Introduction

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The protein-tyrosine kinase family can be divided into subgroups that have similar structural organization and sequence similarity within the kinase domain. The members of the type III group of receptor tyrosine kinases include the platelet-derived growth factor (PDGF) receptors (PDGF receptors  and ), colony-stimulating factor (CSF)-1 receptor (CSF-1R, c-Fms), Flt-3, and stem cell or steel factor receptor (c-Kit). These receptor tyrosine kinases are characterized by five Ig-like domains in the extracellular domain and a cytoplasmic region containing a hydrophilic kinase insert domain (Heldin, 1995). PDGF receptors are normally found in connective tissue and glia but are lacking in most epithelia. However, recent studies have implicated paracrine or autocrine PDGF loops in the growth deregulation of gliomas and sarcomas as well as various human epithelial tumors. Furthermore, PDGF has been implicated in the pathogenesis of several nonmalignant proliferative diseases, including atherosclerosis, restenosis following vascular angioplasty (Ferns et al., 1991), and fibroproliferative disorders such as obliterative bronchiolitis (Hertz et al., 1992).

c-kit is the cellular homolog of the v-kit retroviral oncogene. The c-kit gene product is expressed in hematopoietic progenitor cells, mast cells, germ cells, interstitial cell of cajal, and some human tumors (Nocka et al., 1989; Turner et al., 1992; Ishikawa et al., 1997). Studies with mice with inactivating mutations of c-kit or its ligand have demonstrated that the c-kit gene product is essential for maintenance of normal hematopoiesis, melanogenesis, gametogenesis, and growth and differentiation of mast cells and interstitial cell of cajal. In addition to its normal role, deregulation of c-Kit is thought to plan a role in certain human tumors, including germ cell tumors, mast cell tumors, gastrointestinal stomal tumors, small-cell lung cancers (SCLCs), melanoma, breast cancer, and neuroblastoma. In a number of tumors types, c-Kit-mediated growth has been found to occur via mutation of c-kit, which results in ligand-independent activation of the receptor (Hirota et al., 1998; Longley et al., 1999; Tian et al., 1999).

Recently, we have described a protein-tyrosine kinase inhibitor (STI571/CGP 57148B) of the 2-phenylaminopyrimidine class that has selectivity for the Abl and PDGF receptor tyrosine kinases (Druker et al., 1996; Carroll et al., 1997; Zimmermann et al., 1997). In these reports, STI571 inhibited the Abl and PDGF receptor kinases at the in vitro enzyme, cellular, and in vivo levels. Based on its preclinical activity against Bcr-Abl, STI571 is currently in clinical trials for the therapy of chronic myelogenous leukemia (CML). In the current study, we have further extended the preclinical profile of STI571 against additional protein kinases from the type III group of receptor tyrosine kinases and other related receptor kinases. In addition to its previously reported inhibition of the Abl and PDGF receptor tyrosine kinases, STI571 was found to inhibit c-Kit, but did not affect closely related kinases such as c-Fms, Kdr, Flt-1, Tek, and Flt-3. The data also demonstrate that the compound is a potent inhibitor of c-Kit and PDGF-mediated signal transduction events in cells. In addition to CML, STI571 may have potential clinical utility in cancers and nonmalignant proliferative diseases involving deregulated c-Kit or PDGF receptor activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

CGP 57148 and STI571 (formerly known as CGP 57148B, the methane sulfonate salt of CGP 57148) were synthesized by Novartis Pharma AG (Basel, Switzerland). CGP 57148 is referred to as compound 1 in Zimmermann et al. (1997). No significant difference in results was seen between the two forms of the compound in in vitro studies, and both forms are referred to as STI571 in this study. For in vitro and cellular assays, a stock concentration of 10 mM STI571 was prepared in Me₂SO₄ and stored at 20°C. Dilutions for all assays were freshly made before use. Liquid media, fetal calf serum (FCS), and media additives were from Life Technologies Inc. (Basel, Switzerland). PDGF-BB was from Life Technologies Inc., and PDGF-AA was from Bachem (Bubendorf, Switzerland). Stem cell factor (SCF) was from Genzyme (Cambridge, MA), Flt-3 was provided by Immunex (Seattle, WA), anti-c-Kit antibodies were from Oncogene Science (Cambridge, MA), anti-PDGF receptor  antibodies were from Santa Cruz Biotechnology (sc338; Santa Cruz, CA), and anti-PDGF receptor / antibodies were from Upstate Biotechnology (06-495; Lake Placid, NY). Myo-[2-³H]inositol with PT6 polymer (10-20 Ci/mmol) was from Amersham (Arlington Heights, IL), and the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay was from Promega (Madison, WI).

