CME, BCR/ABL Studies and Myeloproliferative Disorders

Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells

Abstract

Although imatinib mesylate has revolutionized the treatment of chronic myeloid leukaemia (CML), resistance to the drug, manifesting as relapse after an initial response or persistence of disease, remains a therapeutic challenge. In order to overcome this, alternative or additional targeting of signaling pathways downstream of Bcr-Abl may provide the best option for improving clinical response. Bisphosphonates, such as zoledronate, have been shown to inhibit the oncogenicity of Ras, an important downstream effector of Bcr-Abl. In this study, we show that zoledronate is equally effective in inhibiting the proliferation and clonogenicity of both imatinib-sensitive and -resistant CML cells, regardless of their mechanism of resistance. This is achieved by the induction of S-phase cell cycle arrest and apoptosis, through the inhibition of prenylation of Ras and Ras-related proteins by zoledronate. The combination of imatinib and zoledronate also augmented the activity of either drug alone and this occurred in imatinib-resistant CML cells as well. Since zoledronate is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.

Introduction

Chronic myeloid leukaemia (CML) is a malignant haemopoietic stem cell disorder characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the Bcr-Abl oncoprotein.1, 2 Imatinib mesylate (IM), a tyrosine kinase inhibitor that binds to the ATP-binding site of Abl, has been shown to be therapeutically efficient in CML.3, 4, 5, 6 In spite of impressive clinical responses, resistance to IM is common and three mechanisms have been identified: mutations in the Abl kinase domain, overexpression of the Bcr-Abl protein, and upregulation of the P-glycoprotein.7, 8, 9, 10, 11, 12 Therefore, identification of additional targets for molecular therapy among the downstream effectors of Bcr-Abl may provide attractive candidates for treatment of CML.

The Ras pathway plays a key role in Bcr-Abl-mediated leukaemogenesis, and Ras is activated by Bcr-Abl.13, 14 Ras proteins are involved in signal transduction, cell proliferation, and malignant transformation, and their appropriate function is dependent on prenylation, a post-translational modification that anchors the proteins to the plasma cell membrane.15, 16 Prenylation is catalyzed by farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) and when FTase is inhibited by FTase inhibitors, Ras can still be prenylated by GGTase.17, 18

Bisphosphonates (BP) are metabolically stable analogues of pyrophosphate.16 Nitrogen-containing BP inhibit the mevalonate pathway and prevent the prenylation of Ras and other small GTP-binding proteins, through the inhibition of farnesyl diphosphate synthase, thereby reducing levels of the substrates for FTase and GGTase.19 BP can induce apoptosis, inhibit cell cycle progression, and the proliferation in vitro of several types of cancer cells.20, 21, 22, 23, 24, 25, 26, 27 Synergy was also observed when BP were combined with other chemotherapeutic agents.21, 24, 27, 28 Zoledronate (ZOL), a third generation BP, was shown to prolong the survival of immunodeficient mice inoculated with an IM-sensitive human CML cell line both alone and in combination with IM.27

In the present study, we examined whether IM-resistant CML cells exhibit cross-resistance to ZOL, and whether the combination of IM and ZOL offers any advantage for elimination of these cells. We also investigated the effect of ZOL on prenylation, cell cycle and apoptosis in these IM-resistant cells. Our results reveal that resistance to IM is not associated with resistance to ZOL and the combination of both drugs is additive or synergistic, even in IM-resistant cells.

Materials and methods

Cell lines

AR230-s, KCL22-s, AR230-r, and KCL22-r are IM-sensitive and -resistant human CML myeloid cell lines, established as previously described.7 32Dp210-s is an IM-sensitive cell line derived from the transfection of the BCR-ABL cDNA into 32D, a murine myeloid cell line.29 IM resistance was also induced in the 32Dp210 cell line by continuous culture in the presence of gradually increasing doses of IM up to 1 μ M. Ba/F3, a murine lymphoid cell line,30 was transfected with full-length wild-type Bcr-Abl or Bcr-Abl with kinase domain point mutants (Y253F, E255K, T315I, and M351T) generated by site-directed mutagenesis.31 The mechanism of resistance in AR230-r is upregulation of the Bcr-Abl protein associated with amplification of the BCR-ABL gene, while in the IM-resistant 32Dp210, herein referred to as 32Dp210-r, it is a F311V kinase domain mutation.32 The exact mechanism of resistance in KCL22-r is unknown, but it is independent of Bcr-Abl and possibly lies in a down stream effector.7, 33 All cell lines were grown in RPMI 1640 (Gibco, UK) supplemented with streptomycin, penicillin, L-glutamine and 10% fetal calf serum, herein referred to as RF-10. AR230-r, KCL22-r, and 32Dp210-r were maintained in RF-10 containing 1 μ M of IM.

