The Tyrosine Kinase Inhibitor CGP 57148 (ST1 571) Induces Apoptosis in BCR-ABL-Positive Cells by Down-Regulating BCL-X1

1958 Vol. 6, 1958–1968, May 2000 Clinical Cancer Research

The Tyrosine Kinase Inhibitor CGP 57148 (ST1 571) Induces Apoptosis in BCR-ABL-positive Cells by Down-Regulating BCL-X1

Carsten Oetzel, Tarja Jonuleit, Alexander Go¨tz, down-regulation of BCL-X, and (c) is more effective than the Heiko van der Kuip, Heike Michels, inhibition of the downstream signal transduction pathways Justus Duyster, Michael Hallek, and of PI-3 kinase, mitogen-activated protein/extracellular signal-regulated kinase kinase, and Janus-activated kinase 2. Walter E. Aulitzky2 Dr. Margarete Fischer-Bosch Institute for Clinical Pharmacology, INTRODUCTION 70376 Stuttgart [T. J., A. G., H. v. d. K., H. K.]; 2nd Department of 3 Internal Medicine for Oncology, Hematology and Immunology, CML is a clonal stem cell disorder. The hallmark of the Robert Bosch Hospital, 70376 Stuttgart [C. O., W. E. A.]; Department disease is the Philadelphia chromosome caused by a balanced of Internal Medicine, Klinikum rechts der Isar, University of Munich, reciprocal translocation between chromosomes 9 and 22, lead- Munich [J. D.]; and Department of Internal Medicine III, Technical ing to the expression of a chimeric protein BCR-ABL. Fusion of University of Munich, Munich [M. H.] Germany the bcr sequences to the abl gene results in constitutive activation of the ABL kinase. BCR-ABL phosphorylates numerous ABSTRACT cellular targets, leading to the activation of intracellular signal- CGP 57148 is a potent inhibitor of the ABL protein ing pathways. Three biological key processes are affected in tyrosine kinase and a promising new compound for the cells expressing BCR-ABL. BCR-ABL-transformed cells ex- treatment of a variety of BCR-ABL-positive leukemias. We hibit cell cycle entry in the absence of growth factors, decreased used this enzyme inhibitor to characterize the biological susceptibility to induction of apoptosis after exposure to DNA effects of BCR-ABL in primary cells and two growth factor- damage and growth factor withdrawal, and impaired adhesion to dependent BCR-ABL-transfected cell lines. extracellular matrix proteins (1, 2). Numerous substrates of the The effect of CGP 57148 on primary cells is dependent BCR-ABL kinase have been identified in various cell lines. The on the stage of differentiation. The growth of maturing majority of the proteins phosphorylated by BCR-ABL are com- chronic myeloid leukemia cells is independent of BCR-ABL ponents of pathways activated in normal cells by growth factor in the presence of growth factors. However, the proliferation signaling. This includes proteins involved in the RAS pathway of leukemic immature cobblestone-forming area cells is al- (3–5), the JAK/STAT pathway (6), and the PI-3 kinase pathway most completely blocked after the inhibition of the BCR- (7). Other substrates are involved in the process of cell adhesion, ABL kinase. In the BCR-ABL-transfected cell lines, M07/ such as the cytoskeletal proteins paxillin and actin (8). It is not p210 and Ba/F3/p185, CGP 57148 induces apoptosis by clear which of these intracellular transduction pathways are releasing cytochrome c, activating caspase 3, and cleavage of responsible for the antiapoptotic effect of BCR-ABL. The PARP. No alteration of the expression level of the apoptosis BCL-2 family members BCL-2 and BCL-X have been impli- regulator BCL-2 was observed. In contrast, BCL-X was cated in the BCR-ABL-mediated resistance to apoptosis induced down-regulated after exposure to CGP 57148. Inhibitors of by cytokine withdrawal (3) and cytotoxic agents (9). signal transduction proteins such as PI-3 kinase, mitogen- Because the tyrosine kinase activity is the crucial enzy- activated protein/extracellular signal-regulated kinase ki- matic activity driving all known functions of the BCR-ABL nase, and Janus-activated kinase 2 pathways were not capa- protein (10) and is required for the protection from apoptosis by ble of a comparable down-regulation of BCL-X. The Fas/Fas BCR-ABL, targeting this kinase activity is a promising ap- ligand system was not involved either in the induction of proach for therapeutics strategies. Using the known structure of apoptosis by CGP 57148. We conclude that the inhibition of the ATP-binding site of protein kinases, a series of compounds the BCR-ABL kinase by CGP 57148 (a) preferentially in- of the 2-phenylaminopyrimidine have been synthesized and hibits the growth of immature leukemic precursor cells, (b) screened for inhibition of various tyrosine kinases. CGP 57148 efficiently reverts the antiapoptotic effects of BCR-ABL by and CGP 57148 B, the methane sulfonate salt, have been found to be potent inhibitors of the ABL-kinase without affecting various other intracellular tyrosine kinases. The only other ki-

Received 7/22/99; revised 12/3/99; accepted 1/24/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked 3 The abbreviations used are: CML, chronic myeloid leukemia; JAK, advertisement in accordance with 18 U.S.C. Section 1734 solely to Janus-activated kinase; CFU-GM, colony-forming unit-granulocyte/ indicate this fact. macrophage; CAFC, cobblestone area-forming cell; LY, LY 294002; 1 Supported by a research grant of the German research council (Deut- PD, PD 98059; AG, AG 490; GM-CSF, granulocyte macrophage col- sche Forschungsgemeinschaft) project Nr. Au 129/1 and the Robert- ony-stimulating factor; IL-3, interleukin 3; G-CSF, granulocyte colony- Bosch foundation. stimulating factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet- 2 To whom requests for reprints should be addressed, at Robert-Bosch- razolium bromide; BS, blocking solution; TBS, Tris-buffered saline; Krankenhaus, Auerbachstr. 110, 70376 Stuttgart, Germany. E-mail: ERK, extracellular signal-regulated kinase; EGF, epidermal growth [email protected]. factor.

