to grow to 700–800 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 flank of nude mice using a trocar. When the tumor volume reached 100–150 mm3, the mice were randomly assigned into a vehicle control group (n = 12) and treatment groups (n = 6 per group). The control group was given only vehicle, and the treatment groups received SCC244 or INCB28060 at the indicated doses via oral injection once daily for 2–3 weeks. The sizes of the tumors were measured twice per week using a microcaliper.

PDX model.

Animal studies using non–small cell lung cancer(NSCLC) and hepatocellular carcinoma(HCC) PDX models were conducted by WuXi AppTec (Shanghai, China) (LI-03-0022, LI-03-0117, LI-03-0240, LI-03-0317, and LU-01-0439; n = 5 per group for LI-03-0022, LI-03-0117, LI-03-0240, LI-03-0317, n = 8 per group for LU-01-0439) and Crown Bioscience (LI0612, LU1902, LU2071 and LU2503; n = 8 per group). The mice was given only vehicle or SCC244 (10 mg/kg) via oral injection once daily for 18–21 days. The sizes of the tumors were measured twice per week using a microcaliper.

Tumor volume = (length × width2)/2. The tumor volume was shown on indicated days as the median Tumor volume ± SE indicated for groups of mice. Tumor growth inhibition (TGI) (%) values were measured on the final day of study for the drug-treated mice compared with vehicle-treated mice and were calculated as 100 × {1 − [(VTreated Final day − VTreated Day 0)/(VControl Final day − VControl Day 0)]. Response in individual mouse of PDX model was also evaluated on the basis of the Response Evaluation Criteria in Solid Tumors (RECIST guideline, version 1.1; refs. 30, 31).

Study approval.

Animal studies using CDX models were approved by the Institutional Animal Care and Use Committee at Shanghai Institute of Materia Medica (approval no. 2014-03-DJ-13, no. 2015-04-DJ-17). Animal studies using PDX models were approved by the Institutional Animal Care and Use Committee (IACUC) at WuXi AppTec (approval no. R20150728-Mouse and Rat, no. N20160615-Mouse and Rat-B) and at Crown Bioscience (approval No. AN-1507-011-341), respectively.

IHC

Tumor specimens were fixed in 4% paraformaldehyde for 24 hours. The tumor samples were subsequently paraffin-embedded and sliced onto microscope slides. After dewaxing and blocking endogenous peroxidase activity with 3% H2O2, the sections were incubated with 1.5% normal goat serum and then incubated overnight at 4°C with anti-Ki67 or anti-CD31 antibody. Then, the sections were incubated with biotin-conjugated anti-rabbit IgG for 2 hours at 37°C and then with avidin–biotin–peroxidase complex (ABC) for 1 hour using a Vectastain ABC kit (Vector Laboratories). Staining was detected using the DAB (3,3′-diaminobenzidine tetrahydrochloride) Liquid System (ZSGB-Bio) and, in each section, was imaged in 5 different fields. The density of microvessel was assessed by IHC analysis with antibodies to the endothelial marker CD31 and determined according to the method reported previously (32, 33). Briefly, the immunostained sections were initially screened at low magnifications (40×) to identify hotspots, which are the areas of highest neovascularization. Any yellow brown–stained endothelial cell or endothelial cell cluster that was clearly separate from adjacent microvessels, tumor cells, and other connective tissue elements was considered a single, countable microvessel. Within the hotspot area, the stained microvessels were counted in a blinded manner in 12 fields (4 fields per section, 3 sections per tissue) of the tissue using a light microscope at 200× magnification. The number of vessels in each field was averaged.

Human IL8 immunoassay

Serum levels of human IL8 were detected using a Human IL8 Quantikine ELISA Kit (R&D Systems, Inc.) according to the manufacturer's instructions.

