A Selective Small Molecule C-MET Inhibitor, PHA665752, Cooperates with Rapamycin

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2312 Vol. 11, 2312–2319, March 15, 2005 Clinical Cancer Research

A Selective Small Molecule c-MET Inhibitor, PHA665752, Cooperates with Rapamycin

Patrick C. Ma,1 Erik Schaefer,2 PHA665752, rapamycin showed cooperative inhibition to James G. Christensen,3 and Ravi Salgia1 reduce growth of BaF3.TPR-MET- and c-MET-expressing H441 NSCLC cells. 1Section of Hematology/Oncology, Department of Medicine, Pritzker School of Medicine, University of Chicago Medical Center, Chicago, Conclusions: PHA665752 is a potent small molecule– Illinois; 2BioSource International, Inc., Camarillo, California; and selective c-MET inhibitor and is highly active against TPR- 3Research Pharmacology, Pfizer, Inc., La Jolla, California MET-transformed cells both biologically and biochemically. PHA665752 is also active against H441 NSCLC cells. The c-MET inhibitor can cooperate with rapamycin in therapeu- ABSTRACT tic inhibition of NSCLC, and in vivo studies of this combi- Purpose: c-MET is believed to be an attractive receptor nation against c-MET expressing cancers would be merited. target for molecular therapeutic inhibition. TPR-MET, a constitutively active oncogenic variant of MET, serves as excellent model for testing c-MET inhibitors. Here, we char- INTRODUCTION acterized a small molecule c-MET inhibitor, PHA665752, and c-MET is a unique receptor tyrosine kinase (RTK) located tested its cooperation with the mammalian target of rapamy- on chromosome 7p and activated via its natural ligand cin inhibitor as potential targeted therapy. hepatocyte growth factor. c-MET can also be mutated in a Experimental Design: The effect of PHA665752 treat- variety of solid tumors (1), such as in the tyrosine kinase domain ment was determined on cell growth, motility and migration, for hereditary papillary renal cell carcinoma (2, 3) and in the apoptosis, and cell-cycle arrest of TPR-MET-transformed sema and juxtamembrane domains in small cell lung cancer cells. Moreover, the effect of PHA665752 on the phosphor- (SCLC; ref. 4). Many of these are activating mutations and ylation on MET, as well as its downstream effectors, p-AKT potentially targeted by specific c-MET small molecule inhibitors. and p-S6K, was also determined. Finally, growth of TPR- The TPR-MET oncogene is a transforming variant of the MET-transformed cells was tested in the presence of c-MET RTK and was initially identified after treatment of PHA665752 and rapamycin. H441 non–small cell lung a human osteogenic sarcoma cell line transformed by the cancer (NSCLC) cells (with activated c-Met) were also tested chemical carcinogen N-methyl-NV-nitro-N-nitrosoguanidine against both PHA665752 and rapamycin. (5–7). The TPR-MET fusion oncoprotein is the result of a Results: PHA665752 specifically inhibited cell growth in chromosomal translocation, placing the TPR3 locus on chromo- BaF3.TPR-METcells (IC < 0.06 Mmol/L), induced apoptosis 50 some 1 upstream of a portion of the c-MET gene on chromosome and cell cycle arrest. Constitutive cell motility and migration 7 encoding only for the cytoplasmic region (6, 7). Some studies of the BaF3.TPR-MET cells were also inhibited. PHA665752 suggest that TPR-MET is detectable in experimental cancer (8– inhibited specific phosphorylation of TPR-MET as well as 10). Dimerization of the M 65,000 TPR-MET oncoprotein phosphorylation of downstream targets of the mammalian r through a leucine zipper motif encoded by TPR leads to target of rapamycin pathway. When combined with constitutive activation of the c-MET kinase (11, 12). TPR-MET acts similarly to the activated wild-type c-MET RTK and can activate crucial cellular growth pathways, including the Ras pathway (13–16) and the phosphatidylinositol 3-kinase (PI3K)/ Received 8/23/04; revised 12/7/04; accepted 12/23/04. AKT pathway (17, 18). Conversely, in contrast to c-MET RTK, Grant support: American Association for Cancer Research-Astra- TPR-MET is ligand independent, lacks the CBL binding site in Zeneca-Cancer Research and Prevention Foundation fellowship in Translational Lung Cancer Research (P. Ma), American Cancer Society the juxtamembrane region in c-MET, and is mainly cytoplasmic Institutional research grant ACS IRG-58-004-44 (P. Ma), American (5–7, 19, 20). We have recently shown that the TPR-MET Cancer Society (Illinois Division)-LUNGevity Foundation Lung Cancer fusion oncoprotein has potent transforming activity and can be Treatment research award (P. Ma), American Cancer Society Research an excellent model for testing inhibitors as it has tremendous scholar grant RSG-02-244-02-CCE (R. Salgia), NIH/National Cancer Institute R01 grant R01 CA100750-01A1 (R. Salgia), and Institutional constitutively elevated c-MET tyrosine kinase activity that can Cancer research awards from the University of Chicago Cancer Center readily be inhibited (21). along with the American Cancer Society and the V-Foundation When introduced into an interleukin-3 (IL-3)–dependent (R. Salgia). cell line, BaF3, TPR-MET induces oncogenic phenotypes such as The costs of publication of this article were defrayed in part by the growth factor independence and constitutive tyrosine phosphor- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to ylation of multiple cellular proteins, including TPR-MET itself indicate this fact. (21). Recently, a novel-specific small molecule inhibitor of TPR- Requests for reprints: Ravi Salgia, Section of Hematology/Oncology, MET and c-MET kinase activity, SU11274, was identified which Department of Medicine, Room M-255A, University of Chicago has in vitro inhibitory activities in TPR-MET cells and in c-MET Medical Center, 5841 South Maryland Avenue, MC2115, Chicago, IL, 60637. Phone: 773-702-4399; Fax: 773-834-1798; E-mail: rsalgia@ expressing SCLC cells (i.e., H69 and H345) at low micromolar medicine.bsd.uchicago.edu. concentrations (21). SU11274 has cellular IC50 between 2.5 and D2005 American Association for Cancer Research. 5 Amol/L in SCLC.

