A Novel Inhibitor of C-Met and VEGF Receptor Tyrosine Kinases with a Broad Spectrum of in Vivo Antitumor Activities

Home , EPHA7, EPHA8, FGR (gene), TXK (gene), VEGF receptor

Published OnlineFirst April 2, 2013; DOI: 10.1158/1535-7163.MCT-12-1011

Molecular Cancer Chemical Therapeutics Therapeutics

A Novel Inhibitor of c-Met and VEGF Receptor Tyrosine Kinases with a Broad Spectrum of In Vivo Antitumor Activities

Yoshiko Awazu1, Kazuhide Nakamura1, Akio Mizutani1, Yuichi Kakoi1, Hidehisa Iwata2, Seiji Yamasaki2, Naoki Miyamoto3, Shinichi Imamura1, Hiroshi Miki2, and Akira Hori1

Abstract The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are dysregulated in a wide variety of human cancers and are linked with tumorigenesis and metastatic progression. VEGF also plays a key role in tumor angiogenesis and progression by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor tyrosine kinases (VEGFR). Therefore, inhibiting both HGF/c-Met and VEGF/VEGFR signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of tumors. Toward this goal, we generated and characterized T-1840383, a small-molecule kinase inhibitor that targets both c-Met and VEGFRs. T-1840383 inhibited HGF-induced c-Met phosphorylation and VEGF-induced VEGFR-2 phosphorylation in cancer epithelial cells and vascular endothelial cells, respectively. It also inhibited constitutively activated c-Met phosphorylation in c-met–amplified cancer cells, leading to suppression of cell proliferation. In addition, T-1840383 potently blocked VEGF-dependent proliferation and capillary tube formation of endothelial cells. Following oral administration, T-1840383 showed potent antitumor efficacy in a wide variety of human tumor xenograft mouse models, along with reduction of c-Met phosphorylation levels and microvessel density within tumor xenografts. These results suggest that the efficacy of T-1840383 is produced by direct effects on tumor cell growth and by an antiangiogenic mechanism. Furthermore, T-1840383 showed profound antitumor activity in a gastric tumor peritoneal dissemination model. Collectively, our findings indicate the therapeutic potential of targeting both c-Met and VEGFRs simultaneously with a single small-molecule inhibitor for the treatment of human cancers. Mol Cancer Ther; 12(6); 913–24. 2013 AACR.

Introduction c-Met mutations, has been observed in a variety of human The c-Met receptor tyrosine kinase (RTK) is the receptor cancers (1, 2). Clinically, dysregulation of c-Met is correlated with a poor prognostic outcome (1, 2). Recently, for hepatocyte growth factor/scatter factor (HGF; met reviewed in ref. 1). Binding of HGF to c-Met induces c- constitutive activation of c-Met due to ampliﬁcation Met dimerization and phosphorylation of multiple tyro- was also found to be a driver of cell proliferation and sine residues in the intracellular region, leading to acti- survival in several gastric cancer cell lines (3, 4) and has vation of c-Met signaling pathways. c-Met signaling plays additionally been linked to acquired resistance to EGF important roles in cell growth, survival, motility, and inhibitors in lung cancers (5, 6). Thus, c-Met has been morphogenesis during embryonic development as well considered an appealing molecular target for cancer ther- as in cancer cell proliferation, invasion, metastasis, tumor apy (2, 7–9). angiogenesis, and drug resistance (reviewed in refs. 1 The receptors for VEGF, VEGF receptor 1 (VEGFR-1) and 2). Aberrant c-Met signaling, resulting from met and VEGF receptor 2 (VEGFR-2), are members of the RTK genomic ampliﬁcation, c-Met or HGF overexpression, or family; they are mostly located in endothelial cells (reviewed in ref. 10). VEGFR activation occurs through VEGF binding, which triggers receptor dimerization,

Authors' Afﬁliations: 1Oncology Drug Discovery Unit, 2Discovery tyrosine kinase activation, and phosphorylation of tyro- Research Laboratories, and 3Medicinal Chemistry Laboratories, Takeda sine residues. Of the 2 VEGFRs, VEGFR-2 is believed to be Pharmaceutical Company Co. Ltd., Fujisawa, Kanagawa, Japan a major driver of tumor angiogenesis, which plays impor- Note: Supplementary data for this article are available at Molecular Cancer tant roles in tumor malignancy (such as sustaining tumor Therapeutics Online (http://mct.aacrjournals.org/). growth) and in blood-borne metastasis (11). VEGF expres- Y. Awazu, K. Nakamura, and A. Mizutani contributed equally to this work. sion is upregulated by changes associated with cancer, including hypoxia, proto-oncogene activation, loss of Corresponding Author: Kazuhide Nakamura, Oncology Drug Discovery Unit, Takeda Pharmaceutical Company, Ltd., 2-26-1, Muraokahigashi, tumor suppressor gene expression, and growth factor Fujisawa, Kanagawa, 251-8555, Japan. Phone: 81-466-32-2609; Fax: stimuli in tumors (12, 13). Its overexpression has been 81-466-29-4410; E-mail: [email protected] reported to correlate with the degree of vascularity, doi: 10.1158/1535-7163.MCT-12-1011 aggressive disease, and poor prognosis in the majority of 2013 American Association for Cancer Research. human solid tumors (14–17), making the VEGF/VEGFR