Cell Lines. The 32D cell line is a murine myeloid cell line that is dependent on interleukin-3 (IL-3) for proliferation and was obtained from Joel Greenberger, University of Massachusetts Medical Center, Worcester, MA. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Upstate Biotechnology) and 15% WEHI-3B-conditioned medium as a source of IL-3. MO7e cells are a human megakaryocytic leukemia cell line that requires either granulocte macrophage-colony-stimulating factor (GM-CSF), IL-3, or stem cell factor (SCF) for proliferation and were obtained from G. Bagby, Oregon Health Sciences University (Portland, OR). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 2.5 ng/ml GM-CSF.

In Vitro Kinase Assays. The kinase domains of Kdr, Flt-1, c-Met, and Tek were expressed in High Five cells (Invitrogen, San Diego, CA) using the Bac-to-Bac expression system (Life Technologies Inc.). The proteins were then purified to near homogeneity by standard chromatographic techniques. Kinase inhibition was measured by detecting the decrease in phosphorylation of poly(Glu, Tyr) essentially as previously described for the epidermal growth factor receptor (Buchdunger et al., 1994). The in vitro kinase assays were carried out under optimized assay conditions (ATP concentration ~K_m), which allows a comparison of IC₅₀ values for the different kinases.

Jak-2 Kinase Assay. To determine whether Jak-2 was inhibited by STI571, 32Dp210 Bcr-Abl-expressing cells were serum starved for 8 h, and then stimulated for 10 min with 10% WEHI-3B-conditioned medium (IL-3 source). Nonidet P-40 lysates were prepared, and 500 µg of lysate was immunoprecipitated with either anti-Jak-2 antibodies (5 µl; Upstate Biotechnology), or anti-Abl antibodies (20 µl K12; Santa Cruz Biotechnology), overnight at 4°C. Immunoprecipitates were bound to protein G Sepharose for 1 h, washed three times with PBS, and then with kinase buffer (20 mM Tris, pH 7.5, 10 mM MgCl₂, 10 µM sodium vanadate, 1 mM dithiothreitol). Kinase assays were performed with or without 10 µM STI571, run on a 10% acrylamide gel, and exposed on a phosphorimager.

Cell Extraction. Swiss 3T3 cells were grown to confluency in DMEM containing 10% FCS. Medium was replaced by DMEM containing 0.1% (w/v) BSA, and cells were incubated for 90 min with the indicated concentrations of STI571 before stimulation with PDGF-AA or PDGF-BB for 10 min. MO7e were serum starved (growth in serum-free medium for 16 h) and incubated for 90 min at 37°C with the drug before stimulation with recombinant human SCF (50 ng/ml) for 10 min at 37°C. M1 cells were washed twice with RPMI 1640 and resuspended at 2 × 10⁶ cell/ml of RPMI containing 1% (w/v) BSA for 6 to 8 h. The indicated concentrations of inhibitor were added for the final 2 h of incubation. Cells were stimulated with 1 µg/ml recombinant Flt-3 ligand (Immunex, Seattle, WA) for 5 min. c-Fms-expressing NIH 3T3 cells and v-Src-expressing 32D cells were incubated for 4 h in the presence of STI571 before lysis. To measure IL-3-stimulated tyrosine kinase activity, MO7e cells were washed twice with RPMI 1640 and resuspended at 2 × 10⁶ cells/ml of RPMI 1640 containing 1% BSA. Cells were incubated in this medium for 6 to 8 h. Various concentrations of inhibitor were added for the final 4 h of incubation. At the end of this period, cells were stimulated with 10 ng/ml human recombinant IL-3 (gift of G. Bagby, Oregon Health Sciences University).

Immunoprecipitation. c-Kit was immunoprecipitated from MO7e cell extracts containing 500 µg of total protein using 1 µg of anti-c-Kit antibody. Immunoprecipitation was carried out overnight at 4°C, and immunocomplexes were harvested by the addition of Pansorbin (Calbiochem, La Jolla, CA). After extensive washing, precipitates were resuspended in 40 µl of 2× concentrated SDS sample buffer. Similarly, PDGF receptors were immunoprecipitated from Swiss 3T3 cell extracts using anti-PDGF receptor  or anti-PDGF receptor / antibodies.