To further explore the potential of targeted therapy against ‘‘migration index’’ (number of migrating cells treated with the c-MET kinase, we have characterized here a small molecule PHA665752 divided by the number of migrating cells left c-MET inhibitor with relatively improved potency and selectiv- untreated). SE was calculated from the migration indices of ity, PHA665752 (22). PHA665752 was recently identified and independently done experiments. The statistical significance of characterized to be active against several solid tumors (22). We the data was analyzed using the Student’s t test. have recently reported that non-SCLC (NSCLC) tumors can Immunoblotting. Proteins were extracted from whole overexpress c-Met, and the H441 NSCLC cell line has been cells by lysing them in a Tris buffer (50 mmol/L, pH 8.0) shown to overexpress and contain an activated c-Met. Here, we containing NaCl (150 mmol/L), NP40 (1%, v/v), deoxycholic found that PHA665752 specifically inhibited cell growth, acid (0.5%, w/v), SDS (0.1%, w/v), NaF (1mmol/L), Na3VO4 motility, and migration of cells transformed with TPR-MET. (1 mmol/L), and glycerol (10%, v/v; Sigma) supplemented with Inhibition of c-MET kinase activity by the small molecule drug a protease inhibitor cocktail (complete, Roche Diagnostics, induced apoptosis and cell cycle arrest in TPR-MET-transformed Indianapolis, IN). Polyclonal antibodies against p70-S6K BaF3 cells. We also found that the dose-dependent reduction in (Biosource International, Camarillo, CA), total c-MET (C-12, tyrosine phosphorylation of cellular proteins after PHA665752 Santa Cruz Biotechnology, Santa Cruz, CA), PI3K (Upstate treatment correlated with reduced activation of the PI3K/AKT/ Biotechnology, Lake Placid, NY) and phospho-AKT[Ser473]or mammalian target of rapamycin (mTOR) pathway in these cells. phospho-p70-S6K[Thr421/Ser424] (Cell Signaling, Beverly, Finally, we tested the specific mTOR inhibitor rapamycin alone MA), and phosphotyrosine (4G10, Upstate Biotechnology) were or in combination with PHA665752 and found that rapamycin used for immunoblotting. In addition, polyclonal antibodies can cooperate with the c-MET inhibitor in inhibiting cell against phospho-MET [Tyr1230/1234/1235], À[Tyr1349]and viability of BaF3.TPR-MET cells as well as NSCLC H441 cells. À[Tyr1365] (recognizing phospho-[Tyr361/365/366], À[Tyr480], Our results suggest that the combination of c-MET inhibitors and À[Tyr496] respectively in TPR-MET) were obtained from such as PHA665752 with mTOR inhibitor (rapamycin) may be Biosource International for the immunoblotting. The Tyr amino a feasible approach to improve the therapeutic efficacy in c-MET acid numbering of the phosphor-MET antibodies is based on the expressing cancers including NSCLC. shorter splice variant with 100-bp-long exon 10, instead of the full-length 154 bp. Apoptosis Assays. The activity of caspase-3 was mea- MATERIALS AND METHODS sured in cell lysates (CaspACE Assay System, Promega, Cells. The murine pre-B cell line BaF3 was grown in Madison, WI) and Annexin V positive staining was determined RPMI 1640 containing 10% FCS and 10% WEHI-conditioned by fluorescence-activated cell sorting analysis (Annexin-V-Fluos medium as a source of murine