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axis an attractive molecular target for cancer therapy. 96-well plates with 60 ng/mL VEGF (R&D Systems) and Indeed, the monoclonal anti-VEGF antibody bevacizu- designated concentrations of T-1840383 for 7 days. After mab (18) and several RTK inhibitors targeting VEGFRs, fixation with 70% ethanol, the cells were incubated with such as sunitinib (19), sorafenib (20), and pazopanib (21), antihuman CD31 monoclonal antibody (R&D Systems) have proven to be efficacious in clinical settings. followed by Alexa Fluor 488-conjugated antimouse IgG The above-mentioned findings highlight the possibility polyclonal antibody (Invitrogen). Tube formation by that simultaneous inhibition of c-Met and VEGFRs by a endothelial cells was visualized using fluorescence imag- small-molecule inhibitor may result in broader and more ing (Discovery-1 system, Molecular Devices) according to potent antitumor efficacy. Here, we report the character- the manufacturer’s protocol, "Angiogenesis Tube Forma- ization of T-1840383, a novel ATP-competitive multitar- tion." The filament length and number of branch points geted kinase inhibitor that preferentially inhibits c-Met were quantified by using Metamorph software (Molecu- and VEGFR-2, in various functional cellular assays and lar Devices). the evaluation of its in vivo antitumor activities in various tumor xenograft models. Cell scattering assay and E-cadherin expression analysis Materials and Methods A549 cells were seeded onto poly lysine–coated glass- T-1840383 bottom dishes (2,000 cells/dish) and were allowed to form N-[4-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2- colonies of appropriate size for 5 days. Controls or T- a]pyridin-6-yl}oxy)-3-fluorophenyl]-6-methyl-2-oxo-1- 1840383 were added to each dish. After 1-hour incubation, phenyl-1,2-dihydropyridine-3-carboxamide hydrochlo- 50 ng/mL HGF (R&D Systems) was added to the dishes. ride (T-1840383, Fig. 1A) was synthesized at Takeda Phar- Cell scattering was stimulated for 48 hours under a mild maceutical Company, Ltd. For in vitro studies, T-1840383 hypoxic condition (3% O2). Cells were stained using a was dissolved in dimethyl sulfoxide (DMSO) and diluted Diff-Quik stain kit (Sysmex). For E-cadherin expression in culture medium immediately before use. For in vivo analysis, scattering-stimulated cells were fixed with 4% studies, T-1840383 was suspended in vehicle (0.5% methyl paraformaldehyde and were subjected to immunofluo- cellulose in distilled water), and the suspension was rescence staining using anti-E-cadherin primary antibody administered to animals within a week of preparation. and Alexa Fluor 488-conjugated antirabbit IgG antibody. Nucleic acid was stained with propidium iodide. Kinase inhibition assays Kinase inhibition of c-Met, VEGFRs, Tie-2, and Flt-3 was Matrigel invasion assay investigated using AlphaScreen tyrosine kinase technol- Cell invasion was assessed using Matrigel Invasion ogy (PerkinElmer). To evaluate kinase selectivity, a single Chambers (BD Biosciences). A549 cells or SUIT-2 cells concentration (1 mmol/L) of T-1840383 was tested using were placed in the top chamber, and T-1840383 was added 256 kinases by KinaseProfiler (Millipore), and then IC50 to both the top and bottom chambers. Cell invasion was values for the selected 66 kinases were determined using then initiated by adding HGF to the bottom chamber KinaseProfiler IC50 Express (Millipore). (50 ng/mL). After 72 hours, noninvading cells were removed with cotton swabs. Cells that invaded through Cell lines the Matrigel were stained using a Diff-Quik stain kit, and Unless otherwise mentioned, cells were obtained from the number of invading cells was quantified using Win- the American Type Culture Collection. Five gastric cancer ROOF software (Mitani Corp.). cell lines (GSU, Kato III, KE-97, MKN45, and NUGC-4) were obtained from the RIKEN BioResource Center and 4 Cell proliferation assay gastric cancer cell lines (SNU-484, -620, -638, and -668) Cancer cells and HUVECs were seeded in 96-well plates were obtained from the Korean Cell Line Bank. EBC-1 in appropriate media with 10% FBS and human endothe- squamous cell lung cancer cells and SUIT-2 pancreatic lial serum-free medium (Invitrogen), respectively. The cancer cells were obtained from the Health Science following day, serial dilutions of T-1840383 or 0.1% DMSO Research Resources Bank. OE33 esophageal adenocarci- were added to each well. Cancer cells were then incubated noma cells were obtained from the European Collection of for a further 72 hours. Proliferation of HUVECs was Animal Cell Cultures. Human umbilical vein endothelial stimulated with 60 ng/mL recombinant human VEGF for cells (HUVEC) and normal human dermal fibroblasts 120 hours. After incubation, cell proliferation was deter- (NHDF) were purchased from Cambrex. All cells were mined using a Cell Counting Kit-8 (DOJINDO Laborato- cultured in recommended media and serum concentra- ries). IC50 values were calculated by nonlinear regression tion, unless otherwise indicated. No authentication was analysis using GraphPad Prism (GraphPad Software, done by the authors. Inc.).

Tube formation assay Western blotting Human umbilical vein endothelial cells (1,000 cells/ Antibodies were purchased from Cell Signaling Tech- well) and NHDFs (10,000 cells/well) were cocultured in nology. For analysis of A549 cells and HUVECs, c-Met

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A E

800 700 600 500 400 B 300 Cell invasion Cell 0 0 0.03 0.3 100 0.1 1 10 30 1,000 300 T-1840383 (nmol/L) 3 200 HGF - + + + + + + + + + + + per field) (cell number 100 p-Met (Tyr1234/1235) 0 0 1 10 100 T-1840383 concentration (nmol/L) Total Met F

