Farnesyltransferase inhibitor BMS-214662 induces apoptosis ... - Nature

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Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells I Marzo1, P Pe´rez-Galań1, P Giraldo3, N Lo´pez-Royuela1, M Go´mez-Benito1, L Larrad2, P Lasierra2, D Rubio-Fe´lix3, A Anel1 and J Naval1 1

Departamento de Bioquı´mica y Biologıá Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Servicio de Inmunologıá, Hospital Clıńico Universitario, Zaragoza, Spain; and 3Servicio de Hematologıá, Hospital Universitario Miguel Servet, Zaragoza, Spain 2

B-cell chronic lymphocytic leukemia (B-CLL) cells develop resistance to nucleoside analogs over time. This chemoresistance may be caused by selection for B-CLL cells with defects in the particular apoptosis pathway triggered by these drugs. Therefore, anticancer agents that induce apoptosis through alternative pathways might be useful in treating chemoresistant B-CLL. Farnesyltransferase inhibitors (FTIs) are a class of synthetic drugs with definite molecular targets, which have demonstrated cytotoxicity against leukemic cell lines. We have studied the ex vivo effect of the FTI BMS-214662 on cells from 18 patients with B-CLL. Low concentrations (o1 lM) of BMS214662 prevented farnesylation of the chaperone marker HDJ-2 and had no effect on Akt activation. BMS-214662 induced apoptosis in B-CLL cells from all patients studied, including those showing resistance to cladribine and fludarabine ex vivo and in vivo. Treatment with BMS-214662 induced loss of mitochondrial membrane potential (DWm), phosphatidylserine exposure, proapoptotic conformational changes of Bax and Bak, reduction in Mcl-1 levels and activation of caspases 9 and 3. The general caspase inhibitor Z-VAD-fmk did not prevent BMS-214662-induced cell death. These results indicate that BMS-214662 may be a useful drug for treating B-CLL and, in particular, an alternative for the therapy of purine analogresistant or relapsed B-CLL. Leukemia (2004) 18, 1599–1604. doi:10.1038/sj.leu.2403469 Published online 9 September 2004 Keywords: BH3-only proteins; caspases; apoptosis; B-cell chronic lymphocytic leukemia; farnesyltransferase inhibitors

Introduction B-cell chronic lymphocytic leukemia (B-CLL) is the commonest leukemia in the adult, representing nearly 23% of all leukemias.1 This disease is characterized by the accumulation of G0 resting, long-lived, CD5 þ B-cells in blood, which express high levels of the antiapoptotic proteins Bcl-2 and Mcl-1.2 This leukemia has been classically treated with chlorambucil, an alkylating agent, but the purine analogs fludarabine, cladribine and pentostatine are increasingly used as a front-line therapy.3 Treatment with purine analogs usually produces higher response rates, but they do not prolong survival and patients with advanced B-CLL, failing to respond to fludarabine or cladribine, have poor prognosis.4–6 Resistance to apoptosis induction by these drugs may be one of the causes of chemoresistance.7 Therefore, identification of new agents that induce apoptosis in B-CLL cells resistant to purine analogs would be useful to improve the therapy of this disease. BMS-214662 is a tetrahydrobenzodiazepine class of farnesyltransferase inhibitors (FTIs) that is presently under evaluation in Correspondence: Dr I Marzo, Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, pza. San Francisco, s/n, 50009 Zaragoza, Spain; Fax: þ 34 976 762 123; E-mail: [email protected] Received 10 March 2004; accepted 28 March 2004; Published online 9 September 2004

Phase I and II clinical trials for solid tumors.8,9 This and other FTIs in clinical development were originally designed to block oncogenic Ras proteins, preventing their relocation to cell membranes via its farnesyl group.10 Mutations of Ras genes are very rare in B-CLL cells,11–13 and in fact the Ras/Raf/MEK/ERK pathway is not essential for viability of B-CLL cells.14 However, FTIs can act on other molecular targets apart from Ras.15 This could explain why FTIs have shown significant in vivo biological activity in patients with relapsed and refractory acute and chronic myelogenous leukemia, irrespective of their mutational Ras status.9,16 Indeed, it has been recently reported that some FTIs can inhibit the PI3K/Akt pathway, critically involved in survival of B-CLL cells,14 in a Ras-independent way.17,18 We report here that the FTI BMS-214662 efficiently induces apoptosis on B-CLL cells, including cells from patients displaying resistance to fludarabine and cladribine. These results suggest that BMS-214662 could be useful for the treatment of relapsed or purine analog-resistant B-CLL patients.