Drugs

IM was prepared as a 1 mM solution in distilled H2O and stored at 4°C ZOL was prepared as a 10 mM solution in distilled H2O and stored at −20°C.

MTS cell proliferation assay

The MTS assay (methylthiazol-tetrazolium, inner salt) (Promega, UK) was used to determine cell proliferation, as previously reported.34 Cells were plated in quadruplicate and exposed to escalating doses of each drug independently. Three independent experiments were performed. The AR230 and KCL22 cell lines were seeded at 2 × 104; Baf/BCR-ABL cell lines at 4 × 103; and 32D and 32Dp210 cell lines at 2 × 103 cells per well. After 72 h, 20 μl of MTS were added into each well and the absorbance at 490 nm was measured on an automatic microplate reader (MRX; Dynatech, UK).

Data were analysed by the median-effect method using the Calcusyn software (Biosoft, UK)35 to determine the doses of ZOL which would inhibit 50% proliferation (IC50 values) and the effect of combining IM and ZOL. In addition to increasing doses of each drug, the cells were exposed to a constant-ratio combination of both drugs. This was set on the basis of their equipotent ratio, that is, at the ratio between their IC50's. The combination index (CI) at 50, 75, and 90% growth inhibition was calculated to determine if the combination was synergistic (CI<0.9), additive (CI 0.9–1.1) or antagonistic (CI>1.1). Duplicate or triplicate experiments were performed.

Cell cycle analysis

Between 1–2 × 106 cells were centrifuged twice in phosphate-buffered saline (PBS) and incubated in ice-cold 50% ethanol for 30 min. The cells were centrifuged again in PBS and stained with propidium iodide (PI) and RNAse (Sigma-Aldrich, UK). After 1 h, the cells were analysed on a Becton Dickinson FACS Calibur (Becton Dickinson, UK). The proportion of cells in the various stages of the cell cycle were determined using the Cylchred software program, version 1.0.2 for Windows 95 (Cardiff University, UK). Three independent experiments were performed.

Apoptosis assay

Assessment of apoptosis was carried out by Annexin V staining as recommended by the manufacturer (Becton Dickinson). After a 72-h incubation with the drugs, the cells were centrifuged twice in cold PBS, resuspended in Annexin V-binding buffer and incubated with 5 μl of fluorescein-conjugated Annexin V and 10 μl of 50 μg/ml PI for 15 min at room temperature. The cells were then analysed on a Becton Dickinson FACS Calibur. Two independent experiments were performed.

Western blot analysis

After 48-h incubation with ZOL, the cells were centrifuged twice in PBS and lysed in 1% Triton X, 20 mM Tris (tris(hydroxymethyl)aminomethane, pH 8.2), 150 mM NaCl, supplemented with 1 mM phenylmethylsulphonyl fluoride and 1 mM sodium metavanadate. Protein concentrations were determined using the colorimetric DC Protein Assay (BIO-RAD, UK). Protein (60 μg) were resolved onto 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and blotted onto polyvinylidene difluoride membranes (Immobilon™-P, Millipore Corporation, UK) by semidry electrophoretic transfer. The membranes were incubated for 1 h in blocking solution containing 5% dried nonfat milk in TBST (25 mM Tris, 138 mM NaCl, 2.7 mM KCl, pH 8.0 and 0.1% Tween 20) and then incubated overnight with mouse monoclonal anti-Ras antibody (Becton Dickinson) or goat polyclonal anti-Rap1A antibody (Santa Cruz Biotechnology, USA). The membranes were washed three times with TBST and incubated for 1 h with the appropriate secondary antibodies. The bands were detected using the enhanced chemiluminescence kits (Amersham Biosciences, UK).

