[CANCER RESEARCH 60, 3127–3131, June 15, 2000]

Advances in Brief

The Pyrido[2,3-d]pyrimidine Derivative PD180970 Inhibits p210Bcr-Abl TyrosineKinase and Induces Apoptosis of K562 Leukemic Cells1

Jay F. Dorsey, Richard Jove, Alan J. Kraker, and Jie Wu2

Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute [J. F. D., R. J., J. W.] and the Departments of Medical Microbiology and Immunology[J. F. D., J. W.], Biochemistry and Molecular Biology [R. J., J. W.], University of South Florida College of Medicine, Tampa, Florida 33612; Department of Cancer Research,Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105 [A. J. K.]

Abstract

PD180970 is a novel pyrido[2,3-d]pyrimidine class of ATP-competitiveinhibitor of protein tyrosine kinases. We found that PD180970 inhibitedinvivo tyrosine phosphorylation of p210Bcr-Abl (IC50 5 170 nM) and thep210Bcr-Abl substrates Gab2 and CrkL (IC50 5 80 nM) in human K562chronic myelogenous leukemic cells.In vitro, PD180970 potently inhibitedautophosphorylation of p210Bcr-Abl (IC50 5 5 nM) and the kinase activityof purified recombinant Abl tyrosine kinase (IC50 5 2.2 nM). Incubationof K562 cells with PD180970 resulted in cell death. Results of nuclearstaining, apoptotic-specific poly(ADP-ribose) polymerase cleavage, andannexin V binding assays indicated that PD180970 induced apoptosis ofK562 cells. In contrast, PD180970 had no apparent effects on the growthand viability of p210Bcr-Abl -negative HL60 human leukemic cells. Thus,PD180970 is among the most potent inhibitors of the p210Bcr-Abl tyrosinekinase, which is present in almost all cases of human chronic myelogenousleukemia. These findings indicate that PD180970 is a promising candidateas a novel therapeutic agent for Bcr-Abl-positive leukemia.

Introduction

Ph3 is present in almost all cases of human CML and in a subset ofacute lymphoblastic leukemia (1–3). In Ph-positive cells, the c-Abl geneon chromosome 9 is fused to theBcr gene on chromosome 22 andproduces either one of two Bcr-Abl fusion proteins, p210Bcr-Abl orp185Bcr-Abl (1, 4). Fusion of the Bcr sequence to c-Abl constitutivelyactivates the Abl tyrosine kinase and may also alter the substrate speci-ficity of the Abl tyrosine kinase (4, 5). p210Bcr-Abl is found in.95% ofthe cases of human CML (3). It has been demonstrated that p210Bcr-Abl

confers antiapoptotic activity to myeloid cells (6) and induces CML inmice (2). These findings indicate that p210Bcr-Abl is a key protein respon-sible for the pathogenesis of CML. Importantly, the tyrosine kinaseactivity is essential for the transforming activity of p210Bcr-Abl, andinhibition of Abl tyrosine kinase activity blocks cell growth and inducesapoptosis of p210Bcr-Abl-positive human leukemic cells (3, 7). Therefore,p210Bcr-Abl tyrosine kinase is an attractive therapeutic target for thetreatment of human CML. To date, few Abl tyrosine kinase inhibitorshave been identified and characterized (3, 7–9).

Gab2 is a multisite docking protein recently cloned fromp210Bcr-Abl-transformed hematopoietic cells (10). Gab2 is constitutivelytyrosine phosphorylated and associated with the protein tyrosine phos-phatase SHP2 in p210Bcr-Abl-transformed hematopoietic cells such ashuman CML K562 cells. We found that a novel pyrido[2,3-d]pyrimidine

derivative (11), PD180970 (see structure in Fig. 2A), potently inhibitsGab2 tyrosine phosphorylation in K562 cells. Further studies showed thatPD180970 is an inhibitor of the p210Bcr-Abl tyrosine kinase and inducesapoptosis of K562 cells, whereas it has no detectable effect on Ph-negative HL60 human leukemic cells. These findings suggest thatPD180970 is a novel Abl tyrosine kinase inhibitor that can selectivelyaffect Bcr-Abl-positive leukemic cells.