HUVEC (endothelial) 1.8 C SNU-5 (gastric) 3.4 T-1840383 (nmol/L) 0 0 1 10 100 SNU-638 (gastric) 4.2 MKN45 (gastric) 5 HGF -++ + + EBC-1 (NSCLC) 8.4 NUGC-4 (gastric) 10 Hs746T (gastric) 25 OE33 (esophagus) 570 KATO III 2,400 SNU-620 (gastric) >10,000 SNU-668 (gastric) >10,000 D U-87 MG (GBM) >10,000 T-1840383 (nmol/L) 0 0 1 10 100 SNU-484 (gastric) >10,000 AGS (gastric) >10,000 HGF - + + + + A549 (NSCLC) >10,000 Colo 205 (CRC) >10,000 MRC-5 (fibroblast) >10,000 1 10 100 1,000 10,000

IC50 (nmol/L)

Figure 1. T-1840383 inhibits HGF-induced c-Met phosphorylation, cell scattering, and invasion. A, chemical structure of T-1840383. B, A549 cells were treated with T-1840383 for 1 hour before HGF stimulation. c-Met phosphorylation (p-Met) levels were determined by Western blotting. Total c-Met levels are shown as a loading control. Western blot analysis was done in quadruplicate with very similar results. C, T-1840383 was added to the cell culture before HGF stimulation. Scattering-stimulated cells were then stained with a Diff-Quik staining kit. Magnification, 100. D, scattering-stimulated cells were subjected to immunofluorescence stain for E-cadherin expression (green) and nuclear stain with propidium iodide (red). Magnification, 400. E, cell invasion was assessed using a Matrigel invasion chamber for 72 hours under mild hypoxia (3% O2). The invading cells were stained with a Diff-Quik staining kit and quantified. Columns, mean (n ¼ 4); bars, SD. F, cell viabilities were determined in appropriate media containing various concentrations of T-1840383. Shown are the mean of the IC50 values (n ¼ 2–6). Rows labeled as ">10,000" indicate that the IC50 values are more than 10,000 nmol/L. and VEGFR2 phosphorylation were stimulated with in a 1:1 mixture of Matrigel and HBSS and then inoculated. 50 ng/mL HGF and 100 ng/mL VEGF for 10 and 5 After the tumors were established, mice bearing tumors of minutes, respectively. All cells were lysed in RIPA buffer appropriate size were randomized. Once-daily oral (Pierce) with protease and phosphatase inhibitors. In the administration of T-1840383 or vehicle was initiated on case of the tumor samples, tissues were homogenized in the next day after randomization. Tumor volume was RIPA buffer with protease and phosphatase inhibitors. measured twice weekly with Vernier calipers and calcu- Samples were then subjected to Western blot analysis. lated as (length width2) 0.5. Percentage treated/ Detection was carried out using enhanced chemilumines- control ratio (T/C%) was calculated by dividing the cence reagent (GE Healthcare). change in volume of the tumors in the treated mice by the change in volume in the mice treated by vehicle, and Tumor xenograft models statistical analysis was conducted as described previously All animal experiments were conducted according to (22). the guidelines of the Takeda Experimental Animal Care For a peritoneal dissemination model, NUGC-4 cells and Use Committee. Athymic nude mice (BALB/cA Jcl- expressing luciferase (NUGC-4-luc cells) were generated nu/nu) were purchased from Japan CLEA. For tumor by stable transfection and inoculated via intraperitoneal models, cancer cells in Hanks balanced salt solution injection into nude mice (1 106 cells). Daily oral admin- (HBSS; Gibco, Invitrogen Corp.) were subcutaneously istration (5 days per week) of T-1840383 at 5 mg/kg or inoculated into the hind flank of 6- to 7-week-old mice vehicle was initiated 2 weeks after inoculation. Tumor (day 0). For the study with MKN45, cells were suspended growth was monitored on the basis of determination of

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emitted bioluminescence (photons/s) 10 minutes after Results intraperitoneal administration of D-luciferin (1.5 mg/kg). T-1840383 is a potent kinase inhibitor that targets Mice were euthanized when they became moribund. both c-Met and VEGF receptors Statistical analysis was conducted by a log-rank test using The selectivity of T-1840383 (Fig. 1A) was profiled GraphPad Prism. against a panel of protein kinases and lipid kinases (Table 1). T-1840383 inhibited c-Met tyrosine kinase with Immunohistochemistry an IC50 value of 1.9 nmol/L. It also inhibited VEGFR Mice (BALB/cA Jcl-nu/nu) bearing A549 tumors were tyrosine kinases with IC50 values of 7.7, 2.2, and 5.5 treated with T-1840383 at the indicated doses for 3 days. nmol/L for VEGFR-1, -2, and -3, respectively. The struc- The dissected tumors were fixed in paraformaldehyde ture of T-1840383 has little in common with other VEGFR and then paraffin embedded. For visualization of the or c-Met selective inhibitors, except for TAK-593 (Supple- vessels, immunohistochemical staining for CD31 was mentary Fig. S1). T-1840383 was found to inhibit several conducted using rabbit anti-CD31 polyclonal antibody kinases including c-Mer, Ret, Ron, Rse, Tie-2, and TrkA as (Spring Bioscience) and the immunoperoxidase technique well as mutants of Abl, c-Kit, fibroblast growth factor with diaminobenzidine tetrahydrochloride as the chro- receptors 2 (FGFR2), and platelet-derived growth factor a a magen. Ki67 staining for proliferating cells and TUNEL receptors (PDGFR ) with similar IC50 values to those for apoptotic cells was conducted using anti-Ki67 anti- for c-Met and VEGFRs. body (Dako) and ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore), respectively. Stained sections T-1840383 inhibits HGF-induced c-Met were scanned with a NanoZoomer Digital Pathology phosphorylation and HGF-dependent cellular system (Hamamatsu Photonics). Image processing was phenotypes conducted by analyzing 4 or 5 randomly selected fields In cell-based assays using A549 cells, T-1840383 inhib- of each tumor using WinROOF software (Mitani ited HGF-stimulated c-Met phosphorylation with an IC50 Corporation). value of 8.8 nmol/L (Fig. 1B). It also inhibited A549 cell

