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

¹ Section of Hematology/Oncology, Department of Medicine, Pritzker School of Medicine, University of Chicago Medical Center, Chicago, Illinois; ² BioSource International, Inc., Camarillo, California; and ³ Research Pharmacology, Pfizer, Inc., La Jolla, California

Requests for reprints: Ravi Salgia, Section of Hematology/Oncology, Department of Medicine, Room M-255A, University of Chicago Medical Center, 5841 South Maryland Avenue, MC2115, Chicago, IL, 60637. Phone: 773-702-4399; Fax: 773-834-1798; E-mail: rsalgia{at}medicine.bsd.uchicago.edu.

	ABSTRACT

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Purpose: c-MET is believed to be an attractive receptor 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 characterized a small molecule c-MET inhibitor, PHA665752, and tested its cooperation with the mammalian target of rapamycin inhibitor as potential targeted therapy.

Experimental Design: The effect of PHA665752 treatment was determined on cell growth, motility and migration, apoptosis, and cell-cycle arrest of TPR-MET-transformed cells. Moreover, the effect of PHA665752 on the phosphorylation on MET, as well as its downstream effectors, p-AKT and p-S6K, was also determined. Finally, growth of TPR-MET-transformed cells was tested in the presence of PHA665752 and rapamycin. H441 non–small cell lung cancer (NSCLC) cells (with activated c-Met) were also tested against both PHA665752 and rapamycin.

Results: PHA665752 specifically inhibited cell growth in BaF3. TPR-MET cells (IC₅₀ < 0.06 µmol/L), induced apoptosis and cell cycle arrest. Constitutive cell motility and migration of the BaF3. TPR-MET cells was also inhibited. PHA665752 inhibited specific phosphorylation of TPR-MET as well as phosphorylation of downstream targets of the mammalian target of rapamycin pathway. When combined with PHA665752, rapamycin showed cooperative inhibition to reduce growth of BaF3. TPR-MET- and c-MET-expressing H441 NSCLC cells.

Conclusions: PHA665752 is a potent small molecule–selective c-MET inhibitor and is highly active against TPR-MET-transformed cells both biologically and biochemically. PHA665752 is also active against H441 NSCLC cells. The c-MET inhibitor can cooperate with rapamycin in therapeutic inhibition of NSCLC, and in vivo studies of this combination against c-MET expressing cancers would be merited.

Key Words: c-MET • HGF • kinase inhibitor • phosphorylation • tumor

	INTRODUCTION

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c-MET is a unique receptor tyrosine kinase (RTK) located on chromosome 7p and activated via its natural ligand hepatocyte growth factor. c-MET can also be mutated in a variety of solid tumors (1), such as in the tyrosine kinase domain for hereditary papillary renal cell carcinoma (2, 3) and in the sema and juxtamembrane domains in small cell lung cancer (SCLC; ref. 4). Many of these are activating mutations and potentially targeted by specific c-MET small molecule inhibitors.

The TPR-MET oncogene is a transforming variant of the c-MET RTK and was initially identified after treatment of a human osteogenic sarcoma cell line transformed by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (5–7). The TPR-MET fusion oncoprotein is the result of a chromosomal translocation, placing the TPR3 locus on chromosome 1 upstream of a portion of the c-MET gene on chromosome 7 encoding only for the cytoplasmic region (6, 7). Some studies suggest that TPR-MET is detectable in experimental cancer (8–10). Dimerization of the M_r 65,000 TPR-MET oncoprotein through a leucine zipper motif encoded by TPR leads to constitutive activation of the c-MET kinase (11, 12). TPR-MET acts 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)/AKT pathway (17, 18). Conversely, in contrast to c-MET RTK, TPR-MET is ligand independent, lacks the CBL binding site in the juxtamembrane region in c-MET, and is mainly cytoplasmic (5–7, 19, 20). We have recently shown that the TPR-MET fusion oncoprotein has potent transforming activity and can be an excellent model for testing inhibitors as it has tremendous constitutively elevated c-MET tyrosine kinase activity that can readily be inhibited (21).

When introduced into an interleukin-3 (IL-3)–dependent cell line, BaF3, TPR-MET induces oncogenic phenotypes such as growth factor independence and constitutive tyrosine phosphorylation of multiple cellular proteins, including TPR-MET itself (21). Recently, a novel-specific small molecule inhibitor of TPR-MET and c-MET kinase activity, SU11274, was identified which has in vitro inhibitory activities in TPR-MET cells and in c-MET expressing SCLC cells (i.e., H69 and H345) at low micromolar concentrations (21). SU11274 has cellular IC₅₀ between 2.5 and 5 µmol/L in SCLC.