Materials and methods

Cell isolation and culture Patients were diagnosed of B-CLL according to clinical and immunophenotypic criteria. All patients gave informed consent, according to the institutional review board at the University Hospital Miguel Servet of Zaragoza. Peripheral blood was obtained from 18 patients (13 men, five women), who had not been previously treated or remained untreated for the previous 8 months. The median white cell count was 27 109 leukocytes/l (range: 5–95 109 leukocytes/l). Lymphocytes were isolated from blood samples by Ficoll-density centrifugation as described.7 Flow cytometry analysis revealed that 490% purified cells were CD5 þ , CD19 þ , CD23 þ and CD38. B-CLL cells were cultured at 5 106 cell/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, antibiotics and, except when indicated, 20 ng/ml IL-4 (hereafter, complete medium). Patients participating in this study were treated in vivo with fludarabine (9b-D-arabinofuranosyl-2fluoroadenine, F-ara-A) or cladribine (2-chloro-20 -deoxyadenosine, 2CdA) according to established protocols. Patients were termed fludarabine or cladribine resistant when they received a diagnosis of progressive disease after treatment with purine analogs.

Evaluation of cell toxicity and flow cytometry analysis B-CLL cells were treated in 24-well plates (1 ml/well) for the times indicated, with either 0.25 mM BMS-214662 (kindly provided by Bristol-Myers Squibb), 1 mM 2CdA (kindly provided

BMS-214662-induced apoptosis in B-CLL cells I Marzo et al

1600 by Janssen-Cilag) or 10 mM F-ara-A (fludarabine phosphate, Schering) in complete medium. In apoptosis inhibition assays, cells were preincubated for 1 h with the general caspase inhibitor Z-VAD-fmk (100 mM) prior to the addition of drugs. Apoptosis was analyzed by flow cytometry by determining the changes in cell forward/side scatter and through the simultaneous determination of phosphatidylserine (PS) exposure and mitochondrial membrane potential (DCm) loss with annexin VPE (Caltag) and DiOC6(3) (Molecular Probes), respectively.7 In brief, B-CLL cells (5 105) were incubated with 5 nM DiOC6(3) in 200 ml of annexinV-binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at 371C for 15 min. Then, 1 ml of annexin V-PE (0.1 mg/ml) was added and cells incubated for further 20 min at room temperature. Finally, samples were diluted to 1 ml with annexin-binding buffer and analyzed in an EPICS-XL flow cytometer (Coulter). During apoptosis induction, proapoptotic proteins Bax and Bak undergo a conformational change that promotes their oligomerization and causes the release of cytochrome c and other apoptotic mediators from mitochondria. Conformational changes of Bax and Bak induced by BMS-214662 treatment were analyzed with conformation-specific epitope anti-Bax (6A7, BDBiosciences) or anti-Bak (Ab-1, Oncogene) monoclonal antibodies19–21 by intracellular immunostaining and flow cytometry, as well as by immunoprecipitation (see next section). For flow cytometry analysis, cells (1 106) were washed in PBS and fixed with 0.5% paraformaldehyde for 15 min at 41C. Fixed cells were incubated for 25 min at room temperature with 0.5 mg of anti-Bax (6A7), anti-Bak (Ab-1) or an isotype-matched irrelevant mouse IgG in 100 ml of PBS containing 0.1% saponin and 0.25% goat serum. Then, cells were washed two times with 0.03% saponin in PBS and incubated with a 1/250 dilution of a FITC-labeled rabbit anti-mouse IgG antibody (Caltag). Finally, cells were washed with PBS/saponin, resuspended in PBS and analyzed by flow cytometry.

and 1% CHAPS) for 30 min at 41C. Cell lysates were cleared by centrifugation for 10 min at 6000 g and incubated with 2 ml of monoclonal anti-Bax or anti-Bak at 41C for 5 h. Then, 30 ml of agarose–Protein G beads (Amersham Biosciences) were added and incubation prolonged for a further 2 h. Beads were washed three times and proteins were finally released by boiling in nonreducing SDS sample buffer and analyzed by SDS-PAGE and Western blotting.