Table 1. Inhibitory activities of T-1840383 against 72 kinases

Kinase IC50 (nmol/L) Kinase IC50 (nmol/L) Kinase IC50 (nmol/L) Abl (H396P) 2.8 EphB1 13 Mnk2 100 Abl (M351T) 1 EphB2 19 MuSK 51 Abl (Q252H) 4.9 EphB4 79 PDGFRa (D842V) 3 Abl 13 Fer 110 PDGFRa 270 Abl(T315I) 1.6 FGFR1 370 PDGFRa (V561D) 2 Arg 26 FGFR2 270 PDGFRb 290 Aurora-B 210 FGFR2(N549H) 5.6 PTK5 93 Axl 19 Fgr 81 Pyk2 590 Bmx 110 Flt3a 19 Ret (V804L) 58 BRK 100 Fms 11 Ret 2 c-Kit(D816H) 200 Fms(Y969C) 36 Ret (V804M) 160 c-Kit 2,300 Fyn 180 Ron 3 c-Kit(V560G) 2.7 Hck 34 Rse 2 c-Kit(V654A) 1,100 IKKb 110 SIK 290 c-Mer <1 IR 1,400 Tie2a 1.2 c-RAF 100 IRAK1 870 Tie2 (R849W) 10 DDR2 120 Itk 93 Tie2 (Y897S) 14 EphA1 40 Lck 11 TrkA 4 EphA2 12 LIMK1 160 TrkB 10 EphA3 96 LOK 11 Txk 37 EphA4 76 Lyn 65 VEGFR1a 7.7 EphA5 17 MELK 540 VEGFR2a 2.2 EphA7 120 Meta 1.9 VEGFR3a 5.5 EphA8 26 MLK1 43 Yes 73

a For these 6 kinases, the IC50 values were determined by the AlphaScreen assay. For others, the IC50 values were determined using

KinaseProﬁler IC50 Express (Millipore).

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scattering stimulated with HGF (Fig. 1C). Because HGF tive cell lines (IC50 25 nmol/L; SNU-5, SNU-638, signaling has been reported to trigger destabilization of MKN45, NUGC-4, and Hs746T gastric cancer cells as well adherens junctions by downregulation of E-cadherin (23), as EBC-1 non–small cell lung carcinoma cells) were shown expression level of E-cadherin was examined in a similar to overexpress constitutively activated c-Met protein cell-scattering assay. As expected, HGF-stimulated cell (Supplementary Fig. S3A). These cell lines are also known scattering was accompanied by diminished expression of to harbor met genomic amplification (3, 4, 24, 25), suggest- E-cadherin on cell membrane, which was inhibited by T- ing that constitutive activation of c-Met is dependent on 1840383 in a concentration-dependent manner (Fig. 1D). genomic amplification. In MKN45 cells, T-1840383 inhib- T-1840383 also displayed similar potency against HGF- ited activation of c-Met and the downstream effectors induced A549 cell invasion in a Matrigel invasion assay ERK1/2 and AKT in a concentration-dependent manner (Fig. 1E and Supplementary Fig. S2). (Supplementary Fig. S3B). In contrast, OE33 esophageal adenocarcinoma cells and KATOIII and SNU-620 gastric T-1840383 inhibits c-Met–dependent cell cancer cells were much less sensitive to T-1840383 despite proliferation in vitro the fact that c-Met was constitutively active in those cell > The effects of T-1840383 on cell proliferation were lines. Other insensitive cell lines (IC50 10,000 nmol/L) evaluated using a panel of cancer cell lines and HUVECs. did not show constitutively activated c-Met. These results The antiproliferative potency varied widely, with the IC50 suggest that constitutively active c-Met is a necessary values ranging from 1.8 nmol/L to >10,000 nmol/L (Fig. but not sufficient predictor of in vitro cancer cell response 1F). With the exception of HUVECs, all T-1840383–sensi- to T-1840383.

A T-1840383 (nmol/L) 0 0 0.03 0.1 0.3 1 3 10 30 100 300 1,000 VEGF -++++ ++++ +++ p-VEGFR2 (Tyr1175) Total VEGFR2 p-Akt (Ser473) p-ERK1/2 (Thr202/Tyr204) B T-1840383 (nmol/L) 0 0 1 3 10 30 100 VEGF -++++++

C 120 350

100 300 250 80 200 60 150 40 100 Branch points

Total filament (mm) 20 50

0 0 0131030100 0131030100 T-1840383 (nmol/L) T-1840383 (nmol/L)

Figure 2. T-1840383 inhibits VEGFR-2 phosphorylation and VEGF signaling in HUVECs. A, HUVECs were treated with T-1840383 at the indicated concentration for 1 hour. Phosphorylation of VEGFR2 (Tyr1175), Akt (Ser473), and ERK1/2 (Thr202/Tyr204) following VEGF stimulation was analyzed by Western blotting. Western blotting was done in quadruplicate with very similar results. B, HUVECs were cocultured with fibroblasts. T-1840383 was added to the medium followed by VEGF stimulation. Shown are representative images after endothelial cell-specific staining (n ¼ 4). C, total filament length and number of branch points of the stained tube-like structures were measured using image analysis software in 4 to 10 different fields for each condition. Columns, mean; bars, SD.