To further explore the potential of targeted therapy against the c-MET kinase, we have characterized here a small molecule c-MET inhibitor with relatively improved potency and selectivity, PHA665752 (22). PHA665752 was recently identified and characterized to be active against several solid tumors (22). We have recently reported that non-SCLC (NSCLC) tumors can overexpress c-Met, and the H441 NSCLC cell line has been shown to overexpress and contain an activated c-Met. Here, we found that PHA665752 specifically inhibited cell growth, motility, and migration of cells transformed with TPR-MET. Inhibition of c-MET kinase activity by the small molecule drug induced apoptosis and cell cycle arrest in TPR-MET-transformed BaF3 cells. We also found that the dose-dependent reduction in tyrosine phosphorylation of cellular proteins after PHA665752 treatment correlated with reduced activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway in these cells. Finally, we tested the specific mTOR inhibitor rapamycin alone or in combination with PHA665752 and found that rapamycin can cooperate with the c-MET inhibitor in inhibiting cell viability of BaF3.TPR-MET cells as well as NSCLC H441 cells. Our results suggest that the combination of c-MET inhibitors such as PHA665752 with mTOR inhibitor (rapamycin) may be a feasible approach to improve the therapeutic efficacy in c-MET expressing cancers including NSCLC.

	MATERIALS AND METHODS

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Cells. The murine pre-B cell line BaF3 was grown in RPMI 1640 containing 10% FCS and 10% WEHI-conditioned medium as a source of murine IL-3. BaF3 cell lines transfected with a BCR-ABL, TEL-ABL, TEL-JAK2, or TEL-PDGFßR cDNA were grown in the absence of growth factors. A TPR-MET expressing BaF3 cell line was generated by transfection of an expression vector containing the TPR-MET cDNA as previously described (21). NCI-H441 cells were obtained from American Type Culture Collection (Rockville, MD) and were cultured according to the instructions (http://www.ATCC.org). The number of viable cells after treatment with DMSO, PHA665752, and/or rapamycin was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (In vitro Toxicology Assay Kit, Sigma) or trypan blue exclusion.

Chemicals. PHA665752, (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one (Pfizer, Inc., La Jolla, CA), and rapamycin (Calbiochem, La Jolla, CA) were dissolved in DMSO and used at the concentrations as described.

Transwell Migration Assay. The lower chamber of a transwell plate (8-µm pore size polycarbonate membrane, Corning Costar Co., Cambridge, MA) was filled with 600 µL starvation medium (0.5% w/v, bovine serum albumin in RPMI 1640). Cells were counted using a Coulter particle counter (Coulter Counter Z2, Beckman Coulter, Fullerton, CA) and resuspended at 2 x 10⁶ cells/mL in starvation medium; 100 µL of this cell suspension were transferred to the upper chamber. The medium contained either PHA665752 (0.2 µmol/L) or DMSO in the control samples. After 4 hours, cells in the lower compartment were resuspended and counted using a Coulter particle counter. The spontaneous transwell migration of cells was expressed as a "migration index" (number of migrating cells treated with PHA665752 divided by the number of migrating cells left untreated). SE was calculated from the migration indices of independently done experiments. The statistical significance of the data was analyzed using the Student's t test.

Immunoblotting. Proteins were extracted from whole cells by lysing them in a Tris buffer (50 mmol/L, pH 8.0) containing NaCl (150 mmol/L), NP40 (1%, v/v), deoxycholic acid (0.5%, w/v), SDS (0.1%, w/v), NaF (1mmol/L), Na₃VO₄ (1 mmol/L), and glycerol (10%, v/v; Sigma) supplemented with a protease inhibitor cocktail (complete, Roche Diagnostics, Indianapolis, IN). Polyclonal antibodies against p70-S6K (Biosource International, Camarillo, CA), total c-MET (C-12, Santa Cruz Biotechnology, Santa Cruz, CA), PI3K (Upstate Biotechnology, Lake Placid, NY) and phospho-AKT[Ser⁴⁷³] or phospho-p70-S6K[Thr421/Ser424] (Cell Signaling, Beverly, MA), and phosphotyrosine (4G10, Upstate Biotechnology) were used for immunoblotting. In addition, polyclonal antibodies against phospho-MET [Tyr^{1230/1234/1235}], –[Tyr¹³⁴⁹] and –[Tyr¹³⁶⁵] (recognizing phospho-[Tyr^361/365/366], –[Tyr⁴⁸⁰], and –[Tyr⁴⁹⁶] respectively in TPR-MET) were obtained from Biosource International for the immunoblotting. The Tyr amino acid numbering of the phosphor-MET antibodies is based on the shorter splice variant with 100-bp-long exon 10, instead of the full-length 154 bp.