Pharmacodynamic studies

To assess the pharmacodynamics of SCC244 in tumors, mice bearing established xenograft tumors were treated with a single dose of the compound at 10 or 2.5 mg/kg, and tumors were harvested at several time points. At a designated time following administration, mice were humanely euthanized, and their tumors were resected. The tumors were snap-frozen in liquid nitrogen and then homogenized in 500 μL of protein extraction solution (radioimmunoprecipitation assay, RIPA). The tumor extracts were then subjected to Western blot analysis. The individual bands of phospho-c-Met, phospho-AKT, and phospho-ERK were scanned and quantified using Gel Pro Analyzer software (Media Cybernetics, Inc.). The relative tyrosine phosphorylation of each sample at the indicated time points was then calculated, with the average value of vehicle-treated sample used as 100%.

Statistical analysis

Data from in vitro assays are presented as the mean ± SD, whereas data from in vivo efficacy evaluations are presented as the mean ± SE. Significance was determined by Student t test, and differences were considered statistically significant at *, P < 0.05; **, P < 0.01; ***, P < 0.001.

SCC244 is a highly selective c-Met inhibitor with subnanomolar biochemical potency

Through medicinal chemistry studies, we have discovered a series of small-molecule inhibitors of c-Met kinase exemplified by SCC244 (Fig. 1A; ref. 34). SCC244 exhibited high potency (IC50 = 0.42 ± 0.02 nmol/L) against purified c-Met kinase activity using ELISA kinase assay. The potency was comparable with that of INCB28060, the most advanced selective c-Met inhibitor, and much higher than that of crizotinib (Fig. 1B; Supplementary Table S1). According to additional kinetic studies, SCC244 is an ATP-competitive c-Met inhibitor (Fig. 1C). Furthermore, to explore whether this potency was specific for c-Met, SCC244 was evaluated against a panel of 312 other kinases. And we found SCC244 has greater than 2,400-fold selectivity for c-Met over those 312 kinases evaluated, including the c-Met family member RON and highly homologous kinases Axl, Mer, and TyrO3 (Fig. 1D; Supplementary Table S2). These data indicated that SCC244 was a highly specific c-Met kinase inhibitor.

SCC244 blocks c-Met activation and downstream signaling in cancer cells

To investigate whether SCC244 could inhibit c-Met activity in cancer cells, EBC-1 and MKN-45 cells harboring an amplified MET gene and U87MG cells responsive to HGF stimulation were used. SCC244 was found to strongly inhibit c-Met phosphorylation in all tested cell lines (Fig. 1E), as well as AKT and ERK phosphorylation, which are important molecules in the PI3K and Ras signaling 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 increasing concentrations of SCC244 (Fig. 1E). These data indicated that SCC244 significantly inhibits c-Met activation and signaling, regardless of the mechanistic complexity of c-Met activation across 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 induce tumor cell proliferation, the antiproliferative effects of SCC244 were evaluated against a panel of human cancer cell lines with distinct genotypes and normal cell lines. SCC244 was preferentially efficacious against EBC-1, MKN-45, SNU-5, cancer cell lines (Fig. 2A; Supplementary Table S3). The growth of these cells lines is driven by MET amplification, and they have been reported 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 also feature c-Met–addicted cell growth. Moreover, the antiproliferative activity of SCC244 is comparable with that of INCB28060 and much potent than that of crizotinib. In contrast, SCC244 exhibited almost 10,000-fold less potency in other tested cells with low expression or activation of c-Met (Fig. 2A; Supplementary Table S3). c-Met inhibition is known to exert antiproliferative effects via arresting cells in the G1–S phase (35, 38); therefore, SCC244 treatment consistently inducing G1–S cell-cycle arrest (Fig. 2B and C) further confirmed that the potent antiproliferative activity of the compound was because it specifically targeted c-Met signaling. Collectively, the data suggested that SCC244 selectively inhibited c-Met–driven cell proliferation with high potency.

Figure 2.

SCC244 specifically and potently inhibits proliferation of c-Met–addicted human cancer cells. A, The antiproliferative activity of SCC244 against a panel of tumor cell lines and normal cells originating from different tissue types was determined by a sulforhodamine B (SRB) assay, an MTT assay, or a CCK-8 assay. The IC50 values 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-cycle arrest in c-Met–addicted human cancer cells. EBC-1 and MKN-45 cells were treated with the indicated concentrations of SCC244 for 24 hours. The percentages of cells in different cell-cycle phases determined by FACS and analyzed with Modifit LT were plotted. The data shown are the mean ± SD from two independent experiments.