nase inhibited to a similar extent by CGP 57148 is the platelet- using a Miltenyi MACS column (Miltenyi Biotech, Bergisch derived growth factor ␤ receptor tyrosine kinase (11, 12). Fur- Gladbach, Germany) according to manufacturer’s instructions. ther investigations demonstrated a BCR-ABL-specific inhibi- Cell purity was checked by FACScan analysis. The fraction of ϩ tion of cellular proliferation and tumor formation by both pri- CD34 cells ranged from 96% to 99%. mary CML cells and BCR-ABL-expressing cell lines (12). In Cell Culture Techniques. The human M07 cell line was the colony-forming assays of peripheral blood and bone marrow derived from a patient with megakaryocytic leukemia and re- from a patient with CML, a decrease of 92–98% in the number quired GM-CSF, IL-3, or stem cell factor for proliferation. Cells of BCR-ABL colonies was observed, whereas normal colony were cultured in RPMI 1640 medium (Seromed Inc., Berlin, formation was not affected (12). Over a 2-logarithmic dose Germany) supplemented with 10% FCS and 100 ng/ml GM- range with a maximum differential effect at 1 ␮mol/liter, CGP CSF (Leukomax, Sandoz, Switzerland). The M07/p210 cell line 57148 selectively suppressed the growth of CFU-GM and burst- was obtained from Dr. M. Hallek (Mu¨nchen, Germany; Ref. 15). BCR-ABL forming unit-erythroid (13). Analyses of cell lines showed The M07/p210 cell line expressed p210 and was factor- growth inhibition followed by decline in viability of 10 BCR- independent. M07/p210 cells were grown in RPMI 1640 me- ABL-positive cell lines and BCR-ABL-transfected cells (13). dium supplemented with 10% FCS. These experiments showed dephosphorylation of the BCR-ABL The murine pro-B lymphocyte cell line, Ba/F3, expressing protein after exposure to CGP 57148 (14). However, the intra- temperature-sensitive p185 BCR-ABL, was kindly provided by cellular mechanisms by which CGP 57148 inhibits proliferation J. Duyster (Munich, Germany). The construction of plasmids and/or causes decreased viability are not known. and generation of transfected cell lines have been described We show here that CGP 57148-mediated growth inhibition (16). Factor-independent Ba/F3/p185 cultivated at a temperature of primary BCR-ABL-positive cells depends on the stage of of 33°C were grown without IL-3 supplementation. Ba/F3/p185 differentiation of hematopoetic cells. In addition, CGP 57148 cultivated at 39°C showing no BCR-ABL phosphorylation were induces apoptosis in the BCR-ABL-transfected human M07/ used as controls. The growth of factor-independent Ba/F3 clone p210 and the murine Ba/F3/p185 by down-regulating the expressing wtBCR-ABL was transformed with pSLXBcr-Abl BCL-X protein. and cultivated at 37°C. Primary CD34ϩ cells were grown at a density of 0.5 ϫ 106 cells/ml in 24-well plates in RPMI (Seromed Inc., Berlin, Ger- MATERIALS AND METHODS many) supplemented with 10% FCS and 200 ng/ml recombinant Patients. Cells were harvested from newly diagnosed human G-CSF (Amgen Inc., Thousand Oaks) and recombinant CML patients after obtaining informed consent before the ex- human steel factor (50 ng/ml; Amgen Inc.). The culture system periments. All patients studied were in the stable chronic phase has been described in detail (17). All experiments were per- of disease and were not treated previously. Cells from patients formed on days 3–5 of the culture. The majority of these cells with various malignancies in complete remission undergoing lost expression of the CD34 antigen, but remained cycling. blood stem cell harvest were used as controls. All experiments These cells represent maturing myeloid blast cells at an inter- were repeated at least three times. To evaluate the proliferation mediate stage of differentiation. The percentage of S-phase cells of maturing blast cells, cells derived from three different pa- and the expression of the cell surface markers CD34, CD33, and tients were used. The CFU-GM assay was performed five times CD15 were identical in leukemic and normal hematopoietic using cells from four different patients. Accordingly, CD34ϩ cells (17). Because of other groups reporting down-regulation of cells from four patients were investigated in the CAFC assay. the BCR-ABL protein during hematopoietic differentiation, we Reagents. CGP 57148 (now renamed ST1 571) was further checked BCR-ABL expression in our culture system kindly provided by E. Buchdunger Novartis Inc. (Basel, (18). No significant changes in the expression of BCR-ABL Switzerland). A stock solution (10 mg/ml) was prepared by RNA were found between days 3 and 5 of the culture (data not dissolving the compound in DMSO, and it was kept at shown). Cells were washed on day 3 of the culture, and 0.5 ϫ 6 Ϫ20°C. CGP 57148 was used at concentrations ranging from 10 cells were cultured in medium with or without 1 ␮M CGP 0.1 to 10 ␮M. The PI-3 kinase inhibitor, LY, the MEK- 57148 for up to 48 h. inhibitor, PD, and the JAK-2 Inhibitor, AG, were purchased Hematopoietic Progenitor Cell Assay. The assay for from Calbiochem (San Diego, CA). Stock solution of LY detecting CFU-GM was performed as previously described (19). ϩ 3 (25 mM in DMSO), PD (25 mM in DMSO), and AG (25 mM CD34 cells were used at a cell density of 5 ϫ 10 cells/well. in DMSO) were kept at Ϫ20°C, and the final dilution (LY, These cells were suspended in culture medium containing 0.3% 5 ␮M;PD50,␮M; and AG, 20 ␮M) was made immediately agar in a volume of 0.25 ml and plated in multiwell tissue before use. The caspase inhibitor, ZVAD-fluoromethylketone culture plates (Nunc, Wiesbaden, Germany). After solidification (stock solution at Ϫ20°C: 20 mM in methanol; final dilution, of the agar layer, a liquid overlay of culture medium was added 100 ␮M), was purchased from Bachem (Heidelberg, Ger- to the culture plates containing the hematopoietic growth fac- many). The blocking anti-FAS IgG1 monoclonal antibody tors. The final concentration of growth factors was as follows: ZB4 (1 ␮g/ml) and the neutralizing anti-FAS IgG monoclonal G-CSF, 200 ng/ml, and IL-3, 50 ng/ml. At the end of the antibody 4H9 (100 ng/ml) were purchased from Immunotech incubation period, the agar cultures were fixed with glutaralde- (Marseilles, France). hyde, 2.5%, washed with distilled water for 4 h, transferred onto Cell Source. Mononuclear cells were isolated by Ficoll- microscopic slides, dried at 40°C, stained with May-Gru¨nwald- Hypaque density gradient centrifugation (Seromed, Berlin, Ger- Giemsa, and permanently mounted. Colony number was evalu- many). After lysis of residual RBCs, CD34ϩ cells were purified ated by light microscopy.