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T-1840383 inhibits VEGF receptor-2 resulted in substantial inhibition of c-Met phosphoryla- phosphorylation and VEGF signaling in HUVECs tion from 2 to 8 hours, with full recovery by 24 hours. In Because T-1840383 also potently inhibits VEGF RTKs, the addition, dose-dependent inhibition of c-Met, AKT, and effects on VEGF signaling were tested in endothelial cells. ERK1/2 phosphorylation was observed 4 hours after T- As shown in Fig. 2A, T-1840383 inhibited VEGF-induced 1840383 administration (Fig. 3B). Pharmacokinetic anal- ¼ phosphorylation of VEGFR-2 (IC50 0.86 nmol/L). It also ysis showed that the plasma concentration of T-1840383 blocked VEGF-induced activation of the downstream effec- increased in a dose-dependent manner (over the dose tors ERK1/2 and AKT in a concentration-dependent man- range, 1–20 mg/kg; Supplementary Table S1). The tumor ner. Consistent with this, T-1840383 inhibited VEGF-driven concentration of T-1840383 was lower than that in plasma, T HUVEC proliferation with an IC50 value of 1.8 nmol/L (Fig. but relatively sustained (Supplementary Fig. S4). max in 1E). When cocultured with ﬁbroblasts, HUVECs formed tumors was achieved between 2 and 8 hours after dosing. T capillary-like structures in response to VEGF, which was, max and the inhibition of c-Met phosphorylation in however, abolished by the presence of T-1840383 (Fig. 2B). tumors were well correlated (Fig. 3A). In the MKN45 T-1840383 inhibited the VEGF-induced increase in total tumor xenograft mouse model, once-daily 2-week admin- length of tubes and number of blanch points with an istration resulted in a dose-dependent inhibition of tumor IC50 value of 12 and 8.3 nmol/L, respectively (Fig. 2C). growth with the T/C values of 57%, 30%, and 4% at doses of 0.5, 1.5, and 5 mg/kg, respectively (Fig. 3C). T-1840383 T-1840383 inhibits in vivo c-Met signaling and tumor caused tumor regression at 15 mg/kg. Additional tumor growth in mouse xenograft models models were used to assess the potency of T-1840383 To assess the in vivo potency of T-1840383 for inhibiting against c-Met–dependent or independent tumors: EBC- c-Met activation, the level of c-Met phosphorylation in 1 cells expressed elevated levels of constitutively active c- MKN45 tumors was measured at several time points Met and were highly sensitive to T-1840383 in vitro (Fig. 1F following a single oral administration of T-1840383 and Supplementary Fig. S3A), whereas COLO 205 cells (Fig. 3A). T-1840383 administration at a dose of 2 mg/kg showed no sign of constitutively active c-Met and were

A Time after T-1840383 administration (2 mg/kg) 0 h 1 h 2 h 4 h 8 h 24 h

p-Met (Tyr1234/1235)

B T-1840383 Vehicle 0.5 mg/kg 1.5 mg/kg 5 mg/kg

p-Met (Tyr1234/1235)

p-Akt (Ser473)

p-ERK1/2 (Thr202/Tyr204)

Actin

CDEF 2,000 900 1,400 1,200 Vehicle Vehicle Vehicle Vehicle ) ) ) ) 0.5 mg/kg 800 0.5 mg/kg 0.5 mg/kg 3 3 3 0.5 mg/kg 3 1,200 1,000 1.5 mg/kg 700 1.5 mg/kg 1.5 mg/kg 1.5 mg/kg 1,500 5 mg/kg 5 mg/kg * 1,000 5 mg/kg 5 mg/kg 15 mg/kg 600 15 mg/kg 15 mg/kg 800 15 mg/kg 500 800 1,000 600 400 600 300 * 400 * * 400 * 500 200 Tumor volume (mm Tumor volume (mm Tumor volume (mm Tumor volume (mm 200 * * 100 * 200 * * * * 0 0 0 0 81216202428 12 16 20 24 28 32 21 25 29 33 37 41 71115192327 Days after tumor implantation Days after tumor implantation Days after tumor implantation Days after tumor implantation

Figure 3. In vivo inhibition by T-1840383 of c-Met downstream signaling pathways and antitumor efﬁcacy of T-1840383 in tumor xenograft models. A, mice bearing MKN45 tumors were administered a single oral dose of T-1840383 (2 mg/kg). At the indicated time points after administration, the tumors were collected and homogenized. c-Met phosphorylation was determined by Western blotting. B, c-Met signaling in MKN45 tumors from mice that had been received a single oral dose of T-1840383 or vehicle was analyzed by Western blotting. Mice bearing MKN45 (C), EBC-1 (D), U-87MG (E), or COLO 205 (F) tumors were orally administered T-1840383 once daily at the indicated dose levels or vehicle alone for 2 weeks. Data represent the mean SEM for each group over the treatment period (n ¼ 5or6)., P 0.025 versus the vehicle control group by the one-tailed Williams test.