Materials and Methods

Reagents and Cells.PD180970 {6-(2,6-dichloro-phenyl)-2-(4-fluoro-3-methyl-phenylamino)-8-methyl-8H-pyrido [2,3-d]pyrimidin-7-one} was synthe-sized by Parke-Davis Pharmaceuticals (11). The Src family protein tyrosine kinaseselective inhibitors PP1 and PP2 (12) and the recombinant Abl tyrosine kinasewere from Calbiochem. Recombinant c-Src protein and Src substrate peptide werefrom Upstate Biotechnology. Anti-Abl and anti-CrkL antibodies were from SantaCruz Biotech, anti-PARP and antiphosphotyrosine (RC20H) antibodies were fromPharMingen. The sources of other reagents were as given (13) or as indicatedbelow. The K562 human CML cells (1, 3, 6) and the Ph-negative HL60 humanpromyelocytic leukemic cells (1) were maintained in RPMI 1640 containing 10%fetal bovine serum and 100 units/ml penicillin-streptomycin.

Preparation of Anti-Gab2 Antibody. A peptide (CEWTDVRQSSEPSKGAKL)containing the COOH-terminal region of Gab2 (10) was synthesized, conjugated withkeyhole limpet hemocyanin, and used to generate antisera against Gab2 in rabbits. Theantibody was affinity-purified using the antigen peptide. Horseradish peroxidase-conjugated anti-Gab2 antibody was prepared from the affinity-purified antibody usingthe EZ-Link Plus Activated Peroxidase kit (Pierce).

Immunoprecipitation and Immunoblotting. Cells (23 106 cells/sample)were collected by centrifugation (10003 g for 3 min at 4°C), and lysed in bufferA [25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 25 mM NaF, 1 mM benzamidine, 1%Triton X-100, 1 mM Na3VO4, 20 mM p-nitrophenyl phosphate, 2mg/ml leupeptin,2 mg/ml aprotinin, 100mg/ml phenylmethylsulfonyl fluoride]. Immunoprecipita-tions and the subsequent immunoblotting were performed essentially as described(13) using the antibodies indicated in the figure legends.

In Vitro Immune-Complex Kinase Assay.p210Bcr-Abl (2 3 106 cells/sam-ple) was immunoprecipitated from K562 cells using a monoclonal anti-Abl antibody.The immunoprecipitates were washed three times with buffer A and once with bufferB [20 mM Tris-HCl (pH 7.2), 20 mM NaCl]. The autophosphorylation reaction wascarried out by incubating the p210Bcr-Abl immune complex in 30ml of reaction buffer{10 mM Tris-HCl (pH 7.2), 2 mM p-nitrophenyl phosphate, 4 mM MgCl2, 2 mM

MnCl2, 10 mM ATP containing 10mCi of [g-32P]ATP} containing the indicatedconcentrations of PD180970 for 15 min at 30°C. The reaction was stopped by additionof SDS-gel loading buffer and heated at 95°C for 10 min. Proteins were resolved on8% SDS-polyacrylamide gels. Autophosphorylation of p210Bcr-Abl was analyzed witha PhosphorImager and also by autoradiography.