CAFC Assay. CAFC assays were performed on a con- fluent layer of FBMD-1 cells, which were kindly provided by R. Ploemaker (Rotterdam, Germany), in 24-well plates (Falcon; Ref. 20). Indicated numbers of CD34ϩ cells in 0.5 ml of culture media were plated on the FBMD-1 cells in 24-wells and allowed to adhere at 37°C for 6 h. Afterward, the medium was removed, and cells were overlaid with culture medium containing 0.3% agar in a volume of 0.5 ml containing the hematopoietic growth factors. The final concentration of growth factors was as follows: G-CSF, 200 ng/ml, and IL-3, 10 ng/ml. The cultures were

maintained at 33°C and 5% CO2 for 2 weeks. After 1 week, cells were fed with culture medium containing 0.3% agar in a volume of 0.5 ml containing the hematopoietic growth factors in 2-fold access. The number of CAFCs with at least one phase-dark hematopoietic clone of at least five cells (i.e., cobblestone area) beneath the stromal layer was counted on day 14 of culture. Survival Assay. Cells were plated into 96-well plates at 1 ϫ 105/well in their respective media supplemented with 10% FCS. CGP 57148 was added in various concentrations. For measurement of cytotoxic effects, the MTT assay was used. After 16–24 h of exposure, the viable cells in each well were assayed to their ability to transform MTT into a purple formazan (21). Protein Extract Preparation. Protein extracts were prepared as follows: Cells were collected by centrifugation, washed twice with PBS, and dissolved in lysis buffer [50 mM Tris-HCl (pH 7.6); 250 mM NaCl; 0.1% Triton X-100; 5 mM EDTA; 1 ␮g/ml leupeptin; 1 mM phenylmethylsulfonyl fluoride; 1 mM DTT; 1 ␮g/ml aprotinin]. The protein content of the extracts was quantified using the Bradford protein assay with BSA as a protein standard (22). For the analysis of cytosolic cytochrome c, cells were harvested by centrifugation, washed twice, and resuspended in 7 100 ␮l/10 cells lysis buffer [25 mM HEPES (pH 7.5), 5 mM

MgCl2,1mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml pepstatin, 10 ␮g/ml leupeptin]. After centrifugation at 800 g for 10 min at 4°C, supernatants were collected and 5 mM EDTA were added. Subsequently, the extracts were centrifuged Fig. 1 CGP 57148 selectively inhibits the growth of immature CML at 280,000 g for 60 min. Two mM DTT were added to the cells. The proliferation of maturing blast cells is not significantly supernatants (23), and protein concentration was determined by blocked by CGP 57148 (A), whereas a significant CML-specific inhi- Bradford. bition is observed of CML CFU-GM at concentrations ranging from Western Blots. Western blots were performed according 0.05 ␮g/ml to 1 ␮g/ml (B). The most pronounced antiproliferative effect Ϯ to standard techniques. Briefly, proteins (50 ␮g/lane) were run is seen on the growth of CML CAFC (C). Bars, mean SE values of three to five independent experiments. Light bars, experiments studying on SDS-polyacrylamide gels with the Tris glycine buffer system CML cells; dark bars, normal controls. (24). Proteins were then transferred to polyvinylidene difluoride membranes (Boehringer Mannheim, Germany) using a semidry transfer apparatus as recommended by the manufacturer (Bio- Rad). The membranes were blocked by incubating in 1% BS (diluted 1:5000) for 30 min, and proteins were detected by using (Boehringer Mannheim) in TBS (pH 7.6) for 1 h. The blots were a chemiluminescence kit (Boehringer Mannheim). then incubated with anti-BCL-2 [4 ␮l/ml (Santa Cruz, Heidel- berg, Germany) and 10 ␮l/ml (Dako, Glostrup, Denmark)], Caspase 3 Activity. Caspase 3 activity was measured anti-BCL-X (1 ␮g/ml; Transduction, Lexington), anti-p27 using a commercially available assay as described (PharMin- ϫ 6 (2 ␮g/ml; PharMingen), anti-PARP (1 ␮g/ml; Upstate, Lake gen). Briefly, 2 10 cells were lysed in cell lysis buffer [10 Placid), and anticytochrome c (1 ␮g/ml; PharMingen) dissolved mM Tris-HCl (pH 7.5), 27 mM KCl, 100 mM KH2PO4/K2HPO4 in TBS with 0.5% BS overnight. Blots were washed twice in (pH 7.5)] and spun in an Eppendorf centrifuge at 14,000 rpm for TBS containing Tween-20 (0.1%) for 10 min and afterward 10 min. Five ␮l of supernatant were diluted with 80 ␮lof blocked two times with 0.5% BS. Then blots were incubated HEPES buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM with secondary antibody conjugated to horseradish peroxidase DTT], and 5 ␮l of Ac-DEVD-AMC (200 ␮M in DMSO) were

Fig. 2 CGP 57148 selectively induces apoptosis in BCR-ABL-transfected M07 cells. A, the FACS analysis of propidium iodide-stained cells exposed to 5 ␮mol of CGP 57148. Whereas only a few cells with a DNA content Ͻ2N are detectable in M07 cells, the majority of M07/p210 cells undergo ␮ apoptosis after this treatment. B, in a MTT assay, the IC50 of CGP 57148 for M07/p210 cells is 0.069 g/ml, whereas the parental line is largely ␮ resistant, with a IC50 of 32.08 g/ml. Cell death is not inhibited by the addition of exogenous growth factor. C, M07/p210 are cultivated in the presence of indicated CGP 57148 concentrations with and without GM-CSF and IL-3. CGP 57148 selectively inhibits the growth of the BCR-ABL-positive Ba/F3/p185 cell line; however, this effect is antagonized by the simultaneous addition of IL-3.

added. As a control lysate with both 5 ␮l of Ac-DEVD-AMC flow cytometry analysis, cells were rehydrated in cold PBS, (200 ␮M in DMSO) and 5 ␮l Ac-DEVD-CHO (200 ␮M in treated with 50 ␮g/ml RNase A (Boehringer Mannheim) for DMSO), an inhibitor of caspase 3 was used. Fluorescence was at least 15 min at room temperature, and stained with 50 measured using an excitation wavelength of 380 nm and an ␮g/ml propidium iodide. The stained cells were analyzed by emission wavelength of 450 nm. FACScan (Becton Dickinson). The fraction of cells with a Cell Cycle Analysis. Cell cycle analyses were per- DNA content of Ͻ2N, 2N, 2–4N, and 4N was determined by formed by determining the DNA content with propidium means of the Modfit 2.0 software (Becton Dickinson). The iodide. Cells were washed in PBS and fixed in precooled number of viable cells was determined by counting the num- (Ϫ20°C) 70% ethanol for at least1hat4°C. Shortly before ber cells excluding trypan blue.