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insensitive to T-1840383 in vitro (Fig. 1F and Supplemen- body weight between vehicle- and T-1840383–treated tary Fig. S3A). U-87 MG cells are reported to express both EBC-1 tumor xenograft model mice at 15 mg/kg (2.3% HGF and c-Met, and that their proliferation and/or tumor loss vs. 7.9% gain compared with their initial body growth are at least partially driven by the HGF/c-Met weight). However, T-1840383 was generally well tolerated autocrine loop (26); in the present study, T-1840383 at the efficacious doses without obvious signs of animal showed marked antitumor activities in a dose-dependent discomfort in all subcutaneous tumor xenograft models. manner in c-Met–dependent EBC-1 and U-87 MG tumor models (Fig. 3D and E). The T/C values for EBC-1 tumors T-1840383 inhibits tumor angiogenesis were 85% and 26% at 0.5 and 1.5 mg/kg, respectively, and To further understand the mechanism of tumor growth the tumors regressed by 22% and 52% at 5 mg/kg and 15 inhibition by T-1840383, especially in c-Met independent mg/kg, respectively. The T/C values for U-87 MG tumors tumor models, tumor angiogenesis was analyzed. A sin- were 72%, 32%, and 10% at 0.5, 1.5, and 5 mg/kg, respec- gle oral administration of T-1840383 at 2 mg/kg resulted tively, and the tumors were almost static at 15 mg/kg. In in a significant decrease in phosphorylated VEGFR-2 the c-Met–independent COLO 205 tumor model, T- levels in the tumors of ectopically VEGFR-2-expressing 1840383 showed slightly less potent but still substantial 293 cells (Supplementary Fig. S5), suggesting that T- antitumor activity compared with the c-Met–dependent 1840383 inhibits in vivo VEGFR activation at the effica- tumor models, where the T/C values were 30% and 13% at cious doses. We next tested the antiangiogenic activities 5 mg/kg and 15 mg/kg, respectively (Fig. 3F). A slight but in tumor xenografts of the in vitro T-1840383 insensitive statistically significant difference was observed in the cell line A549. Following a 3-day treatment, T-1840383

A B Vehicle 0.5 mg/kg 120 100

80 *

60 *

40 *

1.5 mg/kg 5 mg/kg CD31-positive area

(% of vehicle control) 20

0 Vehicle 0.5 mg/kg 1.5 mg/kg 5 mg/kg

T-1840383

C 160 D 10,000 140 * 8,000 120

100 6,000 80 60 4,000 40 2,000 Ki-67–positive cells TUNEL-positive area (% of vehicle control) of (% (% of vehicle control) of (% 20 0 0 Vehicle 0.5 mg/kg 1.5 mg/kg 5 mg/kg Vehicle 0.5 mg/kg1.5 mg/kg 5 mg/kg

T-1840383 T-1840383

Figure 4. Effects of T-1840383 on tumor microvessel density, tumor cell proliferation, and tumor cell apoptosis in A549 tumors. A, mice bearing A549 tumors were treated with T-1840383 at 0.5, 1.5, and 5 mg/kg/day or vehicle for 3 days. Tumors were fixed, paraffin-embedded, sectioned, and immunostained for CD31. Scale bars, 400 mm. B, quantitative analysis of CD31-positive endothelial cells were conducted. C, tumor specimens were immunostained with anti-Ki67 antibody, and Ki67-positive, proliferating cells were quantified. D, apoptotic tumor cells were visualized and quantified by TUNEL assay. Column, mean (n ¼ 4 or 5); bars, SEM. P 0.025 versus the vehicle control group by the one-tailed Williams test.

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A Vehicle T-1840383 5 mg/kg

Day 14

ﬁ Day 27 Figure 5. Antitumor ef cacy of T-1840383 in a peritoneal dissemination model. A, photon emission from individual mice in which NUGC-4-luc cells had been Day 34 inoculated intraperitoneally on day 0 was visualized before administration of the ﬁrst dose of T-1840383 or vehicle (day 14) and over the course of treatment (days 27, 34, and 41). B, emitted Day 41 bioluminescence from vehicle- treated or 5 mg/kg T-1840383– treated mice at days 14 and 41. C, survival curves of T-1840383– BC treated NUGC-4–bearing mice. 08 1.0 10 Daily administration (5 days a Vehicle ** * 100 week) of T-1840383 or vehicle was 8.0 10 07 T-1840383 initiated at day 14 and lasted until 80 6.0 10 07 the mice were euthanized (up to 60 day 110). 4.0 10 07 Total flux Total 40

2.0 10 07 Percent survival 20

0.0 0 0 20 40 60 80 100 120 41 y 14 y a Days after tumor cell inoculation day 41 , d 3 8 83, da 03 03 4 4 Vehicle, day 14 Vehicle, -18 T-18 T

exhibited significant antitumor activities with T/C values NUGC-4-luc cells in mice. As shown in Fig. 5A and B, of 64%, 51%, and 35% at 0.5, 1.5, and 5 mg/kg, respectively vehicle-treated mice showed a significant increase in (data not shown). T-1840383 treatment resulted in a dose- tumor burden over the monitoring time from day 14 to dependent reduction in the microvessel density in viable day 41 (1.26 107/s vs. 3.63 107/s, P <0.01). In contrast, regions of the tumors by 29%, 50%, and 65% at 0.5, 1.5, and the T-1840383–treated mice did not show a statistically 5 mg/kg, respectively (Fig. 4A and B). In similar dose- significant increase in tumor burdens (1.30 107/s vs. 2.02 dependent manners, T-1840383 treatment also led to a 107/s). In addition, T-1840383 treatment resulted in a decreased tendency of cell mitosis (Ki67) and a significant prolonged survival of mice compared with vehicle treat- increase in apoptosis (TUNEL) in the A549 tumor xeno- ment (Fig. 5C, median survival: 46.5 days vs. 108.5 days, P grafts (Fig. 4C and D). Together, these results suggest that <0.01). In vehicle-treated mice, on the basis of in vivo targeting tumor angiogenesis is one of the mechanisms by imaging analysis, one mouse apparently became tumor- which T-1840383 inhibits growth of c-Met–independent free and showed an exceptionally longer survival than the tumors in vivo. others, probably due to rejection of the tumor challenge.

