ICANCER RESEARCH56. IOO-104.January 1. 19961

ABSTRACT

Oncogenic activation of AbI proteins due to structural modificationscan occur as a result of viral transduction or chromosomal translocation.The tyrosine protein kinase activity ofoncogenic AbI proteins Is known tobe essential for their transforming activity. Therefore, we have attemptedto identify selective inhibitors of the AbI tyrosine protein kinase. Hereinwe describean inhibiter(CGP 57148)of the Abl and platelet-derivedgrowth factor (PDGF) receptor protein-tyrosine kinases from the 2-phenylaminopyrimidine class, which is highly active in vitro and in vivo.Submicromolar concentrations of the compound inhibited both v-Abl andPDGF receptor autophosphorylation and PDGF-induced c-fos mRNAexpression selectively in Intact cells. In contrast, ligand-Induced growthfactor receptor autophosphorylation in response to epidermat growth

factor (EGF), Insulin-like growth factor-I, and insulin showed no or weak

inhibition by high concentrations of CGP 57148. c-fos mRNA expressioninduced by EGF, fibroblastgrowth factor, or phorbolester was alsoinsensitive to inhibition by CGP 57148. In antiproliferative assays, thecompound was more than 30-i®-fold more potent in inhibiting growth ofv-aM-transformed PB-3c cells and v-sis-transformed BALBIC 3T3 cellsrelative to inhibition of EGF-dependent BALB/MK cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. Furthermore, anchorage-independent growth of v-abE- and v-sis-transformed

BALB/c 3T3 cells was inhibited potently by CGP 57148. When tested invivo, CGP 57148 showed antitumor activity at tolerated doses againsttumorigenic v-abl- and v-sis-transformed BALB/c 3T3 cells. In contrast,CGP 57148 had no antitumor activity when tested using src-transformedBALB/c 3T3 cells. These findings suggest that CGP 57148 may havetherapeutic potential for the treatment of diseases that involve abnormalcellular proliferation induced by AbI protein-tyrosine kinase deregulationor PDGFreceptoractivation.

INTRODUCTION

The abl oncogene was isolated originally from the genome of theA-MuLV2 (1) This acutely transforming, replication-defective virusencodes a transforming protein (@1f,()vabI)with tyrosine-specific protein kinase activity (2, 3). A-MuLV, which is able to transformfibroblasts in vitro and lymphoid cells in vitro and in vivo (4, 5),seems to have been formed by recombination between the Moloneymurine leukemia virus and the murine cellular c-abl gene (6). TheN-terminal modification found in v-abl, acquired during transductionby A-MuLV, results in deregulation of its protein-tyrosine kinaseactivity. There is considerable evidence that activated abl genes playan important role in the pathogenesis of specific human leukemias.CML is a hematological stem cell disorder characterized by excessiveproliferation of the myeloid lineage (7). The hallmark of CML is the

Received 7/31195; accepted 10/31/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed, at Ciba Pharmaceuticals Divi

sion, Research Department, Ciba-Geigy Limited, CH-4002 Basel, Switzerland. Phone:061/6962853 or 061/6964498; Fax: 061/6963835.

2 The abbreviations used are: A-MuLV, Abelson murine leukemia virus; CML, chronicmyelogenous leukemia; PDGF, platelet-derived growth factor; IL, interleukin; EGF,epidermal growth factor; IGF, insulin-like growth factor; bFGF, basic fibroblast growthfactor; TIC, treatment versus control; FCS, fetal calf serum.

Philadelphia chromosome (8), which is detected in virtually all casesof CML and 20% of adult acute lymphoblastic leukemia. It is formedby a reciprocal translocation between chromosomes 9 and 22 [t(9;22)and (q34;])J (9—14).The molecular consequence of this translocationis the replacement of the first exon of c-abl with sequences from theBcr gene, resulting in a Bcr-Abl fusion protein with enhanced tyrosinekinase activity (15—17).Recent results have demonstrated that BcrAbl expression also can induce a disease resembling CML in mice(18—21)and providesstrongevidencethat the Bcr-Abl protein is amajor factor in the pathophysiology of CML.