Western Blot Analysis. Equal amounts of protein from cell lysates were analyzed by Western blotting with anti-phosphotyrosine antibodies, anti-c-Kit antibodies, anti-PDGF receptor  or anti-PDGF receptor / antibodies. Bound antibodies were detected by using the ECL Western blotting system from Amersham (Amersham, UK). All experiments were performed at least in triplicate, and the effects of STI571 on cellular tyrosine phosphorylation were analyzed in a semiquantitative manner.

Mitogen-Activated Kinase (MAP) Kinase Analysis. Serum-starved MO7e cells were incubated in the presence of the indicated concentrations of STI571 for 2 h at 37°C and then stimulated with 50 ng/ml human SCF for 5 min at 37°C. A10 smooth muscle cells were grown to 70% confluency in 6-well plates and starved in DMEM containing 0.1% BSA for 18 h. After washing with PBS, cells were incubated in DMEM with the indicated concentrations of drug 2 h before stimulation with 20 ng/ml PDGF-BB for 5 min. After washing with ice-cold PBS containing vanadate, cells were lysed. Samples were analyzed by using the Phosphoplus MAP kinase antibody kit (New England Biolabs, Beverley, MA). In brief, samples were resolved on 10% SDS-polyacrylamide gel electrophoresis, and immunoblotted with phosphoMAP kinase antibodies. To control for equal loading of MAP kinase, samples were immunoblotted in parallel with anti-MAP kinase antibodies that recognize both phosphorylated and nonphosphorylated MAP kinase.

Measurement of [³H]Inositol Phosphate Release. A10 cells were grown in 24-well plates (30,000 cells/well) and incubated with [³H]inositol (1 µCi/well) for 2 days in DMEM (without inositol) containing 10% FBS. On the day before the experiment, quiescence was induced by serum derivation for 24 h by replacing the medium with serum-free DMEM containing [³H]inositol (1 µCi/well). On the day of the experiment, quiescent cells were washed twice with Dulbecco's phosphate-buffered salt solution and then incubated for 5 min with HEPES physiological salt solution (142 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl₂, 3.6 mM NaHCO₃, 1.0 mM MgCl₂, 5.6 mM D-glucose, 30 mM HEPES, pH 7.4) containing 0.1% BSA and 20 mM LiCl. Cells were preincubated with STI571 (50 µl) for 30 min in a shaking (50 oscillations/min) water bath at 37°C. The reaction was initiated by the addition of 10 ng/ml PDGF (50 µl) for a final volume of 500 µl. After 5 min, the reaction was terminated by the addition of 0.2 ml HClO₄ (10% v/v), and the plates were placed on ice for 15 min. The supernatants were transferred to glass test tubes, centrifuged (2000 rpm; 5 min), and neutralized with 1.5 M KOH containing 60 mM HEPES. [³H]Inositol phosphates were separated from the neutralized acid extracts by anion exchange chromatography.

Measurement of Cell Growth. Cells were grown in 96-well culture plates (5000 cells/well) and allowed to adhere overnight. The cells were washed twice with Dulbecco's phosphate-buffered salt solution, and quiescence was induced by replacing the medium 24 h before the start of the experiment with DMEM containing 0.2% FBS. Medium was removed from the wells and replaced with DMEM (without phenol red). Quiescent cells were incubated for 30 min with inhibitors and then stimulated with either 10 ng/ml PDGF or 10% FBS for 24 h. Cell growth was measured by using the CellTiter 96 Aq_ueous Non-Radioactive Cell Proliferation assay that measures the reduction, by living cells only, of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium compound (MTS). Briefly, 20 µl of MTS is added to each well during the last 3 h of stimulation with mitogen. The absorbance at 490 nm was recorded by using a microplate reader.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Selectivity for Inhibition of Protein Kinases. STI571 was tested for inhibition of protein-tyrosine kinases of the PDGF receptor subfamily. Previous studies have shown that STI571 inhibits the PDGF receptor; however, these studies used ligand that activated both PDGF receptor  and , so that is was not possible to determine whether STI571 inhibits each of these receptor subtypes. Because Swiss 3T3 cells possess significant numbers of both - and -PDGF receptors (Kazlauskas et al., 1988), we investigated the effect of STI571 on the response of these cells to PDGF-AA and PGDF-BB. PDGF-AA binds to and activates only the PDGF receptor , whereas PDGF-BB binds to and activates all PDGF receptors, including PDGF receptor - and -homodimers and -heterodimers (Heldin et al., 1988). PDGF-AA induced the tyrosine phosphorylation of a protein with a molecular mass of ~170 kDa, which was identified as the PDGF receptor  by immunoprecipitation with anti-PDGF receptor  antibodies (Fig. 1A). Pretreatment of cells with STI571 caused a concentration-dependent inhibition of PDGF-AA-stimulated PDGF receptor -phosphorylation with an IC₅₀ value of approximately 0.1 µM (Fig. 1A). Tyrosine phosphorylation induced by the PDGF-BB homodimer was inhibited with a similar IC₅₀ value as shown by Western blotting with total cell lysates (Fig. 1B, lanes 1-7) or PDGF receptor -immunoprecipitated lysates (Fig. 1B, lanes 8-14). These data indicate, by inference, that STI571 also inhibits PDGF receptor . The overall levels of expressed PDGF receptor protein did not change after exposure to STI571 (Fig. 1, A and B, bottom).