Results

Induction of apoptosis by BMS-214662 on B-CLL cells Dose- and time-dependent cytotoxicity curves of BMS-214662 were established with B-CLL cells from selected patients (Figure 1). Apoptosis was assayed by simultaneous determination of PS exposure and DCm loss by flow cytometry. Apoptotic cells showed a low DCm and were positive for annexinV-PE labeling, characteristic of PS exposure. A dose of 0.25 mM of BMS-214662 (around the IC50 at 20 h) and an incubation time of about 20 h were selected for testing cytotoxicity in B-CLL cells. We compared ex vivo sensitivity of B-CLL cells from 18 patients to BMS-214662 and 2CdA (Table 1). Cells were incubated with 0.25 mM BMS-214662 for 20 h or 1 mM 2CdA for 48 h. As shown in Table 1, in B-CLL cells from 17 out of 18 patients, incubation with 0.25 mM BMS-214662 for 20 h caused apoptosis in more than 65–90% cells, whereas apoptosis in control cells incubated for the same period of time did not exceed 30% in any case.

Immunoprecipitation and Western blot analysis Akt activation, HDJ-2 farnesylation, caspase activation and levels of Bcl-2 superfamily proteins were analyzed by Western blot essentially as described previously.7 For the detection of the active, phosphorylated form of Akt, B-CLL cells (5 106 cells/ ml) were incubated with either 10 ng/ml PMA for 1 h or 20 ng/ml IL-4 for 6 h in the presence or the absence of 0.25 mM BMS214662. To revert the effect of IL-4, 0.1 mM wortmannin was added as a control. After incubations, cells were lysed in 50 mM Tris/HCl (pH 7.4) buffer containing 0.15 mM NaCl, 10% glycerol, 1 mM Na3VO4, 10 mM Na4P2O7, 50 mM NaF, 1 mM EDTA, 10 mg/ml leupeptin, 1 mM PMSF and 1% Triton X-100. Solubilized proteins from equal numbers of Trypan-bluenegative cells (2 106/lane) were resolved by SDS-PAGE, transferred to nitrocellulose membranes and analyzed by Western Blot. The antibodies used were: anti-HDJ-2 from Neomarkers; anti-phospho-Akt (Ser473) from Cell Signaling; anti-procaspase-3, anti-caspase-3 (active form), anti-caspase-9, anti-Bax from BDBiosciences; rabbit anti-human Bak from UBI; anti-Bcl-2 and anti-Mcl-1 from Santa Cruz Biotechnology. Control of protein loading was achieved by reprobing with anti-b-actin or anti-a-tubulin, both from Sigma (Madrid, Spain). Proapoptotic conformational changes of Bax and Bak in BCLL cells were also analyzed by immunoprecipitation with the conformation-specific anti-Bax and anti-Bak antibodies. Cell extracts (8 106 cells) were prepared in 500 ml CHAPS lysis buffer (10 mM Hepes (pH 7.4) buffer, containing 150 mM NaCl Leukemia

Figure 1 Time-course and dose–response toxicity of BMS-214662 and 2CdA. (a) Cells were incubated with different doses of BMS214662 for 20 h or 2CdA for 48 h and apoptosis was analyzed by simultaneous annexinV-PE/DiOC6(3) staining and flow cytometry. (b) Cells were cultured in the absence (controls) or presence of 0.25 mM BMS-214662 or 1 mM 2CdA for different periods of time and apoptosis was evaluated by annexinV-PE/DiOC6(3) staining and flow cytometry.