Figure 2.

SCC244 specifically and potently inhibits proliferation of c-Met–addicted human cancer cells. A, The antiproliferative activity of SCC244 against a panel of tumor cell lines and normal cells originating from different tissue types was determined by a sulforhodamine B (SRB) assay, an MTT assay, or a CCK-8 assay. The IC50 values 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-cycle arrest in c-Met–addicted human cancer cells. EBC-1 and MKN-45 cells were treated with the indicated concentrations of SCC244 for 24 hours. The percentages of cells in different cell-cycle phases determined by FACS and analyzed with Modifit LT were plotted. The data shown are the mean ± SD from two independent experiments.

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SCC244 inhibited c-Met–dependent neoplastic phenotypes of metastasis and angiogenesis

c-Met is implicated in a variety of tumor cell and endothelial cell functions. Thus, SCC244 was evaluated in a series of cell-based functional assays. Because c-Met activation is important for promoting cancer cell migration and invasion, both contributors to tumor metastasis, we evaluated the effect of SCC244 in this regard. SCC244 strongly suppressed HGF-induced NCI-H441 cell motility and invasion in a dose-dependent manner and was sufficient to block the movement of most cells at a dose of 10 nmol/L (Fig. 3A–D).

Figure 3.

SCC244 inhibited c-Met–dependent neoplastic phenotypes of metastasis and angiogenesis. A, SCC244 suppressed HGF-induced NCI-H441 cell migration. Representative images are shown (scale bars, 1 mm). B, SCC244 suppressed HGF-induced NCI-H441 cell invasion. Representative images are shown (scale bars, 1 mm). C and D, The relative migration (C) and invasion (D) were plotted. The data shown are the mean ± SD from three independent experiments, assuming 100% migration or invasion of cells stimulated with HGF. ***, P < 0.001 versus HGF-stimulating group, determined using t test. E, SCC244 inhibited HGF-induced scattering of MDCK cells. Cells were grown as small colonies at low density and treated with HGF (50 ng/mL) in the presence of increasing concentrations of SCC244 for 18 to 24 hours. Representative images from three separate experiments are shown (scale bars, 50 μm). F, SCC244 significantly inhibited HGF-stimulated invasive cell growth. The MDCK branching morphogenesis in collagen induced by HGF was inhibited by SCC244. Images were obtained 5 days after treatment. Representative images from three separate experiments are shown (scale bars, 20 μm). G, SCC244 inhibited HGF-stimulated primary HUVEC c-Met signaling and proliferation. Prestarved primary HUVECs treated with SCC244 for 2 hours and then stimulated with 100 ng/mL HGF for 15 minutes were lysed and subjected to Western blot analysis. Prestarved primary HUVECs were treated with HGF (100 ng/mL) and increasing concentrations of SCC244 for 48 hours. Cell viability was measured by CCK-8 assay. Representative data are shown from three independent experiments.

Figure 3.

SCC244 inhibited c-Met–dependent neoplastic phenotypes of metastasis and angiogenesis. A, SCC244 suppressed HGF-induced NCI-H441 cell migration. Representative images are shown (scale bars, 1 mm). B, SCC244 suppressed HGF-induced NCI-H441 cell invasion. Representative images are shown (scale bars, 1 mm). C and D, The relative migration (C) and invasion (D) were plotted. The data shown are the mean ± SD from three independent experiments, assuming 100% migration or invasion of cells stimulated with HGF. ***, P < 0.001 versus HGF-stimulating group, determined using t test. E, SCC244 inhibited HGF-induced scattering of MDCK cells. Cells were grown as small colonies at low density and treated with HGF (50 ng/mL) in the presence of increasing concentrations of SCC244 for 18 to 24 hours. Representative images from three separate experiments are shown (scale bars, 50 μm). F, SCC244 significantly inhibited HGF-stimulated invasive cell growth. The MDCK branching morphogenesis in collagen induced by HGF was inhibited by SCC244. Images were obtained 5 days after treatment. Representative images from three separate experiments are shown (scale bars, 20 μm). G, SCC244 inhibited HGF-stimulated primary HUVEC c-Met signaling and proliferation. Prestarved primary HUVECs treated with SCC244 for 2 hours and then stimulated with 100 ng/mL HGF for 15 minutes were lysed and subjected to Western blot analysis. Prestarved primary HUVECs were treated with HGF (100 ng/mL) and increasing concentrations of SCC244 for 48 hours. Cell viability was measured by CCK-8 assay. Representative data are shown from three independent experiments.