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Inhibition of PDGF receptor autophosphorylation by STI571 in intact cells. Confluent Swiss 3T3 cells were incubated for 90 min with the indicated concentrations of STI571 before stimulation with PDGF-AA (20 ng/ml; A, top) or PDGF-BB (100 ng/ml; B) for 10 min. Whole-cell lysates were corrected for protein content and analyzed by Western blotting with anti-phosphotyrosine antibodies. In lanes 8 to 14, PDGF receptors were immunoprecipitated with anti-PDGF receptor  antibodies (A) or anti-PDGF receptor / antibodies (B). Bottom, lysates were analyzed by Western blotting with anti-PDGF receptor  antibodies (A) or anti-PDGF receptor / antibodies (B).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Inhibition of c-Kit autophosphorylation by STI571 in intact cells. Serum-starved MO7e cells were incubated for 90 min with the indicated concentrations of STI571 before stimulation with SCF (50 ng/ml) for 10 min. Whole-cell lysates were corrected for protein content and analyzed by Western blotting with anti-phosphotyrosine antibodies (A). B, c-Kit was immunoprecipitated with anti-c-Kit antibodies before Western analysis with anti-phosphotyrosine antibodies or anti-c-Kit antibodies (bottom).

View larger version (91K): [in this window] [in a new window]   Fig. 3.   Effects of STI571 on the c-Fms tyrosine kinase. NIH/3T3 cells expressing murine c-Fms were incubated with the indicated amount of inhibitor for 4 h before lysis. Lysates, normalized for protein content, were separated on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibodies.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Effects of STI571 on the Flt-3 ligand-stimulated tyrosine phosphorylaion. Serum-starved M1 cells were treated with the indicated concentrations of STI571 for 2 h. Equal amounts of cell lysate protein were analyzed by Western blotting with anti-phosphotyrosine antibodies. Migration of molecular weight markers (MW, Kd) is indicated on the left.

View larger version (68K): [in this window] [in a new window]   Fig. 5.   Effects of STI571 on the v-Src tyrosine kinase. 32D cells expressing v-src were incubated in the presence of the indicated concentrations of STI571 for 4 h. Equal amounts of cell lysate protein were analyzed by Western blotting with anti-phosphotyrosine antibodies.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Effects of STI571 on Jak-2 tyrosine kinase activity. 32D cells expressing Bcr-Abl were starved for 8 h. At the end of this period, cells were stimulated with 10% WEHI-3B-conditioned medium as a source of IL-3. Cells lysates were immunoprecipitated with Jak-2 (left two lanes) or Abl (right two lanes) antisera and in vitro kinase assays were performed in the presence (+) or absence () of 10 µM STI571. The migration of Jak-2 and Bcr-Abl is indicated with an arrow.