In Vitro Abl and Src Kinase Assays.The activity of a purified bacteri-ally expressed Abl protein tyrosine kinase (Calbiochem) was assayed usinga synthetic peptide (EAIYAAPFAKKK) as substrate. The reaction wascarried out at 30°C for 10 min in 40ml of reaction mixture {50 mM

Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.015% Brij35, 0.1 mg/ml BSA, 10mM ATP, 5 mCi of [g-32P]ATP (3000 Ci/mmol),100 mM peptide substrate, and 2 ng of Abl kinase} containing 1–100 nM

PD180970 or DMSO (vehicle). The reaction was stopped by spotting 35mlof the reaction mixture onto p81 phosphocellulose filter (2.5 cm2 andimmediately immersed in 2% phosphoric acid. The filters were washed five

Received 2/15/00; accepted 5/1/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants CA77467 (to J. W.) and CA55652 (to R. J.).2 To whom requests for reprints should be addressed, at Molecular Oncology Program,

MRC3-East, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive,Tampa, FL 33612. Phone: (813) 979-6713; Fax: (813) 903-6817; E-mail: wu@moffitt.usf.edu.

3 The abbreviations used are: Ph, Philadelphia chromosome; CML, chronic myeloge-nous leukemia; Gab2, Grb2-associated binder-2; PARP, poly(ADP-ribose) polymerase;PI, propidium iodide; IL, interleukin.

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times with 2% phosphoric acid, and radioactivity retained on the filters wasmeasured by liquid scintillation counting.

Src kinase activity was determined using a recombinant c-Src produced in Sf9insect cells and a Src substrate peptide (KVEKIGEGTYGVVYK). The reaction wasperformed as above except that 6 units of c-Src and 150mM Src substrate peptide wereused in each reaction and the reaction was carried out at 30°C for 20 min.

Cell Viability, Nuclear Staining, PARP Cleavage, and Annexin V-PIBinding Assays. For determination of cell growth and viability, cells (1.53 105) intriplicate were incubated in 2 ml of RPMI 1640–10% FCS containing tyrosine kinaseinhibitors or DMSO (solvent for the inhibitors). The volume of the DMSO was keptat 0.1% of the medium volume. At the indicated times, cells were collected bycentrifugation (10003 g for 3 min). Cell viability was determined by the trypan blueexclusion assay (14). At least 200 cells were examined in each sample.

For nuclear staining, samples (23 104 cells/each) were collected onto microscopeglass slides by cytospin. The cells were mounted with mounting medium containingDAPI (Vector Laboratories) and examined with a fluorescence microscope.

To analyze PARP cleavage (6), cells (1.53 106) were treated with PD180970(0.5mM) or DMSO for the indicated time. Cells were collected, washed once withcold PBS, and lysed in buffer C [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM

EDTA, 0.5% NP40, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride] for 30min at 4°C. Cell lysate supernatants (40mg protein/each) were resolved on 8%SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and analyzedby immunoblotting with an anti-PARP antibody.

Evaluation of apoptosis by the annexin V-PI binding assay (7) was performedusing the Annexin V-FITC Apoptosis Detection Kit (PharMingen) according to

Fig. 1. Inhibition of tyrosine phosphorylation of p210Bcr-Abl, Gab2, and CrkL in K562cells by PD180970. K562 cells were treated with DMSO (0) or the indicated concentra-tions of PD180970. p210Bcr-Abl, Gab2, and CrkL were immunoprecipitated. Equal por-tions of each immunoprecipitate (IP) were analyzed by immunoblotting (IB) with anantibody against (a) phosphotyrosine (PTyr;B–D, top panels) or with antibodies againstAbl, Gab2, or CrkL (B–D,bottom panels) to confirm equal amounts of protein in eachimmunoprecipitate.Arrowsindicate location of the indicated proteins.A, average levels oftyrosine phosphorylation of each protein from two independent experiments;bars, SD.