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Activation of c-Met drives a complex genetic program termed invasive growth, which is pivotal in driving cancer cell invasion and metastasis (18, 39). In vitro, this morphogenetic program is replicated by stimulating cultured MDCK epithelial cells with HGF in a 3D multicellular-branched morphogenesis model and 2D scattering assay. We therefore chose cell scattering and morphogenesis as two representative models to evaluate the impact of SCC244 on c-Met–mediated invasive growth. As expected, MDCK cells appeared to dissociate and form multicellular-branched structures upon HGF stimulation. Moreover, SCC244 showed strong inhibitory effects on cell scattering (Fig. 3E) and morphogenesis (Fig. 3F), indicating that SCC244 inhibited HGF-induced c-Met–mediated invasive growth.

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

The strong inhibition of c-Met–dependent phenotypes suggests that the pharmacologic activity of SCC244 observed in these functional assays is mediated by the inhibition of c-Met.

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

Encouraged by the potency of SCC244 in reversing c-Met–dependent neoplastic phenotypes in vitro, we proceeded to evaluate its antitumor efficacy in vivo. CDX models representative of cancer indications in which the dysregulation of c-Met is implicated were used, including the gastric carcinoma MKN-45 and SNU-5 models and lung cancer EBC-1 model. In the MKN-45 model, SCC244 significantly inhibited tumor growth with inhibitory rates of 99.3%, 88.6%, and 63.6% at doses of 10, 5, and 2.5 mg/kg, respectively. In addition, tumor stasis was observed following a 21-day treatment with 5 and 10 mg/kg SCC244 (Fig. 4A). Similar results 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 exhibited a greater than 66.0% decrease in tumor mass, and in both the 10 and 5 mg/kg treatment groups, 1 of 6 mice exhibited no evidence of a tumor (Fig. 4A). Moreover, in all the tested models, the efficacy of SCC244 at 10 mg/kg is comparable with that of INCB28060 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 at the highest dose of 50 mg/kg (Supplementary Fig. S1A–S1D).

Figure 4.

SCC244 significantly inhibited c-Met–driven tumor growth in cancer CDX models. A, SCC244 inhibits tumor growth in MKN-45, SNU-5, and EBC-1 xenografts. SCC244 was administered orally once daily after the tumor volume reached 100 to 200 mm3. Tumor volumes were measured twice a week and are shown as the mean ± 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 was determined in the EBC-1 and SNU-5 xenograft models 2 hours after the final administration of SCC244 (scale bars, 100 μm). C, SCC244 reduced secretion of human 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 < 0.001 versus 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 SCC244 only once. Tumors were harvested at several time points, p-c-Met, p-AKT, and p-ERK in tumor samples were detected by immunoblotting, and relative phosphorylation was calculated as described in Materials and Methods and are presented as the mean ± SD.

Figure 4.

SCC244 significantly inhibited c-Met–driven tumor growth in cancer CDX models. A, SCC244 inhibits tumor growth in MKN-45, SNU-5, and EBC-1 xenografts. SCC244 was administered orally once daily after the tumor volume reached 100 to 200 mm3. Tumor volumes were measured twice a week and are shown as the mean ± 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 was determined in the EBC-1 and SNU-5 xenograft models 2 hours after the final administration of SCC244 (scale bars, 100 μm). C, SCC244 reduced secretion of human 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 < 0.001 versus 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 SCC244 only once. Tumors were harvested at several time points, p-c-Met, p-AKT, and p-ERK in tumor samples were detected by immunoblotting, and relative phosphorylation was calculated as described in Materials and Methods and are presented as the mean ± SD.