Fig. 3 Treatment of M07/p210 cells with CGP 57148 induces cytosolic accumulation of cytochrome c, caspase 3 activity, and PARP cleavage. A, a Western blot of cytochrome c in the cytoplasmic fraction of protein extracts before and 4, 16, and 24 h after exposure to CGP 57148. B, the increase in activity of caspase 3 by using a fluorogenic caspase 3 substrate Ac-DEVD-AMC; as a control, an inhibitor of caspase 3, Ac-DEVD-CHO, is used. C, a Western blot of the PARP protein before and 4, 8, and 16 h after treatment with CGP 57148 with the appearance of the characteristic 85-kDa cleavage fragment 16 h after exposure to CGP 57148.

TUNEL Assay. In situ cell death detection was per- leukemic myeloid blast cells with 1 ␮M CGP 57148 exhibited no formed using a commercially available assay as described significant effect on viability of both leukemic and normal cells (Boehringer Mannheim). Briefly, 1 ϫ 106 cells were washed (Fig. 1A). Even prolonged incubation of 48 h lead to a decrease and fixed in 2% paraformaldehyde in PBS for 30 min at room in neither proliferation nor viability of leukemic cells (data not temperature. They were than washed once, permeabilized by shown). Moreover, cell cycle kinetics and apoptosis were also incubating (2 min; 4°C) with 0.1% Triton X-100, and 0.1% not influenced by this treatment (data not shown), and thus in sodium citrate, and then washed twice. The TUNEL reaction the presence of exogenous growth factors, both growth and was carried out by incubating cells (60 min; 37°C) with 50 ␮lof survival of maturing CML cells is not dependent on the BCR- TUNEL mixture (FITC-12-dUTP, dATP, CoCl2, terminal ABL kinase activity. deoxynucleotidyltransferase, and TdT buffer; Boehringer In contrast, the proliferation of leukemic progenitor cells Mannheim). was significantly inhibited after inactivation of the BCR-ABL kinase despite the presence of excess amounts of exogenous RESULTS growth factors. At a concentration of 1 ␮M CGP 57148, Inactivation of the BCR-ABL Kinase Selectively Inhib- CFU-GM colony formation of leukemic cells was inhibited by its the Proliferation of Immature CML Cells. Proliferation 36.14 Ϯ 14%, whereas normal colony formation was not sig- of primary normal and leukemic cells at different stages of nificantly reduced (Fig. 1B). The most pronounced effect of differentiation was studied in the absence or presence of differ- CGP 57148, however, was seen on the formation of immature ent concentrations of the BCR-ABL kinase inhibitor CGP hematopoietic cells. Whereas leukemic CAFCs were efficiently 57148. Twenty-four-h incubation of proliferating normal or inhibited by CGP 57148, normal CAFC formation was not

Fig. 4 The Fas-Fas ligand system is not involved in CGP 57148- induced apoptosis. The figure shows the viability of M07-p210 cells after exposure to CGP 57148. Neither the anti-FAS-receptor antibody 4H9 (100 ng/ml) nor the anti-FAS-ligand antibody ZB4 (1 ␮g/ml) affects CGP 57148-induced apoptosis. Bars, mean values Ϯ SE of triplicate experiments.

affected. Moreover, at lower concentrations of CGP 57148 (0.5 and 1.0 ␮M), normal CAFC formation was even enhanced (Fig. 1C). Inhibition of the BCR-ABL Kinase Efficiently Kills BCR-ABL-expressing M07/p210 and Ba/F3/p185 Cells. The growth and survival of wild-type M07 and Ba/F3 cells are strictly dependent on the presence of exogenous growth factors such as GM-CSF or IL-3. BCR-ABL expression renders these Fig. 5 CGP 57148 down-regulates BCL-X expression in the BCR- cells growth factor-independent (25). Forty-eight-h incubation ABL-positive M07/p210 but not in the maternal M07 without affecting with 1 ␮M CGP 57148 did not affect the viability of wild-type BCL-2. A, Western blotting of BCL-X and BCL-2 protein in cell lysate M07 (Fig. 2) or Ba/F3 cells (data not shown) maintained in the obtained before and 4, 8, 16, and 24 h after exposure to CGP 57148. B, BCL-X down-regulation is specific for the BCR-ABL-positive M07/ presence of growth/survival factors. In contrast, inhibition of the p210. BCL-X is down-regulated subsequent to growth factor withdrawal BCR-ABL kinase with CGP57148 specifically killed BCR- after 24 h. C, this effect is not enhanced by CGP 57148. ABL-positive cells. Incubation of M07/p210 and Ba/F3/p185 cells with 1 ␮M CGP 57148 rapidly resulted in cell death. Both M07/p210 cells (Fig. 2A) and Ba/F3/p185 cells (data not shown) undergo apoptotic cell death after exposure to CGP57148 as to trigger with APAF and caspase 9 the proteolytic activity of indicated by a large proportion of cells with a DNA content caspase 3 (26). Fig. 3A shows increased cytosolic accumulation Ͻ2N. The inhibition of growth of M07/p210 by CGP 57148 is of cytochrome c in M07/p210 after exposure to 1 ␮M CGP dose-dependent with an IC of 0.069 ␮g/ml (Fig. 2B). The two 57148. Furthermore, in M07/p210 after 6-h incubation with 1 cell lines, however, differ in the BCR-ABL dependency of their ␮M CGP 57148, caspase-3 was already active, and the maxi- proliferation. The addition of excess amounts of GM-CSF or mum activation was detected after 16 h (Fig. 3B). As a conse- IL-3 was not capable of preventing cell death from M07/p210 quence of caspase 3 activation, the 85-kDa cleavage product of cells exposed to CGP 57148 (Fig. 2C). Thus, in this cell line, PARP could be shown by means of Western blotting. After 12-h irreversible BCR-ABL dependency of proliferation can be ob- incubation of M07/P210 cells with 1 ␮M CGP 57148, the served. In contrast, the addition of murine IL-3 entirely rescued 85-kDa cleavage product of the 110-kDa PARP was visible Ba/F3-p185 from cell death after inhibition of the BCR-ABL (Fig. 3C). To confirm the significance of caspase activation, we kinase with CGP 57148 (Fig. 2D). analyzed CGP 57148 (1 ␮M)-induced apoptosis in the presence Inhibition of the BCR-ABL Kinase Rapidly Leads to a of a caspase inhibitor ZVAD-fluoromethylketone. The propor- Cytosolic Accumulation of Cytochrome c with Resultant tion of apoptotic cells was determined by FACS analysis of Activation of Caspase-3 and Cleavage of PARP. To further propidium iodide-stained cells. In M07/p210 cells, the fraction investigate the mechanism leading to the induction of apoptotic of cells containing Ͻ2N DNA decreased from 14.3% to 7.1% in cell death after inhibition of the BCR-ABL kinase, we studied the presence of ZVAD after 16 h of incubation and in Ba/F3/ mitochondrial release of cytochrome c, caspase-3 activation, p185 from 36.9% to 9.9%. and degradation of PARP. Recent data indicate that apoptotic Taken together, these data demonstrate that inhibition of stimuli cause a release of cytochrome c from the mitochondria the BCR-ABL kinase by CGP57148 induces apoptotic cell