Apoptosis Assays. The activity of caspase-3 was measured in cell lysates (CaspACE Assay System, Promega, Madison, WI) and Annexin V positive staining was determined by fluorescence-activated cell sorting analysis (Annexin-V-Fluos Staining Kit, Roche Diagnostics) according to the manufacturer's directions in cells that were either treated with PHA665752 or the solvent DMSO.

Cell cycle analysis. Fixed cells were stained with propidium iodide and cell cycle variables analyzed by fluorescence-activated cell sorting analysis.

	RESULTS

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The Small Molecule c-MET Inhibitor PHA665752 Specifically Regulates Cell Growth in TPR-MET-Transformed BaF3 Cells. PHA665752 was identified as a prototype ATP-competitive small molecule inhibitor of the catalytic kinase activity of the c-MET RTK (22). We initially sought to determine if PHA665752 could inhibit cell growth in TPR-MET-transformed BaF3 cells (Fig. 1A). Treatment of BaF3.TPR-MET cells with PHA665752 was found to inhibit cell growth in a dose-dependent manner with an IC₅₀ < 0.06 µmol/L. To further determine if the growth inhibitory effect of PHA665752 on BaF3.TPR-MET cells is maintained over time, cell growth was determined over a 72-hour culture. In the presence of IL-3, PHA665752 had only little effect on cell growth of TPR-MET-transformed cells or BCR-ABL-transformed cells (Fig. 1B, top). In contrast, PHA665752 completely blocked cell growth in the absence of IL-3 in BaF3.TPR-MET and even reduced the number of viable cells (Fig. 1B, bottom). This suggests that IL-3 partially rescues the BaF3.TPR-MET cells from PHA665752-dependent growth inhibition. We did not observe a significant growth inhibitory effect of PHA665752 at 0.2 µmol/L in IL-3 stimulated parental BaF3 cells in a 72-hour culture (data not shown). TPR-MET is therefore implicated in the deregulation of pathways that are independent of or opposed by growth pathways regulated by the IL-3 receptor, similar to the relation between the Abl inhibitor STI-571 and the BCR-ABL oncoprotein in IL-3-treated BaF3 cells. PHA665752 (0.2 µmol/L, 18 hours) also did not inhibit cell growth of BaF3 cells transformed by other oncogenic tyrosine kinases, including BCR-ABL, TEL-JAK2, TEL-ABL, and TEL-PDGFßR (Fig. 1C).

View larger version (23K): [in this window] [in a new window]   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 () or treated () with PHA665752 (1 µmol/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 µmol/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).

Inhibition of c-MET Kinase Activity by PHA665752 Induces Apoptosis and Cell Cycle Arrest in TPR-MET-Transformed BaF3 Cells. Apoptosis is a complex cellular function that is regulated in part through the c-MET tyrosine kinase activity in TPR-MET-transformed cells and inhibition of c-MET kinase is therefore expected to induce an increase in apoptosis. We measured the change in Annexin V positive staining of cells, an indication for increased exposure of phosphatidylserine to the outer cell membrane during apoptosis. Using TPR-MET-transformed BaF3 cells, we found that treatment with PHA665752 (0.2 µmol/L, 18 hours) led to an increase in Annexin V–positive cells compared with DMSO-treated cells (Fig. 2A, top left). In the control cells, 5% of the total population showed signs of apoptosis; however, the number of apoptotic cells increased to 33.1% after PHA665752 treatment. On average, 13.9 ± 1.0% of the cells were in early apoptosis (Annexin V positive) and 19.2 ± 1.8% of the cells were in late apoptosis (Annexin V plus propidium iodide positive). To confirm this observation, we thereafter measured the activation status of caspase-3, a downstream effector of the proapoptotic caspase-9. Similar to the previous data, we observed a consistent increase in caspase-3 activity (3.5 ± 0.7-fold increase; n = 3; P < 0.03) compared with DMSO-treated cells (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   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).