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SCC244 was also evaluated for its effect on tumor mitotic index (Ki67) using IHC methods. A significant dose-dependent decrease in Ki67 levels was observed upon SCC244 treatment in the EBC-1 and SNU-5 models (Fig. 4B). The in vivo antiangiogenic effect of SCC244 was assessed for intratumoral modulation of microvessel density (MVD) by immunostaining for platelet endothelial cell adhesion 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 proangiogenic factor IL8, the release of which was stimulated by c-Met/HGF activation, was also measured. SCC244 significantly reduced human IL8 plasma levels (Fig. 4C). These results indicated that the antitumor activity of SCC244 is mediated by direct effects on tumor cell growth and by antiangiogenic mechanisms.

To evaluate in vivo c-Met inhibition by SCC244, EBC-1 tumors were harvested at several time points after administering a single dose of 2.5 or 10 mg/kg SCC244, and the c-Met–signaling phosphorylation in tumors was examined. Almost complete inhibition of c-Met, ERK, and Akt phosphorylation for 8 hours is observed at the dose of 10 mg/kg, which is consistent with near-complete tumor growth inhibition/cytostasis (104.4% TGI). The dose with suboptimal efficacy (2.5 mg/kg, 66.0% TGI) led to potent inhibition of c-Met signaling for only a portion of the experiment, with almost full recovery by 8 hours (Fig. 4D). These findings suggest that the extent and duration of c-Met targeting inhibition is due to the antitumor efficacy of SCC244.

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

CDXs have proven utility as models for pharmacologic studies and as models for efficacy in cases of oncogene addiction. However, the effectiveness of CDXs is limited by the lack of molecular and cellular heterogeneity. PDX models offer promise as improved disease models through their increased diversity of molecular lesions and preservation of 3D tumor–stromal cell components and interactions. Thus, we studied the antitumor efficacy of SCC244 by interrogating a panel of 4 human NSCLC tumor models and 5 HCC tumor models known to harbor MET-driven mutation [gene amplification or/and Exon 14 skipping mutation (MET ex14)] or c-Met overexpression (Table 1). We found that SCC244 showed robust antitumor efficiency in all these 9 models, with the tumor growth–inhibitory rate from 87.7% to 115.8% at the optimal dose of 10 mg/kg (Fig. 5; Table 1). Tumor shrink was observed in some tumor-bearing mice even during the first 7 days. Moreover, an undetectable tumor (complete response) was observed in some tumor-bearing mice of the LU2503 and LI0612 models (Fig. 5B and E; Table 1). In addition, two NSCLC PDX model (LU2071, LU-01-0439) has MET copy number less than 5, which cannot be considered a clinically relevant gene amplification. But it cannot be ignored that they have conspicuous intratumoral c-Met expression, which level was comparable with that of the MET-amplified LU1902 LU2503 model (Supplementary Fig. S2). Moreover, these two c-Met–overexpressing NSCLC PDX models responded well to SCC244 treatment (Fig. 5C and D). Our result is consistent with the clinical finding that patients with c-Met overexpression not caused by amplification also respond to c-Met inhibitor (41, 42).

Table 1.

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

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

Figure 5.

SCC244 induces tumor regression in NSCLC and HCC PDX models with MET aberration. Nude mice bearing NSCLC PDXs LU1902 (A), LU2503 (B), LU2071 (C), and LU-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.

Figure 5.

SCC244 induces tumor regression in NSCLC and HCC PDX models with MET aberration. Nude mice bearing NSCLC PDXs LU1902 (A), LU2503 (B), LU2071 (C), and LU-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.

Close modal

These data further confirmed the high potency of SCC244 against c-Met–driven tumor growth.

Genotype-based targeted therapies for molecularly selected populations hold promise for improving the therapeutic outcomes of cancer patients. Mutant MET, a clinically relevant molecular oncogenic driver, is found in a diverse spectrum of malignancies, including NSCLC, gastric cancer, liver cancer, and esophageal cancer (4, 12, 14, 15, 23, 43–49). Aberrant c-Met activation is closely associated with tumor formation and metastasis, as well as resistance to approved therapies (19–21). Therefore, inhibiting c-Met signaling could have significant potential for the treatment of human cancers driven by c-Met overactivation.