Primary cells and colony-forming assays

Leukapheresis products were obtained from newly diagnosed IM-naïve CML patients and peripheral blood specimens were obtained from IM-resistant patients. Normal samples were obtained from mobilized peripheral blood stem cell or bone marrow harvests from volunteer donors for allogeneic transplant. Informed consent was obtained from all three groups. Mononuclear cells were separated on Lymphoprep (Axis-Shield PoC, Norway) and plated in Iscoves' methylcellulose medium (Methocult H4230, Stemcell Technologies, Canada) at a density of 1 × 105/ml for newly diagnosed CML patients and normal samples; and at a density of 1–5 × 105/ml for IM-resistant patients. The methylcellulose was supplemented with 5 ng/ml recombinant human (rh) interleukin-3, 100 ng/ml rh stem cell factor (Stemcell Technologies), 1 ng/ml rh granulocyte–macrophage colony-stimulating factor and 100 ng/ml rh granulocyte colony-stimulating factor. Plating was done in quadruplicate wells in flat-bottomed 96-well plates with the drugs added at the required concentrations. Duplicate plates were set up for each experiment. After 14 days, the number of colonies was scored and clusters with more than 100 cells were counted as a colony.

Sequencing of the Abl kinase domain

RNA was extracted using the acid guanidinium thiocyanate method,36 and complementary DNA was synthesized using random hexameric primers. The Abl kinase domain was amplified using a two-step hemi-nested reverse transcriptase/polymerase chain reaction (RT/PCR). In the first step, the forward primer, B2A+ (IndexTerm5′-TTCAGAAGCTTCTCCCTGACAT-3′) anneals onto the exon 13 of the Bcr gene and the reverse primer, NTPE (IndexTerm5′-CTTCGTCTGAGATACTGGATTCCT-3′) anneals onto the exon 9 of the Abl gene, at the end of the tyrosine kinase domain. For the second step, the NTPB+ forward primer (IndexTerm5′-AAGCGCAACAAGCCCACTGTCTAT-3′), anneals onto exon 4 of the ABL gene, at the beginning of the kinase domain, while the reverse primer was the same as for the first step. The PCR products were purified with the QIAquick gel purification kit (Qiagen, UK) and subjected to direct sequencing on a 3700 DNA Analyser (Applied Biosystems, USA).

Statistical analysis

SPSS for Windows version 11.0 statistics package (SPSS Inc, USA) was utilized to analyse differences by the nonparametric two-tailed Mann–Whitney test. Statistical significance was considered as P0.05 in two-tailed analyses.

Results

ZOL exerts a strong antiproliferative effect in IM-sensitive and IM-resistant cell lines

The IC50 for IM and ZOL were determined by the MTS assay and the results are summarized on Table 1. In the Bcr-Abl-overexpressing cell line AR230-r, the IC50 for IM was 18-fold higher than in its sensitive counterpart. In contrast, the IC50 for ZOL was only 1.2-fold higher in AR230-r compared to AR230-s, and this difference was not significant (P>0.05). The difference in IC50 for IM was also significantly different in the KCL22 pair (P=0.05), but not for ZOL (P>0.05). In 32Dp210-r, the IC50 for IM was 14-fold higher than in 32Dp210-s, but the antiproliferative effect of ZOL was similar between the pair. It is also of interest to note that in the non-Bcr-Abl expressing 32D cell line, the IC50 for ZOL was two-fold higher than in the Bcr-Abl expressing 32Dp210-s (P=0.05). This would suggest that ZOL is less toxic to nonleukaemic cell lines.

Table 1 IC50 values of imatinib and zoledronate in human and murine CML cell lines

The Baf/BCR-ABL mutant cell lines studied here contain the most common mutations detected in patients who develop resistance to IM. Baf/BCR-ABLY253F and Baf/BCR-ABLE255K have mutations in the ATP binding site of the kinase domain. Baf/BCR-ABLT315I directly impairs IM binding without affecting the binding of ATP and is highly resistant to IM. Baf/BCR-ABLM351T has a mutation in the catalytic domain and retains some sensitivity to IM although requiring a five-fold higher IC50 than the wild-type Baf/BCR-ABL. In contrast, the antiproliferative effect of ZOL in the mutant cell lines was comparable to or stronger than in the wild-type cell line, and this is reflected by similar or lower IC50's in the former (Table 1). Taken together, the results suggest that cell lines resistant to IM by different mechanisms do not exhibit cross-resistance to ZOL.