Effects on SCF- and PDGF-Mediated Signal Transduction. A common cellular response to a variety of extracellular signals involves the activation of MAP kinase pathways. To assay the phosphorylation of MAP kinases, an antibody that recognizes the tyrosyl phosphorylated forms of MAP kinases was used for immunoblotting cell lysates. Stimulation of MO7e cells with SCF resulted in the phosphorylation of two MAP kinases, known as pp44^erk1 and pp42^erk2. STI571 strongly inhibited the SCF-induced activation of MAP kinases with an IC₅₀ value between 0.1 and 1 µM (Fig. 7A). Similarly, STI571 treatment inhibited PDGF-BB-mediated MAP kinase activation in rat A10 smooth muscle cells (Fig. 7B). The same cell line was used to test the effect of STI571 on PDGF-mediated [³H]inositol phosphate release. The compound inhibited [³H]inositol phosphate release in response to PDGF-BB treatment with an IC₅₀ value of 0.25 µM (Fig. 8A). In addition, this compound inhibited PDGF-BB-stimulated A10 cell proliferation with an IC₅₀ value of 0.2 µM. In contrast, concentrations of STI571 up to 10 µM had no effect on A10 proliferation induced by serum (Fig. 8B).

View larger version (54K): [in this window] [in a new window]   Fig. 7.   Inhibition of MAP kinase phosphorylation by STI571. Serum-starved MO7e cells (A) or serum-starved A10 cells (B) were incubated with the indicated concentrations of STI571 2 h before stimulation with SCF (50 ng/ml; A) or PDGF-BB (20 ng/ml; B) for 5 min. Cells were lysed and analyzed by Western blotting with anti-phosphoerk antibodies.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Inhibition of PDGF-induced inositol phosphate formation and PDGF-induced proliferation of rat A10 smooth muscle cells. A, [3H]inositol-labeled A10 cells were preincubated with increasing concentrations of STI571 for 30 min and then challenged with 10 ng/ml PDGF for 5 min at 37°C. Basal [3H]inositol phosphate release remained unaffected by STI571. Values shown are the mean ± S.E. obtained from three experiments. Where no error bar is shown, the error is within the size of the symbol. B, growth-arrested A10 cells were incubated with increasing concentrations of STI571 for 30 min before stimulation with 10 ng/ml PDGF or 10% serum at 37°C. The effects on proliferation were assessed with the colorimetric MTS reduction assay after 24 h of treatment. Values shown are the mean ± S.E. obtained from three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this article we extended the in vitro profile of the protein-tyrosine kinase inhibitor STI571, which is currently in clinical trials for the treatment of CML. Previous studies have shown STI571 to be a selective inhibitor of Abl and PDGF receptor tyrosine kinases (Druker et al., 1996; Carroll et al., 1997; Zimmermann et al., 1997). However, these previous studies did not determine which of the PDGF receptor subtypes were inhibited by STI571. The current studies demonstrate that STI571 potently inhibits PDGF-AA-stimulated receptor phosphorylation. The finding that STI571 inhibits PDGF-AA-stimulated receptor phosphorylation in Swiss 3T3 cells indicates that the drug is a potent inhibitor of the PDGF receptor . Because PDGF-BB homodimers bind and activate all possible PDGF receptor dimers (Heldin et al., 1988) and the PDGF-BB-stimulated receptor phosphorylation also was inhibited by STI571, we conclude that STI571 is a potent inhibitor of both PDGF receptor subtypes.

In this manuscript, we demonstrate that STI571 also inhibits the c-Kit tyrosine kinase with essentially the same potency. Because c-Kit and PDGF receptors are members of the type III receptor tyrosine kinase family, other members of this family and closely related kinases were evaluated for inhibition by STI571. Interestingly, the related receptor tyrosine kinases Flt-3, Fms, Kdr, Flt-1, Tek, and c-Met were not inhibited. Because STI571 acts as an ATP-competitive inhibitor, this suggests that these tyrosine kinases, although structurally related, have subtle differences in the structure of their ATP-binding domains. These data extend our previous findings where STI571 has been shown to selectively inhibit the Abl tyrosine kinase in vitro (IC₅₀ = 0.025 µM) without affecting other tyrosine kinases such as epidermal growth factor receptor, c-Src, c-Fgr, c-Lyn, tyrosine protein kinase IIB, or serine threonine kinases such as protein kinase A, phosphorylase kinase, casein kinases 1 and 2, and Cdc-2/CycB (Druker et al., 1996). Similarly, a variety of other kinases involved in cytokine signaling (e.g., intracellular tyrosine kinases such as Src or Jak kinases) were not inhibited by STI571. The lack of inhibition of Jak-2 tyrosine kinase activity is in agreement with previously reported cellular results, demonstrating that STI571 lacks antiproliferation activity in IL-3-dependent proliferation and colony-forming assays (Druker et al., 1996). Activation of transmembrane tyrosine kinase receptors is generally associated with a variety of intracellular signaling events, including SH2- and SH3-mediated protein-protein interactions, and activation of signaling enzymes such as phospholipases C, protein kinase C, MAP kinases, and phosphatidylinositol 3-kinase. We have used the aorta vascular smooth muscle cell model to examine interference downstream of the PDGF receptor kinase. Although the exact contribution of these signaling pathways to vascular function remains to be elucidated, it has been demonstrated that activation of distinct signal transduction pathways contributes to the role of PDGF as a potent mitogen and chemotactic factor for vascular smooth muscle cells (Innui et al., 1994). Moreover, PDGF has been implicated as an important factor involved in the vascular response to injury as observed in cardiovascular diseases such as atherosclerosis (Ross, 1995), restenosis (Pauletto et al., 1994), and transplant arteriosclerosis (Gordon, 1992). STI571 was shown to potently inhibit PDGF-induced inositol release, MAP kinase activation, and proliferation of A10 rat aorta smooth muscle cells. This finding suggests that STI571 may have therapeutic use in vascular disease states associated with smooth muscle cell proliferation and migration. In vivo studies also have recently confirmed the activity of STI571 or a structurally related compound (CGP 53716) in such systems (Kallio et al., 1999; Myllarniemi et al., 1999).