ABL-negative M07 and Ba/F3 (Fig. 5, B and C; Fig. 6). More- over, down-regulation of BCL-X after growth factor withdrawal could not be enhanced by CGP 57148 (Fig. 5C). Inhibition of Several Signal Transduction Pathways neither Down-Regulates BCL-X nor Induces Apoptotic Cell Death as Does CGP 57148. Because numerous substrates of the BCR-ABL kinase are components of signal pathways activated in normal cells by growth factor signaling, such as RAS (3–5), JAK/STAT (6), and PI-3 kinase (7), we compared the effect of inhibitors of these pathways with inhibition of BCR- ABL kinase activity by CGP 57148. We studied the effects of the P-I3 kinase inhibitor LY, the MEK inhibitor PD, and the JAK-2 inhibitor AG on induction of apoptosis and regulation of BCL-X in Ba/F3/p185 cells. All Inhibitors had a dose-dependent inhibitory effect on growth of Ba/F3/p185 and the maternal Ba/F3 by means of MTT assay (Fig. 7A). None of these inhibitors revealed a significant induction of apoptosis as demonstrated by means of TUNEL assay (Fig. 7B) nor down-regulated Fig. 6 CGP 57148 down-regulated BCL-X expression without affect- the expression of BCL-X (Fig. 7C). As a control for viability of ing BCL-2 in the BCR-ABL-positive Ba/F3/p185 cell line. The effect of Ba/F3/p185, we investigated the induction of p27 (Fig. 7C). CGP 57148 on BCL-X, however, is antagonized by the simultaneous Induction of p27 expression levels suggests cell cycle arrest addition of IL-3. after exposure to the pathway inhibitors and CGP57148. A combination of LY, PD, and AG with a suboptimal dose of CGP 57148 (0.1 ␮M) did not result in a major increase of CGP 57148-induced apoptosis (Fig. 8A) nor in major variations of death in M07/p210 cells by activation of a mitochondrion- protein levels of BCL-X compared to the effect of CGP 57148 dependent proapoptotic pathway. (0.1 ␮M). CGP 57148-induced Apoptosis Is Not Dependent on Fas-Fas Ligand Interaction. It has been proposed that induction of apoptosis by cytotoxic agents may involve ligand/recep- DISCUSSION tor-driven amplifier systems such as the Fas/CD95 system (27). Protection from apoptosis has been postulated to be one of Also, controversial data exist on the induction of Fas/CD95 the crucial biological consequences of the BCR-ABL transloca- ligand and up-regulation of Fas/CD95 after treatment with cy- tion (1). The antiapoptotic properties of the BCR-ABL protein totoxic drugs. To test whether CGP 57148-induced apoptosis is may be responsible for both the accumulation of malignant cells Fas-CD95 ligand/Fas-CD95-dependent, we used a Fas neutral- as well as the genetic instability leading to clonal evolution of izing and a Fas agonistic antibody in addition to CGP 57148. the malignant clone. Protection from apoptosis after growth Neither of these antibodies could block or enhance CGP 57148- factor deprivation and exposure to DNA-damaging agents has induced apoptosis (Fig. 4). Therefore, we conclude that the FAS been consistently demonstrated in a variety of BCR-ABL- system is not involved in the apoptotic process induced by CGP positive murine and human cell lines. However, the data in 57148 in the human cell line M07/p210. primary cells are less clear. No resistance to apoptosis has been Inhibition of the BCR-ABL Kinase Down-Regulates demonstrated in proliferating myeloid blast cells after ␥-irradi- BCL-X but Has No Effect on BCL-2 Expression. Members ation and growth factor withdrawal (28). Moreover, the results of the BCL-2 protein family crucially regulate programmed cell from studies on the resistance of CFU-GM against DNA dam- death by affecting the mitochondrial membrane. Recently it has age are conflicting (29, 30). The data from our studies on the been described that BCR-ABL-mediated resistance to apoptosis activity of CGP 57148 on primary CML cells may at least is in part attributable to an up-regulation of BCL-X (9). We partially explain this discrepancy. In primary cells, the effect of therefore analyzed the expression of BCL-X and BCL-2 after CGP 57148 depends on the differentiation status of the cell type exposure to CGP 57148 in M07/p210 and Ba/F3/p185 cells. studied. Neither growth inhibition nor induction of cell death BCL-X protein levels substantially decreased within 4–8 h was observed in exponentially growing maturing blast cells after inhibition of the BCR-ABL kinase with CGP 57148 both in when cultured with an excess of exogenous growth factors. The M07/p210 (Fig. 5A) and Ba/F3/p185 (Fig. 6) cells. In contrast, persistent proliferation after removal of growth factors of these no variations in the protein level of BCL-2 was observed in cells, however, was efficiently blocked by CGP 57148 (31). A these cell lines (Fig. 5A; Fig. 6B). The induction of apoptosis is significant antiproliferative activity despite the presence of closely accompanied by the down-regulation of BCL-X. growth factors similar to that described by Druker et al. (12) was Whereas BCL-X down-regulation was not reversed by the ad- seen after exposure of CFU-GM to CGP 57148. The most dition of GM-CSF in the M07/p210 cells (data not shown), the pronounced differential effect on leukemic and normal cells, addition of IL-3 completely blocked down-regulation of BCL-X however, was detected when immature cobblestone forming and apoptosis in the Ba/F3/p185 cell line (Fig. 6B). CGP 57148 cells (CAFCs) were studied. had no effect on the expression levels of BCL-X in the BCR- Therefore, in mature CML cells, BCR-ABL causes a subtle