Combination of IM and ZOL is largely synergistic in both IM-sensitive and -resistant cell lines

Combination studies were next performed and the results of the CI at 50, 75 and 90% growth inhibition are summarized on Table 2. In general, synergy between both drugs, as defined by a CI<0.9,35 was seen in nearly all the paired sensitive and resistant cell lines at high effect levels. An exception was the AR230 pair, where slight antagonism was observed at the IC50, although the combination was nearly additive or synergistic at the IC75 and IC90. Combination studies were not performed in the Baf/BCR-ABLT315I as IM was not active in this cell line at doses below 9 μ M, a dose, which is toxic even to BCR-ABL-negative cell lines.

Table 2 Mean combination index (CI) values at 50, 75, and 90% growth inhibition (IC50, IC75, and IC90)

ZOL effectively inhibits prenylation in IM-sensitive and -resistant cell lines

In order to confirm that ZOL's antiproliferative effect on the BCR-ABL-positive cell lines was associated with its capacity to inhibit post-translational modification of small GTP-binding proteins, we analysed the prenylation status of Ras and Rap1A. Inhibition of Ras prenylation can be detected by the reduced mobility of its unprenylated form in SDS-PAGE. In contrast to Ras, which can be prenylated by both FTase and GGTase, Rap1A is a protein prenylated exclusively by GGTase I, and its unprenylated form is recognized by a specific polyclonal antibody. AR230, KCL22 and Baf/BCR-ABL were treated with 20–120 μ M of ZOL for 48 h. The unprenylated forms of Ras and Rap 1A were detected at doses as low as 20 μ M in the AR230 cell lines and 40 μ M in the KCL22 and Baf/BCR-ABL cell lines (Figure 1).

Figure 1
figure1

Western blot showing the inhibitory effect on prenylation of small GTP proteins. Protein lysates were prepared from the paired IM-sensitive and -resistant AR230, KCL22 and Baf/BCR-ABL cell lines after 48 h of treatment with ZOL.

ZOL induces S-phase arrest and apoptosis in IM-sensitive and -resistant cell lines

With the objective of clarifying the basis of the antileukaemic action of ZOL on the CML-derived and -related cell lines, we investigated its effect on the cell cycle and its capacity to induce apoptosis when combined with IM. An increase in the fraction of cells arrested in S-phase was observed in both the AR230 and KCL22 paired cell lines treated with 100 μ M ZOL between 24 and 72 h. This increase corresponded to a decrease in G1/G0 phase, and there was a small increase in the sub-G1/G0 fraction, which represents an apoptotic population (Figure 2). These results suggested that ZOL induced S-phase arrest and possibly apoptosis as well. More stringent analysis of apoptosis as defined by Annexin-V positivity by flow cytometry confirmed that ZOL did indeed exert this function. After 72 h of treatment with 100 μ M ZOL alone, apoptosis was induced in 38.5% of KCL22-s and 29.2% of KCL22-r. The addition of 1 μ M IM enhanced the level of apoptosis to 88.6% in the KCL22-s. IM at doses of 1 and 2 μ M did not induce apoptosis in the KCL22-r, but when combined with ZOL, the levels of apoptosis were increased to 36.7 and 47.2%, respectively (Figure 3a and b). In AR230, 50 μ M ZOL-induced apoptosis in 40.9% of the sensitive and 37.6% of the resistant cells. The level of apoptosis was also enhanced when IM and ZOL were combined in both AR230-s and AR230-r (90.0 and 77.4%, respectively) (Figure 3c and d). The induction of apoptosis was time- and dose-dependent (data not shown).

Figure 2
figure2

Cell cycle analysis after treatment with ZOL. Cell cycles of (a) AR230-s, (b) AR230-r, (c) KCL22-s, and (d) KCL22-r were analysed at 24, 48, and 72 h after treatment with ZOL at indicated concentrations. The histograms shown are representative of three independent experiments.

Figure 3
figure3

Effect of IM and ZOL as single agents or in combination on inducing apoptosis in the KCL22 and AR230 cell lines at 72 h of treatment. The white columns represent the early apoptotic population (Annexin V positive/PI negative). The black columns represent the late apoptotic/necrotic population (Annexin V positive/PI positive). The figures shown are representative of two independent experiments. IM 1: IM 1 μ M, ZOL 100: ZOL 100 μ M.