Due to its relatively clear etiology, CML represents a disease inwhich therapy using a selective inhibitor of the Abl protein-tyrosinekinase would seem attractive. Inhibition of the AbI tyrosine kinaseactivity has been reported for the benzopyranones and benzothiopyranones (22) and the tyrphostin classes of compounds (23). However,the compounds showed either limited selectivity or potency at thecellular level. Recently, a series of protein-tyrosine kinase inhibitorsof the 2-phenylaminopyrimidine class have been synthesized. CGP53716 has been characterized as a potent inhibitor of the PDGFreceptor tyrosine kinase (24). Although this compound inhibited thev-Abl protein-tyrosine kinase in vitro, it showed preferential inhibition of the PDGF signal transduction pathway at the cellular level(24). Optimization of this compound class for inhibition of the v-Ablkinase has led to the identification of CGP 57148. This derivativeshows potent inhibition of the v-Abl kinase in vitro and in cells andinhibits tumor growth of v-abl-transformed BALB/c 3T3 cells. Inaddition, its activity against the PDGF receptor tyrosine kinase mdicates that the compound could have therapeutic potential in thetreatment of cancers involving deregulated AbI or PDGF receptortyrosine kinase activity.

MATERIALS AND METhODS

Materials. COP 57148 and its methane sulfonate salt (COP 57148B) weresynthesized by CIBA Pharmaceuticals Division, as will be described elsewhere.3 For in vitro and cellular assays, a stock concentration of 10 mMCGP57148 was prepared in Me2SOand stored at —20°C.No significant differencein results could be seen between the two forms of COP 57148. The form usedin in vitro experiments is indicated in the text and legends. All in vivo

experiments were performed using COP 57148B. Monoclonal antiphosphoty

rosine antibody PY2Ocoupled to horseradish peroxidase was from ICN Radiochemicals (Irvine, CA). Monoclonal antibody FRP 5, which acts as a ligandagonist for pl85c.crbB2(25), was kindly provided by N. Hynes (FriedrichMiescher Institute, Basel, Switzerland). Mouse monoclonal antibodies directed

against the c-nb! gene product were from Oncogene Science (Manhasset, NY).PB-3c cl.l5, a IL-3-dependent mouse mast cell line, and the AMuLV.transformed line PB-3c cl.15 v-abl (26) were obtained from A. Nair (Institute forMedical Microbiology, Basel, Switzerland). The cells were cultured in

Iscove's modified Dulbecco's medium supplemented with 10% (v/v) FCS.PB-3c cl.15 was grown in the presence of optimal concentrations of WEHI-3Bcell-conditioned medium as a source of IL-3 (27). The AMuLV-transformedBALB/c AMuLV A.6R.1 cells and MDA-MB 453 human breast carcinomaline (American Type Culture Collection, Rockville, MD) were cultured in

3 J. Zimmermann, manuscript in preparation.

100

Inhibition of the Abi Protein-Tyrosine Kinase in Vitro and in Vivo by a2-Phenylaminopyrimidine Derivative

Elisabeth Buchdunger,' JürgZimmermann, Helmut Mett, Thomas Meyer, Marcel Muller, Brian J. Druker, andNicholas B. Lydon

Ciba Pharmaceuticals Division, Oncology Research Department, Ciba-Geigy Limited@CH-4002 Basel, Switzerland FE. B., J. 1, H. M., T. M., M. M., N. B. Li, and Division ofHematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201 [B. J. D.J

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C0Oil

OOOOCV)r@OOcv)'

12345678

I IIOOOCv)@—OO1@

INHIBITION OF ThE ABL PROTEIN-TYROSINE KINASE

—@ inhibitproteinkinaseC isotypes,or otherserine/threoninekinasessuch as cAMP-dependent protein kinase, phosphorylase kinase, cdc2/

N cyclinBproteinkinase,andproteinkinasesCK1and2(IC50,>100@•44 @.LM). When tested against different protein-tyrosine kinases, COP

57148 did not inhibit the EGF receptor intracellular domain (IC50,>100 jAM) and showed no or weak inhibition of protein kinases of the

src family (c-Src, c-Lyn, c-Fgr, and c-Lck).Selectivitiy of CGP 57148 in Intact Cells. The effect of COP

57148 on v-Abl kinase in intact cells was studied using AMuLVtransformed PB-3c mast cells. COP 57148 inhibited v-Abl tyrosineautophosphorylation with an IC50 value between 0. 1 and 0.3 @M

DMEM supplemented with 10% (v/v) FCS and RPMI 1640 medium containing 20% (v/v) FCS, respectively. Transformed NIH-527-src cells, whichcontain an activated c-src gene carrying a tyrosine-to-phenylalanine mutationat amino acid 527 of the coding sequence,4 were grown in DMEM supple

mented with 10% (v/v) bovine calf serum. Other cell lines and reagents are asdescribed (24).