Fig. 7 In contrast to CGP57148 inhibitors of PI-3 kinase (LY), MEK (PD) and JAK-2 (AG) pathways are not capable of blocking the antiapoptotic BCR-ABL signal. A, viability of Ba/F3 and Ba/F3/p185 after exposure to variable concentrations of LY, AG, and PD in a MTT assay. B, induction of apoptosis in a TUNEL assay. C, Western blotting of BCL-X and p27 of Ba/F3/p185 cells after exposure to LY (5 ␮M), AG (20 ␮M), PD (50 ␮M), and CGP 57148 (B, 0.5 ␮M; C, 0.1 and 1 ␮M).

abnormality of the cell cycle regulation detectable only under itor is sufficient to almost completely block the outgrowth of suboptimal growth factor conditions. In contrast, the growth of CML CAFCs. This observation argues against an antiprolifera- immature CML cells entirely depends on an active BCR-ABL tive effect and supports the view that CGP 57148 may induce kinase. Whether this BCR-ABL effect on immature CML cells apoptosis in these immature cobblestone forming cells. is caused by induction of apoptosis or inhibition of cell cycle The effect of CGP 57148 on cobblestone forming cells is progression cannot be answered thus far because the biochem- leukemia-specific. The growth of normal differentiated and im- ical consequences of CGP 57148 on cobblestone forming cells mature myeloid cells is apparently not affected by inhibition of cannot be studied directly. The addition of CGP 57148 at the the c-ABL kinase. The proliferation of normal CAFCs was even beginning of the culture period without refeeding of the inhib- enhanced by CGP 57148. Accordingly, experiments studying

Fig. 8 Combinations of suboptimal concentrations of CGP 57148 (0.1 ␮M) with inhibitors of PI-3 kinase (LY), MEK (PD), and JAK-2 (AG) pathways do not have an additive effect on induction of apoptosis or BCL-X expression. A, induction of apoptosis in a TUNEL assay. B, Western blotting of BCL-X of Ba/F3/p185 after exposure to combinations of CGP 57148 (0.1 ␮M) with LY (5 ␮M), AG (20 ␮M), and PD (50 ␮M).

the inhibition of the c-ABL kinase with a dominant-negative did not cause a down-regulation of BCL-2 in either the Ba/F3/ mutant demonstrated a stimulatory effect on cell cycle progres- p185 or the M07/p210 cell line. Other investigators reported sion of murine fibroblasts (32). This differential effect on the conflicting results of BCR-ABL transfection on BCL-2 expres- growth of immature normal and leukemic cells offers an intrigu- sion of Ba/F3/p210 cells and HL-60/p 185 cells (34, 35). BCL-X ing mechanism that can be used for selecting BCR-ABL-nega- has been implicated in the antiapoptotic phenotype of BCR- tive precursors, e.g., for autologous transplantation. Experi- ABL-positive cells. Inhibition of the BCR-ABL kinase by CGP ments testing the capacity of CGP 57148 to purge bone marrow 57148 results in a decrease of BCL-X preceding apoptotic cell cells from CML precursors are presently underway at our insti- death both in the murine BA/F3/p185 cell line and the human tution. M07/p210, whereas BCL-2 remains unchanged. The regulation Because the mechanism leading to growth inhibition of of BCL-X closely parallels the induction of cell death. Rescue of primary CML cells could not directly be studied, we used two Ba/F3/p185 after exposure to CGP 57148 with IL-3 abrogates growth factor-dependent BCR-ABL-transfected cell lines for both the down-regulation of BCL-X and the induction of apo- further analysis. CGP 57148 reverses the transformed phenotype ptosis. In contrast, IL-3 is not capable of rescuing M07/p210 of both M07/p210 and Ba/F3/p185. Specific inhibition of the cells after inhibition of the BCR-ABL kinase, and it does not tyrosine kinase of BCR-ABL induces exactly the same pro- prevent down-regulation of BCL-X. We find a close correlation cesses to which BCR-ABL confers increased resistance (33). between the expression of BCL-X and the prevention of apo- The human M07/p210 and the murine Ba/F3/p185 cells undergo ptosis by the BCR-ABL protein. These data confirm previous apoptosis within 24 h after exposure to CGP 57148. Cytochrome reports showing BCL-X regulation in cells overexpressing c release in the cytoplasm with subsequent caspase 3 activation BCR-ABL. A recent report showed that expression of BCR- and PARP cleavage could be observed in M07/p210 subsequent ABL in HL-60, Ba/F3, DAGM, and 3T3 cells induced up- to exposure to CGP 57148. Thus, CGP 57148 induces apoptosis regulation of BCL-X and conferred resistance to apoptosis in the via a pathway involving the disruption of the mitochondrial HL-60 in a variety of agents tested (AR-C, Vp16, VM26, membrane. camptothecin, and actinomycin). Moreover, antisense oligonu- The BCL-2 protein family has been suggested to function cleotides to BCL-X partially restored staurosporine-induced ap- as pore formers and adapter molecules in the mitochondrial optosis to about 28%. However, this cytotoxic effect was less membrane regulating mitochondrial alterations preceding the than that observed in the control HL-60 vector cells, suggesting activation of apoptogenic proteases and nucleases (26). The that BCL-X is one of several targets mediating BCR-ABL- effect of the BCR-ABL oncogene on BCL-2 expression is induced resistance to apoptosis. Accordingly, BCL-X overex- controversial. CGP 57148 treatment of BCR-ABL-positive cells pression caused a lower level of resistance to apoptosis com-