1601 Table 1 Comparative toxicity of BMS-214662 and 2CdA on B-CLL cells ex vivo % PS+/DCmlow

Pat. no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

In vivo Response to treatment treatment in vivo

2CdA

BMS-214662

+

+

10 21 14 30 15 12 18 5 5 6 11 6 18 10 14 3 10 8

26 36 48 44 25 13 52 7 75 75 56 37 67 77 78 45 70 15

13 18 13 29 15 11 25 7 5 4 7 5 10 3 7 3 11 4

70 94 68 65 79 76 69 89 89 74 15 80 66 73 58 77 87 86

2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA 2CdA F-ara-A F-ara-A F-ara-A F-ara-A F-ara-A

PR PR NR CR PR NR NR NR CR PR NR PR CR NR PR PR PR PR

B-CLL cells were incubated in the absence () or presence (+) of 1 mM 2CdA for 48 h or 0.25 mM BMS-214662 for 20 h and apoptosis evaluated by flow cytometry as the percentage of cells with exposed phosphatidylserine (PS+) and low DCm (DClow m ). Data on in vivo response to treatment with 2CdA or F-Ara-A are given for comparison (CR, complete response; PR, partial response; NR, no response).

Under these experimental conditions, cells from one patient (no. 11) showed lower sensitivity to BMS-214662 treatment (Table 1). However, when cells from patient 11 were treated with a higher dose of BMS-214662 (1 mM) or for a longer time (40 h), they readily entered apoptosis (480–90% of PS þ cells after a 20 h incubation with 1 mM BMS-214662). Of note, BMS-214662 induced death of B-CLL cells from patients showing a poor ex vivo response to cladribine (patients no. 1, 5, 6, 8, 12 and 18; Table 1 and Figure 1) and/or fludarabine (Figure 3), and were refractory to purine analog treatment in vivo (patients no. 3, 6, 7 and 14 and Perez-Galan et al7). At doses that induced apoptosis, BMS-214662, but not 2CdA, efficiently prevented farnesylation of the marker chaperone HDJ-2 (Figure 2a). On the other hand, BMS-214662 did not prevent Akt activation induced by IL-4 (via PI3K) or PKC (activated with PMA) (Figures 1, 2b, 3).14

Caspase activation by BMS-214662 BMS-214662 induced activation of caspases 9 and 3 (Figure 4a). Activation of caspase-3 was detected by the appearance of a 17 kDa band corresponding to the processed enzyme. BMS214662 induced processing of caspase-9 and the active p12/p10 fragments could be also detected. Active subunits of caspases 9 and 3 were not observed in cells incubated with BMS-214662 in the presence of the general caspase inhibitor Z-VAD-fmk, although in both cases Z-VAD-fmk did not prevent the decrease in levels of the procaspases (Figure 4a). This was not due to incomplete caspase inhibition, since the cleavage of lamin B1, a specific substrate of caspase-6, a caspase-3-activated caspase, was completely blocked by Z-VAD-fmk (Figure 4a). The reduction in levels of procaspases 9 and 3 in the presence of Z-VAD-fmk could be due to their degradation by the protea-

Figure 2 (a) Inhibition of HDJ-2 farnesylation by BMS-214662. BCLL cells were left untreated or treated with 1 mM 2CdA for 48 h or 0.25 mM BMS-214662 for 20 h. Total cell proteins were analyzed by Western blotting with anti-HDJ-2 antibody. (b) Effect of BMS-214662 on Akt phosphorylation. Cells from three representative patients were incubated for 6 h with 20 ng/ml IL-4 or for 1 h with 20 ng/ml PMA in the presence or absence of 0.25 mM BMS-214662 and 0.1 mM wortmannin, as indicated. Akt phosphorylation in Ser473 was analyzed by Western blot. Results shown correspond to one out of three patients analyzed, who had similar results.