Kinase Assays. Purification of protein kinases and in vitro enzyme assayswere performed as described (24).

Western Blotting. PB-3c cl.15 and PB-3c cl.15 v-ablcells were incubated for90 mm with the indicated concentrations of drugs. Serum-starved MDA-MB 453cells [incubationin culture medium containing0.1% (v/v) FCS for 48 h] wereincubated for 2 h with the compound prior to stimulation with monoclonalantibody FRP 5 (5 @g/ml)for 10 mm. Other cell lines were treated as described(24). Equal amounts of protein of cell lysates were assayed by Western blotting

with 4010 antiphosphotyrosine antibodies (28). MDA-MB 53 cell lysates wereanalyzed by using the PY2O horseradish peroxidase conjugate. Bound antibodies

were detected using EnhancedChemiluminescence(Amersham).Northern Blot Analysis. Isolation of total RNA and analysis by Northern

blotting using afos-specific probe were carried out as described (29). Loadingand integrity of RNA were verified by ethidium bromide staining of the gel

(data not shown).

Antiproliferative Assays. Cell growth inhibition was assayed using methylene blue staining or the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described (30).

Soft Agar Growth. Cells were platedin duplicatein 6-cm dishes in 6 mlculture medium supplemented with 0.3% noble agar and the indicated con

centrations of COP 57 148, overlaying a 0.8% agar layer. Plates were incubated

for 8—14days at 37°C, after which colonies were stained by adding 2 ml PBS

containing 0.5 mg/ml nitro blue tetrazolium for 24 h. Colonies were counted

using an Artek 880 colony counter (Dynatech Laboratories, Inc.).

In Vivo Experiments. in vivo antitumoractivity was tested using AMuLVtransformedBALB/c313 cells, v-sis-transfectedBALB/c3T3 cells,or NIH-527-src cells. One X 106 cells were injected into the left flanks of six female BALB/c

nude mice (Bolmholdtgard, Copenhagen, Denmark) per experimental seL Treat

ment was started 24 h after cell injection, and the compound was administered __________________________________

once daily for up to 30 consecutivedays. SolutionsofCOP 57l48B werepreparedin PBS daily prior to i.p. administration. In all experiments, tumor growth was

followed by measuring perpendicular tumor diameters. Tumor volumes were

calculatedas describedpreviously(31) using the formulap X L X D@/6,whereLis the longestdiameter,and D is the diameter at right angles to iL

RESULTS

In Vitro Selectivity of CGP 57148 for Inhibition of ProteinKinases: Identification of CGP 57148 as an Inhibitor of the v-AblKinase. COP 57148, the structure of which is shown in Fig. 1, wasassayed in vitro for inhibition of a panel of tyrosine and serine/threonine protein kinases and was found to inhibit the v-Abl tyrosinekinase potently, with an IC50 value of 0.038 j.LM.5COP 57148 did not

4 B. J. Druker, unpublished data.

5 B. J. Druker, S. Tamura, E. Buchdunger, S. Ohno, G. M. Segal, J. Zimmermann, and

N. B. Lydon, Effects of a selective inhibitor of the ABL tyrosine kinase inhibitor on thegrowth of BCR-ABL positive cells, submitted for publication.

H H

Fig. 1. Structure of CGP 57148.

A

v-abl-*

B

vabl -*

C

PDGF-R-)

1234567Fig. 2. Inhibition of v-AbI and PDGF receptor autophosphorylation by COP 57148 in

intact cells. PB-3c cI. 15 cells (A and B, Lane 1) and AMuLV-transformed PB-3c cI. 15cells (A and B, Lanes 2—8)were incubated with control medium (A and B, Lanes 1 and2) or the indicated concentrationsof COP 57148 (free base; A and B, Lanes 3—8)for 90ruin. Confluent, quiescent BALB/c 313 cells (C) were incubated with control medium orCOP 57148 (free base) for 90 rain prior to stimulation with PDGF BB (50 ng/ml) for 10mm. Whole-cell lysates were corrected for protein content and analyzed by Westernblotting using antiphosphotyrosine antibodies (A and C) or anti-AbI antibodies (B). R,receptor.