Several studies have revealed that the majority of gliomas coexpress both PDGF and PDGF receptors, suggesting an autocrine mechanism of growth stimulation in this type of malignancy (Hermanson et al., 1992). High-grade primary gliomas express increased levels of PDGF receptors and their ligands compared with low-grade gliomas, indicating that in gliomas the presence of a PDGF autocrine loop correlates with tumor progression (Hermanson et al., 1992). There are several examples of malignant epithelial cells that produce PDGF but do not express PDGF receptors. Expression of PDGF in cultured malignant epithelial cell lines from human patients with breast (Perez et al., 1987) and prostate cancer (Sitaras et al., 1988) has been reported. Recent studies of plasma and tissue PDGF concentration in patients with breast cancer indicate that PDGF levels predict for shorter survival times and have an adverse effect on response to chemotherapy (Seymour et al., 1993). These findings suggest that tumor cell-derived PDGF also plays a role as a paracrine growth factor in tumorigenesis. Because PDGF is a potent mitogen and chemoattractant for both fibroblasts (Seppä et al., 1982) and endothelial cells (Smits et al., 1989), PDGF may have a function in tumor development by stimulating the growth of a supporting connective tissue stroma and contribute to endothelial cell growth. Development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB has been reported (Forsberg et al., 1993). Thus, STI571 also may have utility against such tumors that express PDGF receptors.

SCF is thought to play a central role in the proliferation and differentiation of stem cells (McNiece and Briddell, 1995). Although SCF itself does not promote colony formation in vitro, it works in synergy with other growth factors such as GM-CSF, G-CSF, IL-3, IL-6, IL-7, and Epo to stimulate formation of both differentiated progenitor cells and more primitive multilineage progenitor cells of the myeloid and erythroid lineages (McNiece et al., 1991). In addition to its normal function, deregulation of SCF receptor signaling has been implicated in a number of human cancers, including SCLC (Hibi et al., 1991; Sekido et al., 1991; Krystal et al., 1996), breast carcinomas (Hines et al., 1995), glioblastoma (Berdel et al., 1992), testicular malignancies (Strohmeyer et al., 1991), gastrointestinal stromal tumors (Hirota et al., 1998), gynecological cancers (Inoue et al., 1994) and mastocytomas (Longley et al., 1999). Evidence for the involvement of inappropriate receptor signaling by c-Kit in SCLC comes from the finding that most SCLC cell lines and primary tumors overexpress SCF and c-Kit (Hibi et al., 1991; Sekido et al., 1991; Krystal et al., 1996). Introduction of a dominant-negative kinase-defective mutant of c-Kit into the SCLC cell line NCI-H209 markedly decreased the cells' ability to grow in the absence of growth factors (Krystal et al., 1996), indicating the need for a c-Kit/SCF autocrine loop for growth. Furthermore, STI571 has been found to inhibit signal transduction and growth of SCLC cells (Krystal et al., 2000).

Our findings further suggest that STI571 may have clinical activity in tumors and nonmalignant proliferative disorders with deregulated PDGF receptor and c-Kit signaling. Additionally, the encouraging results seen in clinical trials in CML may result from a combination of the activity of STI571 on Bcr-Abl and c-Kit on leukemia cells.