IL-3. BaF3 cell lines transfected Staining Kit, Roche Diagnostics) according to the manufactur- with a BCR-ABL, TEL-ABL, TEL-JAK2, or TEL-PDGFhR er’s directions in cells that were either treated with PHA665752 cDNA were grown in the absence of growth factors. A TPR- or the solvent DMSO. MET expressing BaF3 cell line was generated by transfection of Cell Cycle Analysis. Fixed cells were stained with an expression vector containing the TPR-MET cDNA as propidium iodide and cell cycle variables analyzed by previously described (21). NCI-H441 cells were obtained from fluorescence-activated cell sorting analysis. American Type Culture Collection (Manassas, VA) and were cultured according to the instructions (http://www.ATCC.org). The number of viable cells after treatment with DMSO, RESULTS PHA665752, and/or rapamycin was determined using a 3-(4,5- The Small Molecule c-MET Inhibitor PHA665752 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay Specifically Regulates Cell Growth in TPR-MET-Trans- (In vitro Toxicology Assay Kit, Sigma) or trypan blue exclusion. formed BaF3 Cells. PHA665752 was identified as a prototype Chemicals. PHA665752, (3Z)-5-[(2,6-dichlorobenzyl)- ATP-competitive small molecule inhibitor of the catalytic kinase sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)- activity of the c-MET RTK (22). We initially sought to determine pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihy- if PHA665752 could inhibit cell growth in TPR-MET- dro-2H-indol-2-one (Pfizer, Inc., La Jolla, CA), and rapamycin transformed BaF3 cells (Fig. 1A). Treatment of BaF3.TPR- (Calbiochem, La Jolla, CA) were dissolved in DMSO and used MET cells with PHA665752 was found to inhibit cell growth in at the concentrations as described. a dose-dependent manner with an IC50 < 0.06 Amol/L. To further Transwell Migration Assay. The lower chamber of determine if the growth inhibitory effect of PHA665752 on a transwell plate (8-Am pore size polycarbonate membrane, BaF3.TPR-MET cells is maintained over time, cell growth was Corning Costar Co., Cambridge, MA) was filled with 600 AL determined over a 72-hour culture. In the presence of IL-3, starvation medium (0.5% w/v, bovine serum albumin in RPMI PHA665752 had only little effect on cell growth of TPR-MET- 1640). Cells were counted using a Coulter particle counter transformed cells or BCR-ABL-transformed cells (Fig. 1B, top). (Coulter Counter Z2, Beckman Coulter, Fullerton, CA) and In contrast, PHA665752 completely blocked cell growth in the resuspended at 2 Â 106 cells/mL in starvation medium; 100 ALof absence of IL-3 in BaF3.TPR-MET and even reduced the this cell suspension were transferred to the upper chamber. The number of viable cells (Fig. 1B, bottom). This suggests that IL-3 medium contained either PHA665752 (0.2 Amol/L) or DMSO in partially rescues the BaF3.TPR-MET cells from PHA665752- the control samples. After 4 hours, cells in the lower compartment dependent growth inhibition. We did not observe a significant were resuspended and counted using a Coulter particle counter. growth inhibitory effect of PHA665752 at 0.2 Amol/L in IL-3 The spontaneous transwell migration of cells was expressed as a stimulated parental BaF3 cells in a 72-hour culture (data not