101

U.....@12345678

(up) (+) PDGF

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0110 01@1@

(Fig. 2A ). The compound was without effect on Abl protein levels,as determined by Western blotting using anti-Abl antibodies (Fig.2B). Due to its structural similarity to COP 53716 (24), COP 57148was tested further for inhibition of the PDGF receptor proteintyrosine kinase activity. Treatment ofcells with COP 57148 causeda concentration-dependent inhibition of ligand-stimulated PDOFreceptor tyrosine phosphorylation with an IC50 value of approximately 0.3 LM(Fig. 2C). Immunoblotting experiments with antiPDGF receptor antibodies showed that COP 57148 did not affectoverall levels of the PDGF receptor (data not shown). To define thespecificity of CGP 57148, the effect of the compound on ligandstimulated EOF receptor autophosphorylation in A43 I cells wastested. The addition of EGF to serum-starved A43 1 cells stimulated EOF receptor autophosphorylation (Fig. 3A, Lane 2). Pretreatment of the cells with up to 100 MM COP 57148 had nosignificant effect on ligand-induced EGF receptor autophosphorylation (Fig. 3A, Lanes 3 and 4). Similarly, p185cerbB2 autophosphorylation in MDA-MB 453 cells was affected only weakly at 10@.LM(Fig. 38). No inhibition of ligand-induced autophosphorylation

of either the /3 chain of the insulin receptor in Rat-i cells (Fig. 3C)or the 13chain of the IGF-I receptor in NIH 3T3 (LISNc4) cells(Fig. 3D) was observed with concentrations of COP 57148 up to100 p@M.The potency and selectivity of COP 57148 were alsoapparent in its effects on c-fos mRNA expression induced bydifferent growth stimuli. The compound inhibited the induction ofc-fos mRNA by PDGF strongly, with an IC50 value between 0.3

and 1 @.LM(Fig. 4, Lanes 3—7).The induction of c-los mRNAexpression by EGF, bFGF, and phorbol 12-myristate 13-acetatewas not affected by the drug even when used at concentrations upto 100 @.LM(Fig. 4, Lanes 8—18).

Antiproliferative Activity of CGP 57148. COP 57148 was testedfor growth inhibition of EOF-dependent BALBIMK cells and theH-ras-transformed T24 bladder carcinoma line using methylene bluestaining. Inhibition of IL-3-dependent growth of FDC-Pl cells wasdetermined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide reduction as described previously (30). The compoundshowed only weak antiproliferative activity against these cell lines,with IC50 values of 12.7 ,.LM,8.4 p@, and 29.2 @M,respectively (Table

IOTIOQ0ocv@@—1@

Table I Antiproliferative activity of CGP57/48Cell

lineIC50(,.tM)―BALB/MK12.7

±1.4FDC-Pl9.4±0.47T2429.2±0.6PB-3c

cI.l5 v-abl (—IL-3)0.1 I ±0.01PB-3c

cI. 15 v-abl (+ IL-3)0.90 ±0.05BALB/cv-sis0.33 ±0.05

IGF-I-R@5> @-

12345Fig. 3. Effect of COP 57148 on growth factor receptor autophosphorylation. Serum

starved A43 I cells (A), MDA-MB 453 cells (B), Rat-I cells (C), and NIH 3D (LiSNc4) cells(D) were incubated for 90 mm (MDA-MB 453 cells for 2 h) with control medium or theindicatedconcentrationsofCGP 57148 (free base) prior to stimulationwith EGF (100 ng/ml;A), monoclonal antibody FPR S(5 @g/ml;B), insulin (5 @g/ml;C), or IGF-l (1 @g/ml;D) for10 mm. Equal amounts of cell lysate protein were analyzed by Westem blotting usingantipbosphotyrosineantibodies.Ins, insulin;mob, monoclonalantibody;R, receptor.

Fig. 4. Selective inhibition of PDGF-induced cfos mRNA expression. Confluent, quiescentBALB/c3T3cellswereincubatedfor90 mmwiththe indicated concentrations of COP 57148 (freebase). After stimulation with PDGF (10 ng/ml;Lanes 2—7),EOF (20 ng/ml; Lanes 8—11). bFGF(100 ng/mJ; Lanes /2—14),or phorbol 12-myristate13-acetate (PMA; 25 ng/ml; Lanes 15—18).samplesof total RNA were analyzed for c-los expression.

F.@_r.11 CV) @IIIIIOO@Cv)'-

c@0@

1@@OO@O@O @-

lNHlB@ON OF THE ABL PROTEIN-TYROSINEKINASE

C ,(—)II@ Ins

aDatarepresentmeanvalues±rangeof at leasttwoindependentexperiments,eachperformed in duplicate. Values were obtained with free-base COP 57l48.

c-fos-*

102

A (-)(+)EGF@Ioo

@iM OCV)1@

EGF-R*@@ ii..]