PHA665752 Inhibits Tyrosine Phosphorylation of Cellular Proteins in TPR-MET-Transformed BaF3 Cells. To determine the biochemical consequences of c-MET kinase inhibition by PHA665752 in BaF3.TPR-MET cells, changes in tyrosine phosphorylation of cellular proteins were evaluated. The tyrosine phosphorylation sites in TPR-MET with the corresponding sites in the tyrosine kinase domain of c-MET are described in Materials and Methods. The extracellular and juxtamembrane domains of c-MET are deleted as a result of the chromosomal translocation resulting in the TPR-MET fusion oncoprotein. Treatment of BaF3.TPR-MET cells with PHA665752 reduced tyrosine phosphorylation of cellular proteins in a dose-dependent manner (Fig. 3A) but did not alter tyrosine phosphorylation of cellular proteins in BCR-ABL-transformed BaF3 cells (data not shown). These data are consistent with the dose-dependent reduction of cell growth in the present studies and suggest that PHA665752 specifically inhibits TPR-MET-induced tyrosine phosphorylation but not BCR-ABL. In addition, using phosphospecific antibodies against tyrosine phosphorylation sites in c-MET, we found that PHA665752 inhibits autophosphorylation in the catalytic tyrosine kinase domain at Tyr^361/365/366 (autophosphorylation site), Tyr⁴⁸⁰ (growth factor receptor binding protein 2 binding site), and Tyr⁴⁹⁶ (important in cell morphogenesis; Fig. 3B).

View larger version (18K): [in this window] [in a new window]   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).

PHA665752 Cooperates with Rapamycin to Inhibit Cell Growth in TPR-MET-Transformed BaF3 Cells through Mammalian Target of Rapamycin–Dependent Pathway. Finally, we determined the significance of mTOR regulation by c-MET in the cells with the specific mTOR inhibitor rapamycin. In the absence of PHA665752, rapamycin reduced cell growth of the BaF3.TPR-MET cells in a dose-dependent manner. In the presence of PHA665752 (0.05 µmol/L), rapamycin cooperated with the c-MET inhibitor in inhibiting cell growth of the TPR-MET-transformed cells (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Fig. 4 PHA665752 cooperates with rapamycin in regulating growth through mTOR-dependent pathway. The relative growth of BaF3 cells transformed by TPR-MET in response to different concentrations of rapamycin (0.01-10 nmol/L) was determined in the presence () or absence () of PHA665752 (0.05 µmol/L) after a 3-day culture (n = 3). Bars, SE.

View larger version (14K): [in this window] [in a new window]   Fig. 5 PHA665752 cooperates with rapamycin in inhibiting growth of NSCLC H441 cells through mTOR-dependent pathway. The cell growth of NSCLC H441 cells, in culture medium containing 2% FCS and HGF (40 ng/mL), in response to different concentrations of rapamycin (0.01-20 nmol/L) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in triplicates in the presence () or absence () of PHA665752 (0.5 µmol/L) after a 3-day culture (n = 3). Bars, SE.

	DISCUSSION

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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 µmol/L (21), compared with studies in this report with PHA665752 that lead to an IC₅₀ of <0.06 µmol/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 at a higher concentration of SU11274 (44.8% inhibition at 1 µmol/L and 80% inhibition at 5 µmol/L) when compared with that observed here with PHA665752 (>90% inhibition at 0.2 µmol/L). Cell motility and migration of a number of cell systems is tightly controlled by PI3K. In this study, we show that PI3K pathway is dramatically affected by c-MET inhibition.

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 by c-MET signaling, among other oncogenic tyrosine kinases. Safe and effective inhibitors of PI3K or AKT have not been well established yet at this time, whereas inhibitors of mTOR such as rapamycin or its analogue CCI-779 are available and have shown antitumor activity (26). Rapamycin acts by binding to the immunophilin FK506-binding protein (FKBP12), with the resultant complex inhibiting the target of rapamycin (TOR).