Figure 3 Cells were cultured in the absence (controls) or in the presence of either 1 mM 2CdA, 10 mM F-ara-A for 72 h, or 0.25 mM BMS214662 for 20 h. Apoptosis was evaluated by annexinV-PE staining and flow cytometry analysis. Results are the means of three independent determinations. s.d. was lower than 15%.

some. It has been recently shown that, during apoptosis, caspase activation results in the cleavage of the regulatory complex of the proteasome and inhibition of proteasomal degradation of cellular substrates, including the own cell proapoptotic molecules.22 Blocking caspase activity allows proteasome to resume its function and degrade caspases in dying cells. In fact, caspases 3, 7 and 9 are ubiquitinated by IAPs,23,24 suggesting their degradation by the proteasome. Z-VAD-fmk also attenuated BMS-214662-induced cell shrinking, as determined by forward/ side scatter measurements (not shown). Caspase inhibition by ZVAD-fmk reduced the amount of annexin V-positive cells but only slightly retarded DCm loss, with a significant portion of cells displaying an intermediate DCm (Figure 4b), and did not prevent cell death, as determined by microscopical evaluation of Trypan blue-stained cells. Z-VAD-fmk had not effect on conformational changes of Bax and Bak induced by BMS214662 (see later). Leukemia


1602

Figure 4 Caspase activation induced by BMS-214662 treatment. (a) Representative immunoblots of caspase activation induced by BMS-214662 treatment. B-CLL cells were cultured in the absence () or presence ( þ ) of 0.25 mM 2BMS-214662 for 20 h and activation of caspases 3 and 9 and cleavage of Lamin B1 were analyzed by Western blotting with the corresponding antibodies. (b) Effect of caspase inhibition on apoptosis induced by BMS-214662. Flow cytometric analysis of PS exposure and DCm of cells from a representative patient treated with 0.25 mM BMS-214662 for 20 h, in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk (100 mM). Numbers refer to the percentages of cells in the different regions.

Effect of BMS-216442 on Bcl-2 superfamily proteins Treatment of B-CLL cells with BMS-214662 induced a pronounced decrease of Mcl-1 levels, while levels of other proteins of the family (Bcl-2, Bax, Bak, Bim) did not change significantly (Figure 5). A proteolytic fragment of Bcl-2 was detected in extracts from BMS-214662-treated cells (Figure 4).25 Caspase inhibition by Z-VAD-fmk did not prevent the BMS-214662induced reduction of Mcl-1 levels, but it inhibited the proteolysis of Bcl-2 (Figure 5). BMS-214662 induced proapoptotic conformational changes of Bax and Bak, as detected by flow cytometry (Figure 6a) and immunoprecipitation (Figure 6b) using conformational-specific antibodies. Conformational changes of Bax and Bak were not prevented by Z-VAD-fmk (Figure 6a), according with the lack of significant protection on cell death.

Discussion Although B-CLL cells exhibit extended viability and resistance to apoptosis compared to normal B cells, most patients usually respond to therapy with purine analogs.4 However, a significant Leukemia

Figure 5 Effect of BMS-214662 on Bcl-2-superfamily protein levels. B-CLL cells were cultured for 20 in the absence (Control) or in the presence of 0.25 mM BMS-214662 for 20 h and levels of Bcl-2 superfamily proteins (Bim, Bak, Bax, Mcl-1 and Bcl-2) were analyzed by Western blot with specific antibodies. Levels of actin or tubulin were also determined as controls for equal loading.

number of patients fail to respond or relapse after treatment with fludarabine and few of these patients obtain a complete remission.3 Salvage therapies with a combination of different drugs and/or monoclonal antibodies are currently under evaluation. Moreover, remissions after salvage therapy are shorter than initial remissions and the prognosis of new relapse is usually worse. Thus, drugs with different molecular targets that can still induce apoptosis are potentially useful. The FTI BMS-214662 may be one of such drugs. BMS-214662 has shown potent in vitro cytotoxic activity against acute leukemia,26 efficiently induces apoptosis in multiple myeloma cell lines (Go´mez-Benito et al, submitted for publication) and is currently under evaluation for solid tumors in Phase II clinical trials.27 Here we show that BMS-214662 is cytotoxic for B-CLL cells from all patients studied, including cells showing resistance to fludarabine and cladribine. The study of the molecular mechanism of this cytotoxicity indicates that BMS-214662 induces apoptosis in B-CLL cells. Upon treatment, cells exhibited all the typical hallmarks of apoptosis, such as cell shrinking, PS exposure and loss of DCm. BMS-214662 also produced activation of the main caspases involved in the mitochondrial apoptotic pathway, namely caspases 9 and 3. In cells from most patients, complete caspase inhibition by Z-VADfmk prevented PS-exposure, a caspase-dependent process,28 but the collapse of DCm was only slightly delayed. These results agree with previous reports showing that caspase activation determines the morphology of dying cells but not the fate of BCLL in response to ex vivo treatment with other chemother-