Fig. 2. Inhibition of p210Bcr-Abl autophosphorylation and the Abl tyrosine kinaseactivity in vitro by PD180970.A, structure of PD180970.B, concentration-dependenteffect of PD180970 on p210Bcr-Abl autophosphorylationin vitro. The average tyrosinephosphorylation from three independent experiments is shown;bars, SD.Inset, repre-sentative autoradiograph. Thearrowhead indicates the p210Bcr-Abl band. C, tyrosinekinase activities of recombinant v-Abl (E) and c-Src (Œ) proteins were assayed byphosphorylation of peptide substrates in the presence of various concentrations ofPD180970. The results represent the average of two experiments performed in duplicate;bars, SD. The radioactivity in the control (100%) samples was 1,231,5756 346,023 cpmfor the v-Abl kinase assay and 322,2326 92,185 cpm for the c-Src kinase assay.

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the supplier’s instructions. At least 13 104 cells in each sample were analyzed.Control cells stained with annexin V-FITC or PI alone were used to compensatefor the flow cytometric analysis. Annexin V and PI double-negative cells aredefined as live cells; annexin V-positive, PI-negative cells are defined as earlyapoptotic cells; and annexin V and PI double-positive cells are defined as lateapoptotic and necrotic cells.

Results

PD180970 Inhibits Tyrosine Phosphorylation of p210Bcr-Abl ,Gab2, and CrkL in K562 cells. We prepared a polyclonal antibodyagainst the newly identified multisite docking protein, Gab2, andbegan to investigate the signaling roles of Gab2. Gab2 is consti-tutively phosphorylated on tyrosine residues in K562 cells (Fig.1C) and in other p210Bcr-Abl-transformed hematopoietic cells (10),suggesting that Gab2 is involved in p210Bcr-Abl-initiated cell sig-naling. Incubation of K562 cells with PD180970 inhibited Gab2tyrosine phosphorylation with an IC50 of 80 nM (Fig. 1, A andC).PD180970 treatment also resulted in dissociation of the Gab2-SHP2 complex in K562 cells (data not shown). PD180970 wasoriginally identified as a Src tyrosine kinase inhibitor (11). Toassess the contribution of the Src tyrosine kinase inhibitor activityof PD180970 on inhibition of Gab2 tyrosine phosphorylation, wetested the effects of two Src family tyrosine kinase-specific inhib-itors, PP1 and PP2 (12), on Gab2 tyrosine phosphorylation in K562cells. In contrast to PD180970, PP1 (10mM) and PP2 (10mM) hadno apparent effects on Gab2 tyrosine phosphorylation and Gab2-SHP2 association in K562 cells (not shown). These observationssuggest that the inhibition of Gab2 tyrosine phosphorylation byPD180970 in K562 cells is unlikely to be due to inhibition of Srcfamily tyrosine kinases.

To determine whether PD180970 can inhibit p210Bcr-Abl tyrosinekinase activity, K562 cells were treated with various concentrations ofPD180970 for 12 h, and p210Bcr-Abl tyrosine phosphorylation was ana-lyzed by immunoblotting with an antiphosphotyrosine antibody (RC20H)after immunoprecipitation with an anti-Abl antibody. Fig. 1,A and B,shows that PD180970 inhibited tyrosine phosphorylation of p210Bcr-Abl

in a concentration-dependent manner with an IC50of 170 nM. Thebottom

panelof Fig. 1Bshows that equal amounts of p210Bcr-Abl protein werepresent in each immunoprecipitate.

CrkL is a well-characterized p210Bcr-Abl substrate (15, 16) and isconstitutively tyrosine-phosphorylated in K562 cells (Fig. 1D). Toconfirm the inhibitory effect of PD180970 on p210Bcr-Abl tyrosinekinase activity in K562 cells, CrkL was immunoprecipitated fromK562 cells that were treated for 12 h with various concentrationsof PD180970 and analyzed by immunoblotting with RC20H. Asshown in Fig. 1,A and D, PD180970 potently inhibited CrkLtyrosine phosphorylation in K562 cells (IC50 5 80 nM). On theother hand, incubation of K562 cells with PP1 and PP2 (10mM for12 h) did not result in any change in CrkL tyrosine phosphorylation(data not shown). Together, these data indicate that PD180970 caninhibit p210Bcr-Abl tyrosine kinase activity in K562 cells.