We have recently shown that PI3K is an important signaling pathway downstream of c-MET (27). PI3K is responsible for diverse cellular regulation, including cell adhesion, motility and migration, proliferation, reduced apoptosis, anchorage independence, and intracellular vesicle trafficking/secretion (28, 29). AKT (30, 31) and FKHR (32, 33) are two downstream targets of PI3K, and phosphorylation of them leads to enhanced cell survival. Constitutive activation of PI3K signaling pathway has been reported in SCLC, mediating anchorage-independent proliferation via protein kinase B/AKT and p70-S6K-dependent pathway (34). Upon activation, c-MET can recruit and associate with PI3K, which eventually lead to downstream pathway activation of AKT (35). AKT in turn regulates cell survival by inhibiting caspase-9, Bad, and the forkhead (FKHR) transcription factors. Here, we show that the c-MET inhibitor PHA665752 treatment of BaF3.TPR-MET cells leads to decreased phosphorylation of AKT at Ser⁴⁷³ (phosphorylated by the kinase PDK2), thereby leading to apoptosis. It has been shown that c-MET/hepatocyte growth factor activation protects against cell death via PI3K- and AKT-dependent pathway in human glioblastoma cells treated with cytotoxic agents (36). It would therefore be useful to determine whether there is any cooperation or synergism between PHA665752 and PI3K inhibition.

mTOR is an important signaling intermediate molecule downstream of the PI3K/AKT pathway that inhibits apoptosis and is important in nutritional status checkpoint (37–40). mTOR is a large (M_r 289,000) multidomain serine/threonine kinase and is a member of the PI3K family of protein kinases based on homology within its catalytic domain. Although signals that activate mTOR have not been well understood, AKT phosphorylation and protein interactions via the mTOR NH₂-terminal multiple repeat HEAT motifs are possible mechanisms. The p70-S6K and the translation inhibitor 4E-BP-1 are the two best-characterized mTOR substrates. Growth factor activation of the PI3K pathway results in phosphorylation and activation of p70-S6K by mTOR or PDK-1. Rapamycin, initially approved by the Food and Drug Administration in 1999 as an immunosuppressant for prevention of allograft rejection, has been shown to have selective antitumor activity in a broad range of human cancers in vitro and in vivo with mutations in PTEN or up-regulation of the PI3K/AKT pathway (41).

In this report, we show that the c-MET inhibitor PHA665752 inhibited c-MET/hepatocyte growth factor pathway–mediated tyrosine phosphorylation of cellular proteins. In addition, there was also a dose-dependent inhibition of the serine phosphorylation of AKT[Ser⁴⁷³] as well as the phosphorylation of the mTOR substrate p70-S6K[Thr⁴²¹/Ser⁴²⁴]. This provides an opportunity for testing the hypothesis that the hepatocyte growth factor/c-MET-PI3K/AKT/mTOR signaling axis can be modulated and inhibited with a combination of both the specific c-MET inhibitor (PHA665752) and also the downstream specific inhibitor of mTOR (rapamycin). Attempts to combine rapamycin and tyrosine kinase inhibitors to improve the treatment of primary/relapsed chronic myelogenous leukemia and/or acute myelogenous leukemia caused by FLT3 mutations have shown some promise in preclinical models (25). Here, we show that this combinational strategy is functional in vitro with the rapamycin cooperating with the c-MET inhibitor in reducing the cell viability of the BaF3.TPR-MET cells as well as NSCLC H441 cells. The c-MET inhibitor PHA665752 may exert its effects downstream of c-MET by modulating the AKT/mTOR pathway. In addition, cooperative effects of PHA665752 and rapamycin seen here may be a result of inhibition of complementary pathways downstream of c-MET that are independent of mTOR. It would now be useful to further test this strategy in an in vivo mouse model. Current data suggest that PHA665752 does have potent in vivo cytoreductive antitumor activity shown in a gastric carcinoma xenograft model (22). Rapamycin or specific drugs similar to it against mTOR pathway may be attractive therapeutic agents to be used in combination therapy with c-MET inhibition by prototype c-MET inhibitors in c-MET expressing cancers.

	FOOTNOTES

 
Grant support: American Association for Cancer Research-Astra-Zeneca-Cancer Research and Prevention Foundation fellowship in Translational Lung Cancer Research (P. Ma), American Cancer Society Institutional research grant ACS IRG-58-004-44 (P. Ma), American Cancer Society (Illinois Division)-LUNGevity Foundation Lung Cancer Treatment research award (P. Ma), American Cancer Society Research scholar grant RSG-02-244-02-CCE (R. Salgia), NIH/National Cancer Institute R01 grant R01 CA100750-01A1 (R. Salgia), and Institutional Cancer research awards from the University of Chicago Cancer Center along with the American Cancer Society and the V-Foundation (R. Salgia).

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.

⁴ Unpublished data.

Received 8/23/04; revised 12/ 7/04; accepted 12/23/04.

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