Antitumor activities of T-1840383 in a gastric tumor c-Met expression and activation under hypoxia peritoneal dissemination model Hypoxia, which is known to be a major stimulator of To extend the ﬁnding that T-1840383 potently inhibits VEGF and consequent angiogenesis, has been shown to the growth c-Met–dependent cancer cells, T-1840383 was promote invasive tumor growth by transcriptional acti- further evaluated in a mouse model of peritoneal dissem- vation of c-met and by sensitizing cells to HGF stimulation ination using c-Met–dependent gastric cancer NUGC-4- (27). To explore this ﬁnding in our systems, we investi- luc cells. In this model, tumor growth was monitored gated c-Met expression and HGF-induced cell invasion noninvasively with bioluminescence emitted from in response to hypoxia. Hypoxia upregulated c-Met

920 Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics

Wide Antitumor Spectrum of a Dual c-Met and VEGFR2 Inhibitor

A Serum (0.1%) Serum (1%) Serum (10%) HHNNHHNNHHNN c-Met p-Met (Tyr1234/1235)

p-Met (Tyr1003)

Figure 6. Hypoxia-induced c-Met Actin expression and activation in cancer cells. A, A549 cells were cultured in media containing 0.1%, 1%, or 10% B Caki-1 SUIT-2 U-87 MG – FBS in collagen-type I coated plates HHNN HHNNHHN N under normoxia or hypoxia (2% O2) for 24 hours. B, Caki-1, SUIT-2, and U-87 MG cells were cultured in c-Met appropriate media containing 10% FBS under normoxia or hypoxia p-Met (Tyr1234/1235)

(3% O2) for 48 hours. c-Met phosphorylation was determined by Actin Western blotting. C, SUIT-2 cell invasion was stimulated with HGF 450 under normoxia or hypoxia (2% O2) C for 72 hours. The invading cells were 400 Normoxia stained with a Diff-Quik staining kit 350 ﬁ Hypoxia and quanti ed. Columns, mean 300 (n ¼ 4); bars, SD. 250 200 150 Cell invasion 100 (Cell number / field) (Cell number 50 0 012.52550100 HGF concentration (ng/mL)

expression compared with normoxia (Fig. 6A). Moreover, marked inhibition of cell growth and survival (3, 28). we found that hypoxia induced c-Met phosphorylation in Thus, the response profile of T-1840383 may well reflect A549 cancer cells without HGF stimulation, whereas the pharmacologic consequence of inhibition of c-Met serum starvation had no such effects on c-Met expression rather than other targets. However, T-1840383 failed to and activation. Similar effects were seen in Caki-1 renal display antiproliferative activities against some c-met– cell carcinoma, SUIT-2 pancreatic cancer, and U-87 MG amplified cancer cells despite the fact that they expressed glioma cells (Fig. 6B). In addition, hypoxia augmented constitutively activated c-Met, suggesting that c-Met SUIT-2 cell invasion induced by HGF compared with amplification/activation alone may not necessarily deter- normoxia (Fig. 6C), suggesting functional relevance for mine the response. Such resistance may be attributed to the c-Met pathway in cancer cell invasive growth under collateral activation of HER family members (29, 30), hypoxia. amplification of wild-type KRAS (31), or c-Met mutations that affect the inhibitor binding (32, 33), although these speculations need to be further investigated. The response Discussion profile we observed here may provide a tool to help Here, we have characterized T-1840383, a novel multi- identify reliable predictive markers, sensitive patient targeted kinase inhibitor, in various functional assays. populations for T-1840383 or other c-Met inhibitors cur- T-1840383 potently blocked c-Met activation and down- rently under clinical evaluation (reviewed in refs. 2, 7–9), stream signaling in cancer cells, whereas cell proliferation and effective combination therapy for specific patients in assay revealed that T-1840383 selectively inhibited cancer the clinical setting. cells that harbored c-met gene amplification. Recent stud- We have also shown the in vivo antitumor efficacies of ies have shown that some gastric and lung cancer types T-1840383. In the tumor xenograft models using in vitro T- harboring c-met amplification are "addicted" to the con- 1840383–sensitive cancer cells, T-1840383 treatment stitutively activated c-Met signals and that targeting such resulted in potent tumor growth inhibition, in agreement activated c-Met with small-molecule inhibitors results in with attenuation of c-Met phosphorylation levels and

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Awazu et al.