Fig. 1 PHA665752 inhibits cells growth of TPR-MET-transformed BaF3 cells. BaF3 cells lines transformed by tyrosine kinase oncogenes were used to determine cell growth (A-C) or transwell migration (D) in response to the small molecule c-MET kinase inhibitor PHA665752. A, relative growth of BaF3 cells transformed by TPR-MET in response to different concentrations of PHA665752 was determined after 18 hours (n = 3). B, TPR-MET- transformed BaF3 cells were either left untreated (x) or treated (E) with PHA665752 (1 Amol/L) for the indicated time in the presence or absence of IL- 3(n = 3). C, BaF3 cells transformed by tyrosine kinase oncogenes were treated for 18 hours with PHA665752 (1 Amol/L; n = 3). D, cells were treated for 18 hours with the indicated dose of PHA665752 and the spontaneous transwell migration relative to DMSO-treated cells determined (n = 4).

shown). TPR-MET is therefore implicated in the deregulation of inhibited with 0.2 Amol/L PHA665752 (92.5 F 3% inhibition of pathways that are independent of or opposed by growth the cell migration) compared with DMSO-treated cells indicating pathways regulated by the IL-3 receptor, similar to the relation the ability to inhibit this phenotype of malignant transformation. between the ABL inhibitor STI-571 and the BCR-ABL onco- This shows that the TPR-MET kinase activity regulates cell protein in IL-3-treated BaF3 cells. PHA665752 (0.2 Amol/L, growth, motility, and migration of the transformed BaF3 cells. 18 hours) also did not inhibit cell growth of BaF3 cells Inhibition of c-MET Kinase Activity by PHA665752 transformed by other oncogenic tyrosine kinases, including Induces Apoptosis and Cell Cycle Arrest in TPR-MET- BCR-ABL, TEL-JAK2, TEL-ABL, and TEL-PDGFßR (Fig. 1C). Transformed BaF3 Cells. Apoptosis is a complex cellular Untransformed BaF3 cells do not migrate through a function that is regulated in part through the c-MET tyrosine transwell membrane. However, cells transformed by TPR-MET kinase activity in TPR-MET-transformed cells and inhibition of display spontaneous transwell migration consistent with en- c-MET kinase is therefore expected to induce an increase in hanced cell motility. Migration of BaF3.TPR-MET cells was apoptosis. We measured the change in Annexin V positive