PD180970 Inhibits p210Bcr-Abl Autophosphorylation and Re-combinant Abl Tyrosine Kinase Activity in Vitro. To assesswhether PD180970 can directly affect p210Bcr-Abl tyrosine kinaseactivity, p210Bcr-Abl was immunoprecipitated from K562 cells, andin vitro autophosphorylation of p210Bcr-Abl was performed in thepresence or absence of PD180970. As shown in Fig. 2B, PD180970potently inhibited autophosphorylation of p210Bcr-Abl in vitro. TheIC50 obtained from three independent experiments for inhibition ofp210Bcr-Abl autophosphorylation by PD180970 was 5 nM.

To further confirm that PD180970 can directly inhibit Abl tyrosinekinase activity, we determined the effect of PD180970 on the kinaseactivity of purified recombinant Abl tyrosine kinase (17). ThisEsche-richia coli-expressed recombinant protein is a truncated form of v-Abl(18) that contains an Abl tyrosine kinase domain identical to that ofc-Abl. As illustrated in Fig. 2C, PD180970 inhibited the tyrosine kinaseactivity of the recombinant Abl protein, with an IC50 of 2.2 nM. A similarIC50 value was obtained when the kinase assay was performed with a10-fold lower concentration of Abl protein (not shown). These resultsdemonstrate that PD180970 can directly and potently inhibit Abl tyrosinekinase activity. Fig. 2Calso shows that PD180970 inhibited c-Src tyro-sine kinase activity with an IC50 of 0.8 nM, confirming that PD180970 isa potent inhibitor of c-Src tyrosine kinase (11).

Fig. 3. Effects of PD180970, PP1, and PP2 on cellgrowth and viability of K562 and HL60 cells. K562 or HL60cells (1.53 105/ml) in triplicate were mock-treated withDMSO (0.1%), PD180970 (0.5mM), PP1 (10mM), or PP2(10 mM). Viable and total cell numbers were determinedevery 24 h after staining with trypan blue.A andB, effectsof PD180970 on K562 and HL60 cells.C andD, effects ofPP1 and PP2 on K562 and HL60 cells.DM, DMSO; PD,PD180970. The data represent the averages of two triplicateexperiments;bars, SD.

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PD180970 Causes Apoptotic Cell Death of K562 Cells.We nextdetermined the effects of PD180970 on cell growth and viability of K562cells. The Bcr-Abl-negative human HL60 leukemic cell line was used asa control. K562 cells and HL60 cells were incubated with PD180970 (0.5mM) or DMSO (solvent) for 1–4 days, and cell numbers were countedeach day after trypan blue staining. Fig. 3Ashows that the viable cellnumber of PD180970-treated K562 cells was below the initial plating cellnumber after day 1. Fig. 3B shows that this was due to cell death. Instriking contrast to the effects on K562 cells, PD180970 had no apparenteffects on cell proliferation and viability of HL60 cells (Fig. 3).

To assess whether the K562 cell killing effect of PD180970 wasattributed to inhibition of Src-like tyrosine kinases, we examinedthe effects of PP1 and PP2 on K562 cells. As shown in Fig. 3D,PP1 and PP2 (10mM) had moderate effects on K562 cell viability:79 – 80%, 65–72%, and 38 –55% of K562 cells treated with PP1and PP2 were still viable on days 2, 3, and 4, respectively. Incomparison, only 346 2%, 156 2%, and 66 3% of cells treated

with PD180970 (0.5mM) remained viable on days 2, 3, and 4,respectively (Fig. 3B). Thus, there were considerable differences inthe viability of K562 cells treated with PD180970 and with the Srckinases inhibitor PP1 or PP2.