downstream signaling in the tumors, suggesting that invasion may have important implications for treatment such potent efficacies primarily depend on direct inhibi- of the invasive growth and metastasis of cancer, tion of c-Met–driven tumor cell growth. In clinical set- although this remains to be confirmed experimentally. tings, patients with gastric cancers having scirrhous-type In the tumor xenograft models we used here, the HGF/ stroma particularly showed poor prognosis even after c-Met paracrine loop was nearly absent due to the low curative resection; they also showed highly progressed affinity of mouse HGF derived from host stromal cells to peritoneal dissemination (34). This type of cancer has been the human c-Met on implanted cancer cells (41). It is observed to often possess c-met gene amplification (35). T- therefore necessary to use relevant preclinical models, 1840383 showed a potent antitumor efficacy in a perito- such as tumor allograft models or xenograft models in neal tumor dissemination model for a c-met–amplified human HGF-transgenic or knock-in mice (42), where the gastric cancer, leading to a survival benefit in those mice. HGF/c-Met paracrine loop is functional between stro- These results provide a rationale for further investigation ma and cancer cells. of T-1840383 as an antitumor agent for patients with such We further investigated c-Met signaling in response to types of gastric cancer. hypoxia because hypoxia has been reported to promote In addition, our findings indicate that in vivo efficacy invasive growth by transcriptional activation of c-Met may also depend on the antiangiogenic properties. (27). In agreement with previous studies on this subject, Indeed, T-1840383 potently inhibited endothelial cell hypoxia indeed upregulated c-Met expression and pro- responses to VEGF stimuli in vitro and decreased tumor moted HGF-induced cell invasion. Moreover, we found microvessel density in vivo at the efficacious doses, even in that hypoxia elevated c-Met phosphorylation levels even the c-Met–independent tumor models. It is therefore clear in the absence of HGF stimuli, although this may be partly that T-1840383 affects tumor angiogenesis as a VEGFR explained through the recent findings that hypoxia pro- inhibitor. In addition, the c-Met inhibitory activity may motes interaction between c-Met and neuropilin-1, a contribute to angiogenesis inhibition because HGF/c-Met coreceptor for VEGF-A, which in turn facilitates VEGF- signaling induces tumor angiogenesis by directly induc- induced c-Met activation (43). These results indicate a key ing proliferation and migration of endothelial cells, by role of c-Met activation in response to the hypoxic stress of inducing expression of VEGF, as well as by downregulat- cancer cells. Importantly, it has been suggested that hyp- ing thrombospondin 1, a negative regulator of angiogen- oxia generated by inhibition of angiogenesis triggers path- esis (36). T-1840383 has a strong inhibitory effect on other ways that make tumors more aggressive and metastatic angiokinases such as Tie-2 and PDGFR and thus such (44, 45). Thus, it is possible that c-Met activation under inhibitory activities may also be involved in the antian- hypoxia is a mechanism of refractoriness to antiangio- giogenic property of T-1840383. We believe that inhibition genic treatment by inducing a tumor-invasive switch (46). of angiogenesis represents a mechanism by which T- In addition, a recent study suggests that an alternative 1840383 manifests a wide spectrum of its antitumor activ- angiogenic pathway, via c-Met activation in endothelial ities. Indeed, compared with the selective inhibitors of cells, is involved in resistance/low-responsiveness to VEGFR or c-Met, T-1840383 showed a broad in vivo anti- antiangiogenic treatment (47). Collectively, pharmacolog- tumor spectrum (Supplementary Table S2). ic intervention of c-Met signaling is a potential strategy to Several small-molecule inhibitors, including cabozan- prevent tumors from progressing under hypoxia, an tinib/XL184 (37) and foretinib/XL880 (38), that simulta- adverse tumor microenvironment condition, and to cir- neously target c-Met and VEGFRs have been previously cumvent resistance to angiogenesis inhibitors. This strat- reported. Cabozantinib seems to have a stronger inhibi- egy clearly warrants further exploration. tory activity against VEGFR2 than against c-Met (e.g., IC50 In summary, T-1840383 is a novel, highly potent inhib- values: 0.035 vs. 1.3 nmol/L, respectively), whereas T- itor of c-Met and VEGFR tyrosine kinases. Through this 1840383 has very similar activities against them (2.2 vs. 1.9 unique spectrum of dual kinase inhibition, T-1840383 nmol/L). Similar to T-1840383, foretinib has similar inhib- showed in vivo broad and potent antitumor activities by itory activities against VEGFR2 and c-Met (0.4 vs. 0.86 targeting tumor epithelial cells and vasculature. Our nmol/L). While foretinib is required to be administered at findings indicate the therapeutic potential of targeting a dose of approximately 100 mg/kg to effectively sup- both c-Met and VEGFRs simultaneously with a single press c-Met and VEGFR activity in vivo, T-1840383 sup- small-molecule inhibitor for treatment of various human presses their activities in similar in vivo models at much cancers. lower doses (around 2 mg/kg). This finding may be attributed to more potent cellular activities and/or favor- Disclosure of Potential Conflicts of Interest able pharmacokinetic profile of T-1840383. No potential conflicts of interest were disclosed. Hepatocyte growth factor is a potent inducer of epi- Authors' Contributions thelial–mesenchymal transition (EMT; ref. 39) that facil- Conception and design: Y. Awazu, K. Nakamura, S. Imamura, A. Hori itates most of the invasive growth functions of cancer, Development of methodology: Y. Awazu, K. Nakamura, Y. Kakoi, such as loss of adhesive junctions, motility, angiogen- H. Iwata, A. Hori Acquisition of data (provided animals, acquired and managed patients, esis, and survival (reviewed in ref. 40). Thus, the ability provided facilities, etc.): Y. Awazu, K. Nakamura, A. Mizutani, Y. Kakoi, of T-1840383 to block HGF-induced cell scattering and H. Iwata, S. Yamasaki, A. Hori

922 Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics

Wide Antitumor Spectrum of a Dual c-Met and VEGFR2 Inhibitor

Analysis and interpretation of data (e.g., statistical analysis, biostatis- for helpful discussion; Ryujiro Hara for critical reading of the manuscript; tics, computational analysis): Y. Awazu, K. Nakamura, Y. Kakoi, A. Hori and Yuichi Hikichi, Osamu Nakanishi, Syuichi Furuya, and Hideaki Writing, review, and/or revision of the manuscript: K. Nakamura, A. Hori Nagaya for their guidance and support during the course of this work. Administrative, technical, or material support (i.e., reporting or orga- The costs of publication of this article were defrayed in part by the nizing data, constructing databases): N. Miyamoto, H. Miki, A. Hori payment of page charges. This article must therefore be hereby marked Other: Design and synthesis of T-1840383: S. Imamura advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Study supervision: A. Hori this fact.

Acknowledgments The authors would like to thank Yuka Yarita for excellent technical Received October 17, 2012; revised March 25, 2013; accepted March 25, assistance; Shigemitsu Matsumoto, Hideyuki Oki, and Takeshi Watanabe 2013; published OnlineFirst April 2, 2013.

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924 Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics

A Novel Inhibitor of c-Met and VEGF Receptor Tyrosine Kinases with a Broad Spectrum of In Vivo Antitumor Activities

Yoshiko Awazu, Kazuhide Nakamura, Akio Mizutani, et al.

Mol Cancer Ther 2013;12:913-924. Published OnlineFirst April 2, 2013.

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