pared to BCR-ABL despite a higher level of expression of the thorough understanding of both the mechanisms of the antiapo- BCL-X protein (9). ptotic function of BCR-ABL is essential for a further develop- Thus far little is known about the regulation of BCL-X. ment of an effective strategy using CGP 57148 for treatment Several intracellular pathways have been implicated. The IL-2- of CML. receptor has been shown to induce BCL-2 and BCL-X via the adapter molecule, Shc, promoting proliferation but not viability ACKNOWLEDGMENTS in a murine T-cell line (36). In Ba/F3 cells, R-Ras and insulin- like growth factor I showed synergistic inhibition of IL-3 with- CGP 57148 was kindly provided by E. Buchdunger Novartis Inc. (Basel, Switzerland). drawal-induced apoptosis. Insulin-like growth factor I and R- Ras activated the ERK and the Akt kinase, respectively, and cooperatively induced BCL-X expression (37). In our experi- REFERENCES ments, the addition of a selective ERK kinase inhibitor or LY, a 1. Deininger, M. W., and Goldman, J. M. Chronic myeloid leukemia. PI-3 kinase inhibitor, did not block BCR-ABL-mediated sur- Curr. Opin. Hematol., 5: 302–308, 1998. vival, and no decrease of BCL-X expression after exposure to 2. Gotoh, A., and Broxmeyer, H. E. The function of BCR/ABL and CGP 57148 was observed. related proto-oncogenes. Curr. Opin. Hematol., 4: 3–11, 1997. Recently, the JAK kinase-dependent pathway has been 3. Sanchez-Garcia, I., and Martin-Zanca, D. Regulation of Bcl-2 gene expression by BCR-ABL is mediated by Ras. J. Mol. Biol., 267: shown to regulate BCL-X(L). 32D.3, FDC-P1.2, and primary 225–228, 1997. fetal liver-derived myeloid cells were shown to down-regulate 4. Cortez, D., Stoica, G., Pierce, J. H., and Pendergast, A. M. The BCL-X(L) in protein and RNA levels after IL-3 deprivation. BCR-ABL tyrosine kinase inhibits apoptosis by activating a Ras- Furthermore, transfected cells with an erythropoietin receptor dependent signaling pathway. Oncogene, 13: 2589–2594, 1996. defective in activation of JAK2 (EpoR-PB) died when it was 5. Goga, A., McLaughlin, J., Afar, D. E., Saffran, D. C., and Witte, shifted to medium containing Epo, whereas transfectants har- O. N. Alternative signals to RAS for hematopoietic transformation by boring EGF receptor-JAK2 kinase domain chimeric receptor the BCR-ABL oncogene. Cell, 82: 981–988, 1995. maintained viability in the absence of IL-3 but were activated 6. Chai, S. K., Nichols, G. L., and Rothman, P. Constitutive activation of JAKs and STATs in BCR-Abl-expressing cell lines and peripheral blood with EGF. BCL-X levels decreased in the dying EpoR-PB cells cells derived from leukemic patients. J. Immunol., 159: 4720–4728, 1997. and increased in EGF receptor-JAK2 transfectants activated 7. Skorski, T., Kanakaraj, P., Nieborowska-Skorska, M., Ratajczak, with EGF, suggesting that the JAK kinase pathway plays a M. Z., Wen, S. C., Zon, G., Gewirtz, A. M., Perussia, B., and Calabretta, crucial role in the cytokine-regulated BCL-X regulation as cell B. Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and death antagonist (38). In our experiments, a JAK kinase inhib- is required for the growth of Philadelphia chromosome-positive cells. Blood, 86: 726–736, 1995. itor did not induce cell death of Ba/F3/p185, and there was no 8. Sattler, M., and Salgia, R. Activation of hematopoietic growth factor decrease of BCL-X in response to CGP 57148. signal transduction pathways by the human oncogene BCR/ABL. Cy- Combined treatment with inhibitors of the RAS-ERK, the tokine Growth Factor Rev., 8: 63–79, 1997. PI-3 kinase, the JAK, and a suboptimal dose of CGP 57148 also 9. Amarante-Mendes, G. P., McGahon, A. J., Nishioka, W. K., Afar, did not abolish the antiapoptotic effect of BCR-ABL. Moreover, D. E., Witte, O. N., and Green, D. R. Bcl-2-independent Bcr-Abl- the inhibitors were also not capable of down-regulating BCL-X mediated resistance to apoptosis: protection is correlated with up regu- in the Ba/F3/p185 cell lines. Therefore, direct inhibition of the lation of Bcl-xL. Oncogene, 16: 1383–1390, 1998. BCR-ABL kinase is apparently the most effective approach to 10. Lugo, T. G., Pendergast, A. M., Muller, A. J., and Witte, O. N. Tyrosine kinase activity and transformation potency of bcr-abl oncogene block the biological effects of the BCR-ABL protein. In addi- products. The BCR-ABL oncogene transforms Rat-1 cells and cooper- tion, these experiments indirectly support the view that either ates with v-myc. Science (Washington DC), 247: 1079–1082, 1990. unknown or redundant biochemical pathways are responsible 11. Buchdunger, E., Zimmermann, J., Mett, H., Meyer, T., Muller, M., for both the down-regulation of BCL-X and the antiapoptotic Druker, B. J., and Lydon, N. B. Inhibition of the Abl protein-tyrosine function of BCR-ABL. kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res., 56: 100–104, 1996. The discrepant results of the experiments analyzing the 12. Druker, B. J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G. M., effect of exogenous growth factors on BCR-ABL-positive cells Fanning, S., Zimmermann, J., and Lydon, N. B. Effects of a selective are not clear. Whereas Ba/F3/p185 cells can be readily rescued inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive by IL-3 after exposure to CGP 57148, neither IL-3 nor GM-CSF cells. Nat. Med., 2: 561–566, 1996. nor TPO rescued the M07/p210 cells. Both species differences 13. Deininger, M. W., Goldman, J. M., Lydon, N., and Melo, J. V. The of the biological activities of the BCR-ABL protein or CGP tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of 57148 as well as differing clonal selection of the cell lines might BCR-ABL-positive cells. Blood, 90: 3691–3698, 1997. be responsible for this phenomenon. 14. Gambacorti-Passerini, C., le, C. P., Mologni, L., Fanelli, M., Ber- tazzoli, C., Marchesi, E., Di, N. M., Biondi, A., Corneo, G. M., Belotti, In conclusion, our experiments demonstrate that inhibition D., Pogliani, E., and Lydon, N. B. Inhibition of the ABL kinase activity of the BCR-ABL kinase by CGP 57148 predominantly affects blocks the proliferation of BCR/ABLϩ leukemic cells and induces immature primary CML cells. In growth factor-dependent BCR- apoptosis. Blood Cells Mol. Dis., 23: 380–394, 1997. ABL-transfected cell lines, CGP 57148 induces apoptosis by 15. Hallek, M., Danhauser-Riedl, S., Herbst, R., Warmuth, M., Win- activation of mitochondrial cytochrome c release, activation of kler, A., Kolb, H. J., Druker, B., Griffin, J. D., Emmerich, B., and Ullrich, A. Interaction of the receptor tyrosine kinase p145c-kit with the caspase 3, and PARP cleavage. This proapoptotic effect is p210bcr/abl kinase in myeloid cells. Br. J. Haematol., 94: 5–16, 1996. accompanied by down-regulation of the BCL-X protein. These 16. Bai, R. Y., Jahn, T., Schrem, S., Munzert, G., Weidner, K. M., experiments therefore further support the role of BCL-X as a Wang, J. Y., and Duyster, J. The SH2-containing adapter protein target for the transforming function of BCR-ABL. However, a GRB10 interacts with bcr-abl. Oncogene, 17: 941–948, 1998.