To determine whether PD180970 causes apoptosis of K562 cells, wefirst examined the nuclei of K562 cells by fluorescence microscopy afterDAPI staining. As illustrated in Fig. 4A, many PD180970-treated K562cells had condensed and fragmented nuclei, which is characteristic ofapoptotic cells. We next analyzed the PARP protein in K562 cells. It hasbeen demonstrated previously that the 116-kDa PARP is specificallycleaved into 85- and 25-kDa fragments by caspase-3 in cells, includingK562 cells, that are undergoing apoptosis (6). Fig. 4B shows that little85-kDa PARP was present in mock (DMSO)-treated K562 cells, whereasPD180970 treatment resulted in the apoptotic-specific cleavage of PARP.

To further assess whether PD180970 induces apoptosis of K562cells, we performed an annexin V-PI binding assay (7). Redistri-bution of the plasma membrane annexin V-binding phosphatidyl-

Fig. 4. Apoptosis of K562 cells induced by PD180970.K562 cells were treated with or without PD180970 (0.5mM)for the indicated times.A, cytospins of K562 cells wereexamined by nuclear staining with DAPI.B, PARP proteincleavage was examined by immunoblotting with an anti-PARP antibody.C, cells were analyzed by the annexin V-PIbinding assay. The results are representative of triplicate andduplicate experiments.

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serine to the outer leaflet of plasma membrane is a hallmark ofearly apoptotic cells, whereas permeability to PI occurs in the laterstage of apoptosis or in necrotic cells. In untreated K562 cells,4.9 6 0.6% of cells were annexin V-positive/PI-negative (lowerright quadrant), whereas 2.66 0.9% of cells were annexin V-PIdouble-positive (upper right quadrant). These values were essen-tially unchanged in mock-treated cells (Fig. 4C, top panels). In-cubation of K562 cells with PD180970 (0.5mM) for 24 and 48 hincreased annexin V-positive/PI-negative cells to 15.16 3.1% and34.5 6 7.4%, respectively (Fig. 4C, bottom panels). A consider-able increase in annexin V-PI double-positive cells (22.56 2.9%)was also detected in K562 cells treated with PD180970 for 48 h.Parallel experiments showed no change in annexin V-PI binding ofHL60 cells treated with PD180970 (data not shown). These resultsdemonstrate that PD180970 induced apoptosis of K562 cells.

Discussion

Because nearly all cases of CML are related to p210Bcr-Abl and itstyrosine kinase activity is essential for neoplastic transformation,inhibiting the p210Bcr-Abl tyrosine kinase activity represents an im-portant therapeutic approach for p210Bcr-Abl-positive CML. The keyto this approach is the availability of protein tyrosine kinase inhibitorsthat can specifically kill p210Bcr-Abl-positive CML cells at submicro-molar concentrations. Only one such compound, the 2-phenylamin-opyrimidine derivative CGP57148, has been characterized thus far (3,7). We demonstrate here that the novel pyrido[2,3-d]pyrimidine de-rivative PD180970 inhibits p210Bcr-Abl tyrosine kinase activity andselectively induces apoptosis of p210Bcr-Abl-positive K562 cells atnanomolar concentrations.

PD180970 inhibits p210Bcr-Abl autophosphorylationin vitro with an IC50

of 5 nM. In cellular tyrosine phosphorylation assays, PD180970 inhibitstyrosine phosphorylation of p210Bcr-Abl, with an IC50 of 170 nM, whereas itinhibits tyrosine phosphorylation of p210Bcr-Abl substrates Gab2 and CrkL,with an IC50 of 80 nM. The higher concentration required for inhibition oftyrosine kinase autophosphorylation than for inhibition of phosphorylation ofexogenous substrates is a general feature of competitive inhibitors of proteintyrosine kinases (19). In particular, this has been observed previously with thep210Bcr-Abl tyrosine kinase inhibitor CGP57148(7).