staining of cells, an indication for increased exposure of proapoptotic caspase-9. Similar to the previous data, we phosphatidylserine to the outer cell membrane during apoptosis. observed a consistent increase in caspase-3 activity (3.5 F 0.7- Using TPR-MET-transformed BaF3 cells, we found that fold increase; n =3;P < 0.03) compared with DMSO-treated treatment with PHA665752 (0.2 Amol/L, 18 hours) led to an cells (Fig. 2B). increase in Annexin V–positive cells compared with DMSO- We also determined if inhibition of the TPR-MET treated cells (Fig. 2A, top left). In the control cells, 5% of the tyrosine kinase would induce cell cycle arrest. Cells were total population showed signs of apoptosis; however, the number treated with DMSO or increasing concentrations of the c-MET of apoptotic cells increased to 33.1% after PHA665752 kinase inhibitor and the different phases of cell cycle treatment. On average, 13.9 F 1.0% of the cells were in early distribution were then determined by propidium iodide apoptosis (Annexin V positive) and 19.2 F 1.8% of the cells staining and fluorescence-activated cell sorting (Fig. 2C). were in late apoptosis (Annexin V plus propidium iodide The percentage of cells in G1 phase increased from 42.4% to positive). To confirm this observation, we thereafter measured 77.0% in PHA665752-treated cells (0.2 Amol/L), whereas the the activation status of caspase-3, a downstream effector of the percentage of cells in S phase (reduced from 45.4% to 17.5%)

Fig. 2 PHA665752 induces apoptosis and cell cycle arrest in TPR-MET-transformed BaF3- cells. TPR-MET-transformed BaF3 cells were treated for 18 hours with either DMSO or the indicated amount of PHA665752 (n =3).A, Annexin V and propidium iodide staining was determined by flow cytometry. B, activity of caspase-3 was determined in cell lysate (n = 3). C, % cells in different cell cycle phases was determined by flow cytometry after propidium iodide staining (n = 3).

Fig. 3 PHA665752 inhibits c-MET-mediated tyrosine phosphorylation and TPR-MET autophosphorylation and regulates cell growth through the mTOR-dependent pathway. Phosphorylation of cellular proteins was determined by immunoblotting in whole cell lysate as indicated using anti- phosphotyrosine antibody (4G10, A), total c-MET antibody, anti-pY1230/1234/1235-MET antibody (recognizing the corresponding pY361/365/366 sites in TPR-MET), anti-pY1349-MET (recognizing the pY480 site in TPR-MET) and anti-pY1365-MET (recognizing the pY496 site in TPR-MET) phospho-antibodies (B), and phospho-AKT and phospho-S6K antibodies (C). TPR-MET-transformed BaF3 cells were treated with the indicated amount of PHA665752. Blots were probed for equal loading with antibodies against p85 PI3K or p70-S6K (A-C).