ZOL inhibits clonogenicity of IM-resistant primary CML cells

Having shown the antiproliferative and proapoptotic effects of ZOL in BCR-ABL-positive cell lines resistant to IM, we next evaluated its performance on primary progenitor cells from IM-naïve and -resistant CML patients and normal individuals. In samples from newly diagnosed IM-naïve CML patients, treatment with 10, 20, and 40 μ M of ZOL reduced CFU-GM colony formation to 72.0, 50.9, and 30.9% of control values, respectively. Treatment with 0.5 μ M IM confirmed that these CML samples were sensitive to IM in vitro. Using the same doses of ZOL in normal progenitors, a differential effect in CFU-GM colony reduction was seen when compared to the CML samples and this was significant at 20 and 40 μ M (P<0.05) (Figure 4a). These results suggest that ZOL was less toxic to normal progenitors.

Figure 4
figure4

Effect of 0.5 μ M IM and increasing doses of ZOL on formation of CFU-GM colonies at D14 (a) normal (n=6) and newly diagnosed IM-naïve CML patients in chronic phase (n=5), (b) IM-naïve patients (n=5), patients resistant to IM due to unknown mechanism (n=3) and patients resistant to IM due to a kinase mutation (n=3). Vertical bars show 1 s.d. (*P0.05).

The efficacy of ZOL was next tested in six samples from CML patients who had never attained a cytogenetic response or who had lost the response while on IM treatment. Sequencing of the Abl kinase domain revealed that of the six patients, one had a Y253F, one a H396R, and one a G250E mutation. Additional chromosomal abnormalities were also present in three patients. The patient with the Y253F mutation had a double Philadelphia chromosome and the patient with the H396R mutation had an isochromosome 17. The remaining patient had an interstitial deletion in chromosome 18. Whereas, as expected, 0.5 μ M IM produced a significant differential effect in inhibiting CFU-GM colony formation between IM-naïve and -resistant samples, ZOL treatment yielded only a small difference between the two groups. When compared to the IM-naïve group, there was a trend towards increased colony formation in IM-resistant samples, which did not have a mutation, and a reduction in samples, which had mutation (Figure 4b). Although these differences were not significant (P>0.05) probably due to the small number of samples analysed, the increased sensitivity of the mutation-harbouring primary cells to ZOL was consistent with the proliferation assay results which also showed an increased sensitivity to ZOL among the Baf/BCR-ABL mutant cell lines.

CFU-GM assays were also performed in the presence of both IM and ZOL. In IM-naïve samples, the combination of 0.5 μ M IM and 20 μ M ZOL reduced CFU-GM colony formation to 13.2% of the control, and the combination was significantly more effective than IM or ZOL alone (31.6 and 50.1%, respectively; P=0.05) (Figure 5a). The enhancement of combination therapy was also observed in samples from IM-resistant patients in whom the mechanism of resistance was not known (Figure 5c): at the same doses, IM and ZOL reduced CFU-GM colony numbers to 24.2% of the control and this was significantly better than the effect of IM (84.0%; P=0.05) or ZOL alone (70.0%; P=0.05). In samples, which harboured a mutation, combination therapy was marginally more effective than single agent therapy but the difference was not significant (Figure 5d). Similar to earlier results that ZOL or IM alone were less toxic to normal haemopoietic progenitors, the toxicity was not increased when both drugs were combined (Figure 5b).

Figure 5
figure5

Effect of combination therapy with IM and ZOL on formation of day-14 CFU-GM colonies (a) newly diagnosed CML patients in chronic phase (n=4), (b) normal (n=3), (c) patients resistant to IM by unknown mechanisms (n=3), (d) patients resistant to IM due to kinase domain mutations (n=3). The black columns represent IM or ZOL treatment alone. The white columns represent combined treatment with IM 0.5 μ M and ZOL. Vertical bars show 1 s.d. (*P0.05).

Discussion

Although the introduction of IM has represented a major achievement for the therapy of CML, one-quarter of the patients treated upfront with this inhibitor fail to achieve complete cytogenetic response,3 and the majority persists with molecular evidence of residual disease.37 In addition to this important component of primary resistance, relapse of the leukaemia after an initially successful response is a major problem.4, 5, 6 It is clear, therefore, that monotherapy with IM may not be the best option in CML, and that addition of drugs, which can inhibit alternative signal transduction pathways and synergize with IM may enhance the effect of the targeted therapy.