1234B (-)i@(+)mabFPR5

p185@°@@@@ .‘@@

I 2345

Ins-R@-*

I 2345

D (-) (.1-)IGF-IF&o 0 oo@

@iM ______

(-),, PDGF •@EGF IbFGF, PMA

I 2@ 4@ 6 @891ohh12131@5161@8

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Table 2 Inhibition of soft agar growth by CGP57148BALB/c

AMuLV cells (3 X lO3/plate) and BALB/c 3T3 v-sis cells (5 X lO―/plate)were plated in culture medium supplemented with 0.3% agar in the presence of theindicatedconcentrationsof COP 57148. After 8 days (BALB/c AMuLV cells) or 14 days (BALB/c 3T3 v-sis cells), colonies >50 ,.am were counted. The mean colony numbers (±range)ofone

typical experiment are shown.Control

I @M 10@aMCells

No. colonies % inhibition No. colonies % inhibition No. colonies %inhibitionBALB/c

AMuLV 335 ±22 0 97 ±3 71 20 ±0.594BALB/c3T3 v-sis 963 ±25 0 152 ±16 84 25 ±0.5 97

1 6 1013 17 21 27 31

days after ceOinjection

INHIBITION OF THE ABL PROTEIN-TYROSINEKINASE

A B1.50

1.25

1.00C,)

I 0.75@ 0.50

0.25

0.00

Cl)

@010

E

Fig. 5. In S'IW)antitumor activity of CGP 57l48B. In vivoantitumor activity was tested using AMuLV-transformed (A)and v-sis-transformed (B) BALB/c 3T3 cells. Treatment wasstarted 24 h after cell injection. COP 57l48B was administeredi.p. once daily for 30 days (A) or 27 days (B). •.placebocontrol; 0. 50 mg/kg; & 12.5 mg/kg; 0, 3.13 mg/kg.

7 14 21 28

daysafter cell injection

1). However, when tested on v-abl-transformed PB-3c cells, incubation with CGP 57148 resulted in potent growth inhibition even in thepresence of exogenous IL-3 (IC50 values, 0. 1 @.tMand 0.9 @.LM,respectively). Similar results were obtained using v-sis-transformed BALB/c3T3 cells, which grow in response to autocrine PDGF production(IC50, 0.3 ELM).

Inhibition of Anchorage-independent Growth. A characteristicfeature of tumor cells is their anchorage-independent growth. BALB/cAMuLV and BALB/c 3T3 v-sis cells develop large colonies in softagar within 1—2weeks. CGP 57148 was found to inhibit colonyformation of both cell lines potently (Table 2).

In Vivo Antitumor Activity. The maximally tolerated dose for asingle p.o. or i.p. administration of COP 57148B in BALB/c mice was>500 mg/kg. BALB/c AMuLV and BALB/c 3T3 v-sis cells, which

were sensitive in the colony-forming assay, were used to test COP57148B for antitumor activity in female BALB/c nude mice. Oncedaily i.p. applications of 50, 12.5, or 3. 13 mg/kg CGP 57148B givenfor 30 consecutive days resulted in a strong antitumor effect againstAMuLV-transformed BALB/c 3T3 tumors (Fig. 5A). Similarly, antitumor experiments using v-sis-transformed BALB/c 3T3 cells revealed dose-dependent antitumor activity (Fig. SB). Maximal T/C(X 100%) values of 4% (AMuLV tumors) and 1 1% (v-sis tumors)

were obtained when CGP 57148B was administered at SOmg/kg bodyweight. In contrast, COP 57148B showed no antitumor activityagainst tumors derived from NIH-S27src cells when 50 mg/kg wereadministered p.o. once daily for 30 days (T/C, 102%). Using the sameroute of application, T/C values of 7 and 22% against AMuLV andv-sis tumors, respectively, were obtained when 50 mg/kg COP

S7l48B were given.

103

DISCUSSION

Bcr-Abl expression is thought to play an initiating role in thepathogenesis of CML. To test the hypothesis that selective inhibitionof the Abl tyrosine kinase activity might be useful for chemotherapy ofPhiladelphia chromosome-positive leukemias, we have searched for solective protein-tyrosine kinase inhibitors from the 2-phenylaminopyrimidine class. We describe the biological profile of COP 57148, whichinhibits the AbI tyrosine kinase potently and which, additionally, showspotent inhibition of the PDOF receptor tyrosine kinase.