PD180970 was originally identified as a Src tyrosine kinase inhibitor (11).Although the Src kinase inhibitory activity of PD180970 may contribute tothe effects of PD180970 on K562 cells to some degree, several lines ofevidence suggest that the effects of PD180970 on K562 cells is largely dueto inhibition of the p210Bcr-Abl tyrosine kinase. (a) PD180970 inhibited(IC50 5 2.2 nM) recombinant Abl tyrosine kinase purified fromE. coli (Fig.2C). This result unequivocally demonstrates that PD180970 is a potentinhibitor of the Abl tyrosine kinase. (b) The Src tyrosine kinase inhibitors PP1and PP2 (12) had no apparent effect on tyrosine phosphorylation of Gab2 andCrkL in K562 cells. (c) The effects of PP1 and PP2 on K562 cell viabilitywere relatively small compared with that of PD180970 (Fig. 3B). Thedifference between the extent of K562 cell death caused by P180970 and byPP1/PP2 is likely due to inhibition of p210Bcr-Abl by PD180970 (Fig. 3,BandD). This view is further supported by our observations in 32Dcl3 murinemyeloid cells. Src tyrosine kinase is known to be involved in IL-3-stimulated32Dcl3 proliferation (20). We found that PD180970 (0.5mM), PP1 (10mM),and PP2 (10mM) cause a similar 50% inhibition of 32Dcl3 cell proliferation,whereas all three compounds have little effect on the IL-3 dependent cellviability (data not shown). These results suggest that PD180970, PP1, andPP2 have similar effects on Src-like kinases at the concentrations used in ourstudies.

It has been suggested that p210Bcr-Abl renders myeloid cells resistant toapoptosis (6). Our finding that PD180970 causes apoptosis of K562 cells

is consistent with this notion. Apoptosis of K562 cells and other Bcr-Ablpositive cells induced by the Abl kinase inhibitor CGP57148 has alsobeen reported (7). PD180970 had no apparent effect on cell proliferationand viability of Bcr-Abl-negative HL60 leukemic cells. In the normal32Dcl3 myeloid cells, PD180970 partially inhibited IL-3-dependent cellproliferation but had little effect on cell viability (data not shown). Thesefindings are very important in terms of the development of novel CMLtherapeutic agents. Studies of the Abl tyrosine kinase inhibitorCGP57148 also demonstrate that inhibition of Abl tyrosine kinase activ-ity causes cell death only in Bcr-Abl-positive leukemic cells (3, 7). Takentogether, our results demonstrate that PD180970 is among the mostpotent p210Bcr-Abl tyrosine kinase inhibitors identified and representsanother potential therapeutic agent for CML.

Acknowledgments

We thank Jodi Kroeger of the Moffitt Cancer Center Flow Cytometry Corefor flow cytometric analysis.

References

1. Clark, S. S., McLaughlin, J., Crist, W. M., Champlin, R., and Witte, O. N. Uniqueforms of the abl tyrosine kinase distinguish Ph1-positive CML from Ph1-positiveALL. Science (Washington DC),235: 85–88, 1987.

2. Daley, G., ven Etten, R., and Baltimore, D. Induction of chronic myelogenous leukemiain mice by the p210bcr/abl gene of the Philadelphia chromosome. Science (WashingtonDC), 247: 824–830, 1990.

3. Druker, B. J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G. M., Fanning, S.,Zimmermann, J., and Lydon, N. B. Effects of a selective inhibitor of the Abltyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med.,2: 561–566,1996.

4. McWhirter, J. R., and Wang, J. Y. J. Effect of Bcr sequences on the cellular function ofthe Bcr-Abl oncoprotein. Oncogene,15: 1625–1634, 1997.

5. McWhirter, J. R., and Wang, J. Y. J. Activation of tyrosine kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins. Mol. Cell. Biol.,11:1553–1565, 1991.

6. Dou, Q. P., McGuire, T. F., Peng, Y., and An, B. Proteasome inhibition leads to significantreduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 humanchronic myelogenous leukemia cells. J. Pharm. Exp. Ther.,289: 781–790, 1999.