In this study, we reported SCC244, a c-Met inhibitor with superior efficacy. SCC244 has subnanomolar biochemical and nanomolar cellular potency against c-Met kinase and c-Met–dependent neoplastic phenotypes, including cancer cell proliferation, migration, invasion, and invasive growth. In 3 c-Met–driven CDX models, SCC244 significantly inhibited tumor growth, causing tumor stasis or regression. The efficacy of 10 mg/kg SCC244 is comparable with that of 15 mg/kg INCB28060, which is the most advanced c-Met selective inhibitor. PDX models are more informative than standard CDX models because they maintain tumor heterogeneity and preserve. We also investigated the in vivo efficacy of SCC244 in 9 NSCLC and HCC cancer PDX models harboring MET aberration. SCC244 showed significant antitumor efficiency in all these models at the optimal dose of 10 mg/kg. SCC244 induced rapid tumor shrink in 2 CDX and 7 PDX models. In these models, tumors rapidly decreased in size during the first 7 days.

Furthermore, most c-Met inhibitors currently undergoing clinical trials are multitarget inhibitors, which may cause unwanted off-target toxicity. In the new era of personalized medicine, highly selective c-Met inhibitors could largely avoid toxicity arising from targeting extra molecules and thus could provide a better treatment option for the subpopulation of c-Met–driven cancers and serve as a “clean” component for rational combination. Another advantageous characteristic of SCC244 is its high selectivity for c-Met, SCC244 showed at least a 2,400-fold selectivity for c-Met over a panel of 312 kinases, establishing it as one of the most selective c-Met inhibitors described to date. Consistent with this observation, cancer cells with low c-Met activation or expression were markedly less sensitive to SCC244 than c-Met–addicted cells (almost 10,000-fold less potency). Lacking the troublesome issue of off-target kinase inhibition, SCC244 may have a favorable safety profile. Indeed, SCC244 demonstrated a broad therapeutic window in preclinical studies and induced tumor stasis and shrink at oral doses of 5 or 10 mg/kg with no body weight loss, even at a dose of 50 mg/kg. The preclinical safety profile of SCC244 was further characterized through repeated-dose studies up to 1 month in duration in rats and dogs. The SD rats were given once daily oral administration with SCC244 at 10, 30, or 100 mg/kg for 28 days. A No-Observed-Effect-Level (NOEL) of 10 mg/kg/day was established and the MTD was more than 100 mg/kg/day. Only slight increases in WBC, mainly lymphocytes and neutrophils, were noted by the end of the dosing phase at the dose of 30 and 100 mg/kg/day while recovered by the end of the 28-day dosing-free phase. For repeated-dose study in dog, the Beagle dogs were given once daily oral administration with SCC244 at 5, 20, or 60 mg/kg for 28 days. The NOAEL was considered to be 5 mg/kg/day and the MTD was 60 mg/kg/day. The main findings noted by the end of the dosing phase at dose of 20 and 60 mg/kg/day included changes in ECG parameters, gastrointestinal adverse effects, lymphoid atrophy in thymus as well as increased myeloid cells and megakaryocytes in the bone marrow while they were not present by the end of the 28-day recovery phase. SCC244 achieved a greater than 40-fold therapeutic index when comparing its efficacious dose (tumor stasis) with its MTD in both rats and dogs. Because of its superior potency and favorable preclinical safety window, SCC244 could maximize the therapeutic potential of c-Met inhibition alone in human cancers addicted to c-Met aberrations.

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

No potential conflicts of interest were disclosed.

Conception and design: J. Ai, J. Ding, M. Geng

Development of methodology: X. Yang

Acquisition 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. Gao

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Ai, Y. Chen, X. Peng, M. Geng

Writing, review, and/or revision of the manuscript: J. Ai, X. Peng, M. Geng

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Yang, Y. Ma

Study supervision: J. Ai, J. Ding, M. Geng

Other (compound design and synthesis): B. Xiong, J. Shen

This research was supported by grants from the “Personalized Medicines –Molecular Signature-based Drug Discovery and Development,” Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDA12020000, for M. Geng; XDA12020103, for J. Ai), and the Natural Science Foundation of China (no. 81473243 and 81773762, for J. Ai). We thank Dr. H. Eric Xu (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) for kindly providing MDCK cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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