421 424 and G2-M phase (reduced from 12.2% to 5.5%) decreased. mTOR substrate p70-S6K[Thr /Ser ] (Fig. 3C). This would This suggests that inhibition of TPR-MET kinase activity suggest that inhibition of c-MET kinase activity leads to reduced leads to G1 cell cycle arrest in the transformed cells. In activation of the PI3K/AKT/mTOR pathway in these trans- addition, there was an increase of cells in sub-G1 phase, formed cells. which was consistent with apoptotic cells. These data show PHA665752 Cooperates with Rapamycin to Inhibit Cell that PHA665752 induces cell cycle arrest as well as apoptosis, Growth in TPR-MET-Transformed BaF3 Cells through and both events in combination are likely to contribute to the Mammalian Target of Rapamycin–Dependent Pathway. reduced cell growth of the PHA665752-treated TPR-MET- Finally, we determined the significance of mTOR regulation transformed cells. by c-MET in the cells with the specific mTOR inhibitor PHA665752 Inhibits Tyrosine Phosphorylation of rapamycin. In the absence of PHA665752, rapamycin reduced Cellular Proteins in TPR-MET-Transformed BaF3 Cells. cell growth of the BaF3.TPR-MET cells in a dose-dependent To determine the biochemical consequences of c-MET kinase manner. In the presence of PHA665752 (0.05 Amol/L), inhibition by PHA665752 in BaF3.TPR-MET cells, changes in rapamycin cooperated with the c-MET inhibitor in inhibiting tyrosine phosphorylation of cellular proteins were evaluated. cell growth of the TPR-MET-transformed cells (Fig. 4). The tyrosine phosphorylation sites in TPR-MET with the PHA665752 can also Cooperate with Rapamycin in corresponding sites in the tyrosine kinase domain of c-MET Inhibiting Non–Small Cell Lung Cancer H441 Cells. The are described in Materials and Methods. The extracellular and significance of the regulation of mTOR pathway by c-MET and juxtamembrane domains of c-MET are deleted as a result its potential for therapeutic targeting in NSCLC was assessed. of the chromosomal translocation resulting in the TPR-MET PHA665752 was active against NSCLC H441 cells in vitro (22). fusion oncoprotein. Treatment of BaF3.TPR-MET cells with H441 cells were recently identified as sensitive to PHA665752 PHA665752 reduced tyrosine phosphorylation of cellular inhibition in soft agar colony growth, cell proliferation, and cell proteins in a dose-dependent manner (Fig. 3A) but did not alter migration, abrogating signaling through extracellular signal- tyrosine phosphorylation of cellular proteins in BCR-ABL- regulated kinase 1/2, AKT, and FAK (22). Here, we show that transformed BaF3 cells (data not shown). These data are PHA665752 (0.5 Amol/L) can cooperate with rapamycin in consistent with the dose-dependent reduction of cell growth in inhibiting cell viability of H441 cells (Fig. 5), similar to that seen the present studies and suggest that PHA665752 specifically with BaF3.TPR-MET cells. inhibits TPR-MET-induced tyrosine phosphorylation but not BCR-ABL. In addition, using phosphospecific antibodies against DISCUSSION tyrosine phosphorylation sites in c-MET, we found that c-MET RTK has been convincingly shown in many PHA665752 inhibits autophosphorylation in the catalytic human cancers to be involved in oncogenesis, tumor progres- tyrosine kinase domain at Tyr361/365/366 (autophosphorylation sion with enhanced cell motility and invasion, and metastasis site), Tyr480 (growth factor receptor binding protein 2 binding (1, 23). c-MET can be activated through overexpression or site), and Tyr496 (important in cell morphogenesis; Fig. 3B). mutations in various human cancers, including SCLC (4) and In addition, we sought to determine if inhibition of TPR- NSCLC.4 We have characterized here a novel small molecule– MET would reduce the phosphorylation and alter the activation selective c-MET inhibitor, PHA665752, that inhibited cell status of pathways that are involved in cell growth and growth, motility and migration of BaF3 cells transformed with proliferation. We found that the dose-dependent reduction in TPR-MET. We had previously shown that the BaF3 cell line tyrosine phosphorylation of cellular proteins after PHA665752 treatment correlated with reduced serine phosphorylation of 473 AKT[Ser ] as well as the reduced phosphorylation of the 4 Unpublished data.