In this study, we evaluated ZOL as a potential agent for overcoming IM resistance in patients with CML. ZOL is an attractive candidate as it is already available for clinical use in the treatment of hypercalcemia of malignancy, bone metastases and multiple myeloma.38, 39 It has also been shown to be active in two IM-sensitive CML cell lines, BV173 and K562, and to synergize with IM in inhibiting proliferation in vitro and prolonging the survival of mice xenotransplanted with BV173.27 The effect of ZOL on cells resistant to IM, a most relevant issue, had not yet been investigated.

The cell lines, we selected for demonstration of the biological effects of ZOL model the most common mechanisms of resistance reported in patients who have become refractory to IM, namely mutations in the Abl kinase domain, Bcr-Abl overexpression and defects on Bcr-Abl downstream effectors. In our study, none of the resistant cell lines exhibited cross resistance to ZOL, regardless of their mechanism of resistance. In fact, the antiproliferative effect of ZOL seemed to be stronger in some of the mutant cell lines. The exact reason for this is unclear but we speculate that certain mutations of the Abl kinase domain may alter the oncogenic potency of Bcr-Abl itself and consequently, its downstream effectors (Griswold IJ et al. Blood 2004; 104: 161, abstract; Gorre M et al. Blood 2004; 104: 161, abstract). A significant finding in this study, in agreement with previous reports,27, 40 was that ZOL was less toxic to normal haemopoietic cells, as shown by proliferation and clonogenecity assays. Nitrogen-containing BP alter the function of Ras by inhibiting its prenylation through the abrogation of FTase and GGTase, essential enzymes in the mevalonate pathway.19 We have, in fact, observed that ZOL blocked the prenylation of Ras, as well as Rap 1A, a substrate exclusively prenylated by GGTase, in a dose-dependent manner, and that this phenomenon occurred in both IM-sensitive and -resistant CML cell lines. While the unprenylation of Ras was not complete in two cell lines that were studied, we hypothesize that partial inhibition of prenylation may be sufficient to induce synergistic antiproliferative and apoptotic effects when combined with IM. It is important to note that the dual action of ZOL on FTase and GGTase may render it a more potent antileukaemic drug than FTase inhibitors.

A consequence of Ras inhibition is alteration of the cell cycle,41, 42 and BP are reported to induce S-phase arrest in tumour cells.20, 22, 26 An increase in the proportion of cells in the S-phase was also seen in our study in both IM-sensitive and -resistant cells, in a time-dependent manner. In addition to the increase in S-phase, there was also a detectable rise in the apoptotic sub G1/G0 cells. IM, on the other hand, caused a G1 arrest with a decrease in the proportion of cells in S-phase (data not shown), a phenomenon also previously observed by our group.43

Induction of apoptosis by BP in tumour cells has been described in several malignancies.20, 22, 23, 24, 25 Our data further supports this notion by showing that at doses required to inhibit 50% of the cell proliferation, ZOL was able to induce apoptosis in 30–40% of the cells in both IM-sensitive and -resistant cells. A noteworthy finding was that the addition of IM further increased the degree of apoptosis, and this was most evident in KCL22-r. In this cell line, the combination of IM and ZOL resulted in a five-fold and 1.5-fold increase compared to IM or ZOL monotherapy, respectively.

In our cell proliferation assays, the combination of IM and ZOL was shown to be additive or synergistic in most of the cell lines tested. Since each drug acts at a different level in the Bcr-Abl signalling pathway, it is not surprising that both drugs when combined displayed additivity or synergism in IM-sensitive cells. What is intriguing, however, is that the IM-resistant cells also respond in a similar way to the combination of the two drugs. This is not an unusual occurrence, as it has also been observed for other drugs in IM-resistant cells.34, 44, 45 A possible reason for this phenomenon is that at low doses that do not induce apoptosis but are suffcient to reduce Bcr-Abl kinase activation, IM is able to sensitize the resistant cells to the antiproliferative or cytotoxic effect of the second drug. An alternative explanation is that these resistant cells still retain some quantitative sensitivity to IM,46 and synergism with a second drug can only be achieved when combined with a dose of IM high enough to overcome resistance.44 Both mechanisms are evident in this study. In KCL22-r (resistant due to defects downstream of Bcr-Abl), when low doses of IM insuffcient per se to induce apoptosis were combined with ZOL, the killing effect was increased compared to ZOL alone. On the other hand, in AR230-r (resistant due to Bcr-Abl overexpression), a higher dose of IM was required to increase the level of apoptosis in combination with ZOL. It is likely that this increase in apoptosis, while modest, may be one of the mechanisms accounting for the synergism arising from combination therapy seen in the cell proliferation assays. Another possible mechanism contributing to the synergism is the effect on different phases of the cell cycle of these two drugs, with IM inducing cell cycle arrest at the G1-phase and ZOL at the S-phase. A similar situation was observed in the clonogenicity of primary leukaemic cells, where combination of IM with ZOL was also superior to single agent therapy for inhibition of CFU-GM colony formation, and this was especially evident in the IM-sensitive and IM-resistant CML cells for which the mechanism of resistance was not known. Regardless of the mechanism leading to synergism, the combination of IM and ZOL is certainly encouraging and promising.