1603 apoptosis induction in B-CLL cells. This and other FTIs were initially designed to block mutated Ras function, but other cell molecules could also be targeted by these drugs.15 Most B-CLL cells express wild-type Ras,13 which does not play a critical role in cell survival,14 according to its quiescent status. It has been recently shown that some FTIs,17 but not others,33 inhibit Akt activation/activity in tumor cells. Akt activity is important for BCLL survival,14 but the time course of B-CLL cell death induced by Akt inhibition is much slower than that induced by BMS214662.14,34 Anyway, BMS-214662 did not inhibit Akt activation/activity either basal or induced via PKC or PI3K. Additional work is needed to characterize the molecular targets of BMS214662 in B-CLL cells. In any case, BMS-214662 induces bona fide apoptosis in B-CLL cells and would be a good candidate for the treatment of relapsed or purine analog-resistant B-CLL. Further studies on the effectiveness and in vivo action of this drug would also be interesting.

Acknowledgements We thank Dr Veeraswamy Manne (Bristol-Myers Squibb) for providing us with BMS-214662. This work was supported in part by grants PIO20065 and G03/136 Thematic Network from Fondo de Investigaciones Sanitarias (Ministerio de Sanidad, Spain) and P24/2000 from Diputacioń General de Aragoń.

References Figure 6 Proapoptotic conformational changes of Bax and Bak induced by BMS-214662 in B-CLL cells. (a) Cells were treated with 0.25 mM BMS-214662 for 20 h in the absence or in the presence of 100 mM Z-VAD-fmk, as indicated. Labeling with conformation-specific antibodies was performed as described in Materials and methods and cells were analyzed by flow cytometry. (b) Immunoprecipitation of activated Bax and Bak. Cells were cultured for 20 h in the absence (control) or in the presence of 0.25 mM BMS-214662. Cells extracts were prepared in CHAPS lysis buffer, immunoprecipitated with conformation-specific anti-Bax 6A7 or anti-Bak Ab-1 antibodies and analyzed by Western Blot. Asterisk corresponds to Protein G band.

apeutic drugs.7,29 The present results also demonstrate a key role for proteins of the Bcl-2 superfamily in the commitment phase of BMS-214662-induced apoptosis of B-CLL cells. First, BMS214662 induces a decrease in levels of the antiapoptotic protein Mcl-1. It has been recently shown that Mcl-1 levels regulate cell susceptibility to UV irradiation in HeLa cells30 and that Mcl-1 disappearance might be necessary for the induction of apoptosis in Jurkat cells.31 In B-CLL cells, there seems to be a correlation between Mcl-1 levels and chemoresponse.2,7 Second, BMS214662 induces proapoptotic conformational changes of Bax and Bak proteins. In living cells, native Bax and Bak remain in an inactive conformation but upon the induction of apoptosis, both proteins undergo a conformational change that causes their oligomerization in mitochondrial membrane and the release of cytochrome c and other apoptotic mediators.20 Cytochrome c in cytosol triggers a caspase cascade responsible for cell structure disassembly and the morphological manifestations of apoptosis. Caspases can engage an amplification loop, acting on mitochondria and enhancing DCm loss and cytochrome c release,32 as it seems to be the case in B-CLL cells treated with BMS214662. However, it is unclear as to what are the mechanisms linking inhibition of protein farnesylation by BMS-214662 and

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