Molecular targeted therapies against protein tyrosine kinases, such as BCR-ABL (non-RTK) in chronic myelogenous leukemia and epidermal growth factor receptor (RTK) in NSCLC, have gained much progress in recent years. However, resistance to tyrosine kinase inhibitors has also become an important emerging issue. The mechanisms of tyrosine kinase inhibitor resistance includes oncogene amplification and overexpression, as well as resistant mutations in the kinase oncoprotein, not limited to the kinase domain (24). Hence, attempts to combine additional inhibitory agents together with tyrosine kinase inhibitors in the quest for curative therapy, or at least a more durable response, have taken on new momentum (25). The PI3K/AKT/mTOR pathway is an attractive target for this combinational approach because it has been shown activated Fig. 4 PHA665752 cooperates with rapamycin in regulating growth by c-MET signaling, among other oncogenic tyrosine kinases. through mTOR-dependent pathway. The relative growth of BaF3 cells transformed by TPR-MET in response to different concentrations of Safe and effective inhibitors of PI3K or AKT have not been well rapamycin (0.01-10 nmol/L) was determined in the presence (E)or established yet at this time, whereas inhibitors of mTOR such as absence (n) of PHA665752 (0.05 Amol/L) after a 3-day culture (n = 3). rapamycin or its analogue CCI-779 are available and have shown Bars, SE. antitumor activity (26). Rapamycin acts by binding to the immunophilin FK506-binding protein (FKBP12), with the resultant complex inhibiting the target of rapamycin (TOR). system can serve as an excellent model for studying trans- We have recently shown that PI3K is an important signaling forming tyrosine kinase oncogenes (21). The specificity of pathway downstream of c-MET (27). PI3K is responsible for PHA665752 was further shown by its inability to inhibit diverse cellular regulation, including cell adhesion, motility and growth mediated by transforming forms of the ABL, JAK2, or migration, proliferation, reduced apoptosis, anchorage indepen- PDGFh receptor. Inhibition of c-MET kinase activity by the dence, and intracellular vesicle trafficking/secretion (28, 29). small molecule drug induced apoptosis and cell cycle arrest in AKT (30, 31) and FKHR (32, 33) are two downstream targets of TPR-MET-transformed BaF3 cells. We also found that the PI3K, and phosphorylation of them leads to enhanced cell dose-dependent reduction in tyrosine phosphorylation of survival. Constitutive activation of PI3K signaling pathway has cellular proteins after PHA665752 treatment correlated with been reported in SCLC, mediating anchorage-independent reduced activation of the PI3K/AKT/mTOR pathway in these proliferation via protein kinase B/AKT and p70-S6K-dependent cells. The specific mTOR inhibitor rapamycin was found able pathway (34). Upon activation, c-MET can recruit and associate to cooperate with the c-MET inhibitor PHA665752 in the with PI3K, which eventually lead to downstream pathway reduction of cell growth of BaF3.TPR-MET and H441 NSCLC cells. PHA665752 serves as an example of specific small molecule c-MET inhibitor. Recently, we have reported the effects of the c-MET inhibitor SU11274 on the TPR-MET-transformed BaF3 cell system and showed that this constitutively activated oncogenic c-MET allows the identification and characterization of small molecule–selective c-MET inhibitors (21). Unlike Gleevec for chronic myelogenous leukemia, (targeting BCR/ABL) and gastrointestinal stromal tumor (targeting c-KIT), targeted small molecule inhibitors against c-MET have not yet come to clinical fruition. In our previous study with BaF3.TPR-MET cells, SU11274 inhibited the IL-3-independent cell growth in a dose- dependent manner with an IC50 of <3 Amol/L (21), compared with studies in this report with PHA665752 that lead to an IC50 of <0.06 Amol/L. Evidently, in the current results, PHA665752 is more potent than SU11274. Similar to our current findings with PHA665752 in this study, we also found dramatic inhibitory effects on cell motility and migration of BaF3.TPR-MET cells when treated with SU11274. However, the effects were observed Fig. 5 PHA665752 cooperates with rapamycin in inhibiting growth of at a higher concentration of SU11274 (44.8% inhibition NSCLC H441 cells through mTOR-dependent pathway. The cell growth at 1 Amol/L and 80% inhibition at 5 Amol/L) when compared of NSCLC H441 cells, in culture medium containing 2% FCS and HGF with that observed here with PHA665752 (>90% inhibition (40 ng/mL), in response to different concentrations of rapamycin (0.01- A 20 nmol/L) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- at 0.2 mol/L). Cell motility and migration of a number of cell nyltetrazolium bromide assay in triplicates in the presence (E)or systems is tightly controlled by PI3K. In this study, we show that absence (n) of PHA665752 (0.5 Amol/L) after a 3-day culture (n = 3). PI3K pathway is dramatically affected by c-MET inhibition. Bars, SE.

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Patrick C. Ma, Erik Schaefer, James G. Christensen, et al.

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