A possible caveat to the use of ZOL is that the maximal plasma levels range from 0.5 to 5 μ M depending on the dosage and duration of infusion.47, 48 This is 10–100-fold less than the levels required to inhibit cell growth or induce apoptosis in vitro. There are no data available, as yet, on the bioavailability of ZOL in bone marrow cells. However, BP accumulate rapidly in the bone and high levels of between 100 and 1000 μ M have been estimated for alendronate at the osteoclastic resorption site,49 and clinical pharmacological studies suggest that ZOL is bound to bone for prolonged periods.39 It is this avidity for the bone and possibly bone marrow, which makes BP ideal candidates for the treatment of bone marrow disorders, such as CML. Indirect evidence already exists which shows the utility of BP in multiple myeloma and CML. ZOL has been shown to delay the appearance of leukaemic cells in mice transplanted with BV173, and to improve survival.27 ZOL was also shown to reduce tumour-induced osteolysis and skeletal tumour burden, as well as to prolong survival in a 5T2 murine model of myeloma.50 The preferential concentration of BP in the bone may itself be a double-edged sword as it may allow the proliferation of leukaemic cells in extramedullary sites. While this may preclude the use of ZOL as monotherapy in CML, combination with IM will definitely be an attractive option in newly diagnosed CML patients and in patients who are resistant to IM-monotherapy. This is currently being tested in our centre in an ongoing phase I–II clinical trial with this combination in CML patients who failed to achieve a complete remission with IM alone.

References

  1. 1

    Goldman JM, Melo JV . Chronic myeloid leukemia – advances in biology and new approaches to treatment. N Engl J Med 2003; 349: 1451–1464.

    CAS  Article  Google Scholar 

  2. 2

    Sawyers CL . Chronic myeloid leukemia. N Engl J Med 1999; 340: 1330–1340.

    CAS  Article  Google Scholar 

  3. 3

    O'Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004.

    CAS  Article  Google Scholar 

  4. 4

    Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002; 99: 3530–3539.

    CAS  Article  Google Scholar 

  5. 5

    Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, Guilhot F et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99: 1928–1937.

    CAS  Article  Google Scholar 

  6. 6

    Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645–652.

    CAS  Article  Google Scholar 

  7. 7

    Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM et al. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000; 96: 1070–1079.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001; 293: 876–880.

    CAS  Article  Google Scholar 

  9. 9

    von Bubnoff N, Schneller F, Peschel C, Duyster J . BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet 2002; 359: 487–491.

    CAS  Article  Google Scholar 

  10. 10

    Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002; 99: 3472–3475.

    CAS  Article  Google Scholar 

  11. 11

    Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002; 2: 117.

    CAS  Article  Google Scholar 

  12. 12

    Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc Natl Acad Sci USA 2002; 99: 10700–10705.

    CAS  Article  Google Scholar 

  13. 13

    Puil L, Liu J, Gish G, Mbamalu G, Bowtell D, Pelicci PG et al. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway. EMBO J 1994; 13: 764–773.

    CAS  Article  Google Scholar 

  14. 14

    Druker B, Okuda K, Matulonis U, Salgia R, Roberts T, Griffin JD . Tyrosine phosphorylation of rasGAP and associated proteins in chronic myelogenous leukemia cell lines. Blood 1992; 79: 2215–2220.

    CAS  PubMed  Google Scholar 

  15. 15

    Rebollo A, Martinez A . Ras proteins: recent advances and new functions. Blood 1999; 94: 2971–2980.

    CAS  PubMed  Google Scholar 

  16. 16

    Rogers MJ, Gordon S, Benford HL, Coxon FP, Luckman SP, Monkkonen J et al. Cellular and molecular mechanisms of action of bisphosphonates. Cancer 2000; 88 (12 Suppl): 2961–2978.