17. Schwab, R., Peschel, C., Despres, D., Derigs, G., Fischer, T., 29. Amos, T. A., Lewis, J. L., Grand, F. H., Gooding, R. P., Goldman, Huber, C., and Aulitzky, W. E. Deficient cell cycle control in myeloid J. M., and Gordon, M. Y. Apoptosis in chronic myeloid leukaemia: cells of patients with newly diagnosed chronic myeloid leukemia. Cy- normal responses by progenitor cells to growth factor deprivation. tokines Mol. Ther., 1: 281–288, 1995. X-irradiation and glucocorticoids. Br. J. Haematol., 91: 387–393, 1995. 18. Wetzler, M., Talpaz, M., Van, E. R., Hirsh-Ginsberg, C., Beran, M., 30. Bedi, A., Zehnbauer, B. A., Collector, M. I., Barber, J. P., Zicha, and Kurzrock, R. Subcellular localization of Bcr, Abl, and Bcr-Abl M. S., Sharkis, S. J., and Jones, R. J. BCR-ABL gene rearrangement and proteins in normal and leukemic cells and correlation of expression with expression of primitive hematopoietic progenitors in chronic myeloid myeloid differentiation. J. Clin. Invest., 92: 1925–1939, 1993. leukemia. Blood, 81: 2898–2902, 1993. 19. Peschel, C., Paul, W. E., Ohara, J., and Green, I. Effects of B cell 31. Jonuleit, T., Peschel, C., Schwab, R., van, d. K., Buchdunger, E., stimulatory factor-1/interleukin 4 on hematopoietic progenitor cells. Fischer, T., Huber, C., and Aulitzky, W. E. Bcr-Abl kinase promotes Blood, 70: 254–263, 1987. cell cycle entry of primary myeloid CML cells in the absence of growth 20. Gordon, M. Y., Dowding, C. R., Riley, G. P., and Greaves, M. F. factors. Br. J. Haematol., 100: 295–303, 1998. Characterisation of stroma-dependent blast colony-forming cells in hu- 32. Sawyers, C. L., McLaughlin, J., Goga, A., Havlik, M., and Witte, O. man marrow. J. Cell. Physiol., 130: 150–156, 1987. The nuclear tyrosine kinase c-Abl negatively regulates cell growth. Cell, 21. Mosmann, T. Rapid colorimetric assay for cellular growth and 77: 121–131, 1994. survival: application to proliferation and cytotoxicity assays. J. Immu- 33. Amarante-Mendes, G. P., Naekyung, K. C., Liu, L., Huang, Y., nol. Methods, 65: 55–63, 1983. Perkins, C. L., Green, D. R., and Bhalla, K. Bcr-Abl exerts its antiapo- 22. Bradford, M. M. A rapid and sensitive method for the quantitation ptotic effect against diverse apoptotic stimuli through blockage of mi- of microgram quantities of protein utilizing the principle of protein-dye tochondrial release of cytochrome C and activation of caspase-3. Blood, binding. Anal. Biochem., 72: 248–254, 1976. 91: 1700–1705, 1998. 23. Martins, L. M., Mesner, P. W., Kottke, T. J., Basi, G. S., Sinha, S., 34. Nishii, K., Kabarowski, J. H., Gibbons, D. L., Griffiths, S. D., Tung, J. S., Svingen, P. A., Madden, B. J., Takahashi, A., McCormick, Titley, I., Wiedemann, L. M., and Greaves, M. F. ts BCR-ABL kinase D. J., Earnshaw, W. C., and Kaufmann, S. H. Comparison of caspase activation confers increased resistance to genotoxic damage via cell activation and subcellular localization in HL-60 and K562 cells under- cycle block. Oncogene, 13: 2225–2234, 1996. going etoposide-induced apoptosis. Blood, 90: 4283–4296, 1997. 35. Sanchez-Garcia, I., and Grutz, G. Tumorigenic activity of the BCR- 24. Laemmli, U. K. Cleavage of structural proteins during the assembly ABL oncogenes is mediated by BCL2. Proc. Natl. Acad. Sci. USA, 92: of the head of bacteriophage T4. Nature (Lond.), 227: 680–685, 1970. 5287–5291, 1995. 25. Sirard, C., Laneuville, P., and Dick, J. E. Expression of bcr-abl 36. Lord, J. D., McIntosh, B. C., Greenberg, P. D., and Nelson, B. H. abrogates factor-dependent growth of human hematopoietic M07E cells The IL-2 receptor promotes proliferation, bcl-2 and bcl-x induction, but by an autocrine mechanism. Blood, 83: 1575–1585, 1994. not cell viability through the adapter molecule Shc. J. Immunol., 161: 26. Zamzami, N., Brenner, C., Marzo, I., Susin, S. A., and Kroemer, G. 4627–4633, 1998. Subcellular and submitochondrial mode of action of Bcl-2-like onco- 37. Suzuki, J., Kaziro, Y., and Koide, H. Synergistic action of R-Ras proteins. Oncogene, 16: 2265–2282, 1998. and IGF-1 on Bcl-xL expression and caspase- 3 inhibition in Ba/F3 27. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K. M. Involve- cells: R-Ras and IGF-1 control distinct antiapoptotic kinase pathways. ment of the CD95 (APO-1/FAS) receptor/ligand system in drug- FEBS Lett., 437: 112–116, 1998. induced apoptosis in leukemia cells. Nat. Med., 2: 574–577, 1996. 38. Packham, G., White, E. L., Eischen, C. M., Yang, H., Parganas, E., 28. Albrecht, T., Schwab, R., Henkes, M., Peschel, C., Huber, C., and Ihle, J. N., Grillot, D. A., Zambetti, G. P., Nunez, G., and Cleveland, Aulitzky, W. E. Primary proliferating immature myeloid cells from CML J. L. Selective regulation of Bcl-XL by a Jak kinase-dependent pathway patients are not resistant to induction of apoptosis by DNA damage and is bypassed in murine hematopoietic malignancies. Genes Dev., 12: growth factor withdrawal. Br. J. Haematol., 95: 501–507, 1996. 2475–2487, 1998.

Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2000 American Association for Cancer Research. The Tyrosine Kinase Inhibitor CGP 57148 (ST1 571) Induces Apoptosis in BCR-ABL-positive Cells by Down-Regulating BCL-X

Carsten Oetzel, Tarja Jonuleit, Alexander Götz, et al.

Clin Cancer Res 2000;6:1958-1968.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/6/5/1958

Cited articles This article cites 37 articles, 13 of which you can access for free at: http://clincancerres.aacrjournals.org/content/6/5/1958.full#ref-list-1

Citing articles This article has been cited by 17 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/6/5/1958.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/6/5/1958. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.