Inhibitors of the 2-phenylaminopyrimidine class have been shownto act as competitive inhibitors of protein kinases with respect toATP.6 Therefore, they are likely to bind in the AlP-binding cleft ofthe Abl and PDOF receptor enzymes. The selectivity of such inhibitors toward different classes of protein-tyrosine kinases, therefore,could be explained by interactions between the inhibitor and theamino acid residues lining the AlP-binding cleft. Recently, we haveprovided a rational basis for selectivity of ATP competitive inhibitorsfor the EOF receptor kinase using a model of the kinase domain (32).Inspection of the residues that interact with ATP, as predicted fromthe X-ray crystal structure of the cyclic AMP-dependent proteinkinase, reveals a large degree of similarity between the Abl and PDGFreceptor kinases. However, a final understanding of the molecularbasis of selectivity awaits the resolution of a crystal structure of anenzyme-drug complex.

COP 57148 selectively inhibited the in vitro activity of the v-Ablprotein-tyrosine kinase and showed preferential inhibition of v-Ablautophosphorylation in cells. We have examined the specificity of

6 T. Meyer and J. Zimmermann, unpublished results.

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INHIB@ON OF THE ABL PROTEIN-TYROSINE KINASE

COP 57148 by analyzing its effects on signal transduction via different tyrosine kinase receptor-mediated pathways. Although the ligandinduced activation of the EGF, bFGF, insulin, and IGF-I receptortyrosine kinases were not affected by CGP 57148, the PDGF pathwaywas sensitive to inhibition by the compound. The antiproliferativeactivity of COP 57148 against both v-abl- and v-sis-transformedBALB/c 3T3 support the selectivity profile of COP 57148 further.

COP 57148 was tested for in vitro and in vivo antitumor activityusing v-abl- and v-sis-transformed BALB/c 3T3 cells. Inhibition ofanchorage-independent growth was achieved at concentrations ofCOP 57148 that were similar for inhibition of v-Abl and PDGFreceptor autophosphorylation. in vivo antitumor efficacy against AMuLV-transformed BALB/c 3T3 tumors was obtained at doses betweenSo and 3.13 mg/kg. In antitumor studies using v-sis-transformedBALB/c 3T3 cells, which grow in response to autocrine PDGF production, COP 57148 was well tolerated and showed similar antitumoractivity. Our finding that COP 57148 did not inhibit tumor growth ofsrc-transformed NIH 3T3 cells is compatible with the lack of inhibition of Src kinase activity by COP S7i48@ and demonstrates furtherthat the compound is relatively selective for inhibition of Abl- andPDGF-driven tumor growth in vivo.

Cytogenic studies established CML as the first human neoplasm tobe associated consistently with a particular chromosomal abnormality,known as the Philadelphia chromosome. The Philadelphia translocation juxtaposes c-abl proto-oncogene sequences on chromosome 9with the Bcr gene on chromosome 22 (33). This results in a Bcr-Ablfusion protein, which resembles the@ oncogene product ofthe A-MuLV. Both oncogenic proteins have a tyrosine-specific activity that is indispensable for its capacity to transform cells. TheBcr-Abl protein, which has elevated tyrosine kinase activity relative toc-Abl, has been shown to induce various hematopoietic malignanciesin transgenic mice and mice reconstituted with Bcr-abl retrovirusinfected bone marrow (18, 19, 21, 34).

PDGF has been suggested to be a mediator ofpathological cell growth,e.g.,carcinogenesis,aswell asnonmalignantproliferative disorders,suchas atherosclerosis and fibrosis (for review, see Ref. 35).

The reported findings with CGP 57148 suggest that it may be adevelopment candidate for use in the treatment of Philadelphia chromosome-positive leukemias. Additional potential applications forCOP 57148 may include proliferative diseases that involve abnormalPDGF receptor activation.

ACKNOWLEDGMENTS

We would like to acknowledge the assistance of L. Duboc, C. Koelbing,R. Reuter, V. Rigo, A. Schmidt, and F. Wenger.

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1996;56:100-104. Cancer Res   Elisabeth Buchdunger, Jürg Zimmermann, Helmut Mett, et al.   by a 2-Phenylaminopyrimidine Derivative

in Vivo and in VitroInhibition of the Abl Protein-Tyrosine Kinase

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