7. Gambacorti-Passerini, C., Courtre, P. I., Mologni, L., Fanelli, M., Bertazzoli, C.,Marchesi, E., Nicola, M. D., Biondi, A., Corneo, G. M., Belotti, D., Pogliani, E., andLydon, N. B. Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL1 leukemic cells and induces apoptosis. Blood Cells Mol. Dis.,23: 380–394, 1997.

8. Anafi, M., Gazit, A., Zehavi, A., Ben-Neriah, Y., and Levitzki, A. Tyrphostin-inducedinhibition of p210bcr-abl tyrosine kinase activity induces K562 to differentiate. Blood,12:3524–2529, 1993.

9. Kaur, G., Gazit, A., Levitzki, A., Stowe, E., Cooney, D. A., and Sausville, E. A.Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinaseactivity in K562 chronic myelogenous leukemia. Anticancer Drugs,5: 213–222, 1994.

10. Gu, H., Pratt, J. C., Burakoff, S. J., and Neel, B. J. Cloning of p97/Gab2, the majorSHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Mol. Cell2: 729–740, 1999.

11. Kraker, A. J., Hartl, B. G., Amar, A., M., Barvian, M. R., Showalter, H. D. H., and Moore,C. W. Biochemical and cellular effects of c-src selective pyrido[2, 3-d]pyrimidine tyrosinekinase inhibitors. Biochem. Pharmacol., in press, 2000.

12. Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H.,Weringer, E. J., Pollok, B. A., and Connelly, P. A. Discovery of a novel, potent,and Src family-selective tyrosine kinase inhibitor. J. Biol. Chem.,271: 695–701,1996.

13. Cunnick, J. M., Dorsey, J. F., Standley, T., Turkson, J., Kraker, A. J., Fry, D. W., Jove,R., and Wu, J. Role of tyrosine kinase activity of epidermal growth factor receptor in thelysophosphatidic acid-stimulated MAP kinase pathway. J. Biol. Chem.,273: 14468–14475, 1998.

14. Freshney, R. I. Culture of Animal Cells, a Manual of Basic Technique, Ed. 2, pp.245–256. New York: Wiley-Liss, 1987.

15. Ten Hoeve, J., Arlinghaus, R. A., Guo, J. Q., Heisterkamp, N., and Groffen, J. Tyrosinephosphorylation of CrkL in Philadelphia1 leukemia. Blood,84: 1731–1736, 1994.

16. Uemura, N., Salgia, R., Li, J-L., Sattler, M., and Griffin, J. D. TheBCR/ABLoncogenealters interaction of the adapter proteins CRKL and CRK with cellular proteins. Leukemia,11: 376–385, 1997.

17. Foulkes, J. G., Chow, M., Gorka, C., Frackelton, A. R., Jr., and Baltimore, D. Purificationand characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemiavirus. J. Biol. Chem.,260: 8070–8077, 1985.

18. Wang, J. Y. J., and Baltimore, D. Localization of tyrosine kinase-coding region in v-abloncogene by the expression of v-abl-encoded proteins in bacteria. J. Biol. Chem.,260:64–71, 1985.

19. Levitzki, A., and Gazit, A. Tyrosine kinase inhibition: an approach to drug development.Science (Washington DC),267: 1782–1788, 1995.

20. Chaturvedi, P., Reddy, M. V. R., and Reddy, E. P. Src kinases and not JAKs activateSTATs during IL-3 induced myeloid cell proliferation. Oncogene,16: 1749–1758, 1998.

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2000;60:3127-3131. Cancer Res   Jay F. Dorsey, Richard Jove, Alan J. Kraker, et al.   Leukemic Cells

Tyrosine Kinase and Induces Apoptosis of K562Bcr-Abl]pyrimidine Derivative PD180970 Inhibits p210dThe Pyrido[2,3-

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