    CAS  Article  Google Scholar 

  17. 17

    Lerner EC, Zhang TT, Knowles DB, Qian Y, Hamilton AD, Sebti SM . Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines. Oncogene 1997; 15: 1283–1288.

    CAS  Article  Google Scholar 

  18. 18

    Whyte DB, Kirschmeier P, Hockenberry TN, Nunez-Oliva I, James L, Catino JJ et al. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 1997; 272: 14459–14464.

    CAS  Article  Google Scholar 

  19. 19

    Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ . Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 1998; 13: 581–589.

    CAS  Article  Google Scholar 

  20. 20

    Shipman CM, Rogers MJ, Apperley JF, Russell RG, Croucher PI . Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour activity. Br J Haematol 1997; 98: 665–672.

    CAS  Article  Google Scholar 

  21. 21

    Tassone P, Forciniti S, Galea E, Morrone G, Turco MC, Martinelli V et al. Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cell lines. Leukemia 2000; 14: 841–844.

    CAS  Article  Google Scholar 

  22. 22

    Iguchi T, Miyakawa Y, Yamamoto K, Kizaki M, Ikeda Y . Nitrogen-containing bisphosphonates induce S-phase cell cycle arrest and apoptosis of myeloma cells by activating MAPK pathway and inhibiting mevalonate pathway. Cell Signal 2003; 15: 719–727.

    CAS  Article  Google Scholar 

  23. 23

    Hiraga T, Williams PJ, Mundy GR, Yoneda T . The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases. Cancer Res 2001; 61: 4418–4424.

    CAS  Google Scholar 

  24. 24

    Jagdev SP, Coleman RE, Shipman CM, Rostami H, Croucher PI . The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer 2001; 84: 1126–1134.

    CAS  Article  Google Scholar 

  25. 25

    Tassone P, Tagliaferri P, Viscomi C, Palmieri C, Caraglia M, D'Alessandro A et al. Zoledronic acid induces antiproliferative and apoptotic effects in human pancreatic cancer cells in vitro. Br J Cancer 2003; 88: 1971–1978.

    CAS  Article  Google Scholar 

  26. 26

    Lee MV, Fong EM, Singer FR, Guenette RS . Bisphosphonate treatment inhibits the growth of prostate cancer cells. Cancer Res 2001; 61: 2602–2608.

    CAS  Google Scholar 

  27. 27

    Kuroda J, Kimura S, Segawa H, Kobayashi Y, Yoshikawa T, Urasaki Y et al. The third-generation bisphosphonate zoledronate synergistically augments the anti-Ph+ leukemia activity of imatinib mesylate. Blood 2003; 102: 2229–2235.

    CAS  Article  Google Scholar 

  28. 28

    Caraglia M, D'Alessandro AM, Marra M, Giuberti G, Vitale G, Viscomi C et al. The farnesyl transferase inhibitor R115777 (Zarnestra) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa) and Pamidronate. Oncogene 2004; 23: 6900–6913.

    CAS  Article  Google Scholar 

  29. 29

    Laneuville P, Heisterkamp N, Groffen J . Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line. Oncogene 1991; 6: 275–282.

    CAS  PubMed  Google Scholar 

  30. 30

    Daley GQ, Van Etten RA, Baltimore D . Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 1990; 247: 824–830.

    CAS  Article  Google Scholar 

  31. 31

    La Rosee P, Corbin AS, Stoffregen EP, Deininger MW, Druker BJ . Activity of the Bcr-Abl kinase inhibitor PD180970 against clinically relevant Bcr-Abl isoforms that cause resistance to imatinib mesylate (Gleevec, STI571). Cancer Res 2002; 62: 7149–7153.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Barnes DJ, Palaiologou D, Panousopoulou E, Schultheis B, Wong A, Pattacini L et al. Bcr-Abl expression levels determine the rate of development of resistance to imatinib mesylate. Cancer Res 2005, in press.

  33. 33

    Tipping AJ, Deininger MW, Goldman JM, Melo JV . Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate. Exp Hematol 2003; 31: 1073–1080.

    CAS  Article  Google Scholar 

  34. 34

    Tipping AJ, Mahon FX, Zafirides G, Lagarde V, Goldman JM, Melo JV . Drug responses of imatinib mesylate-resistant cells: synergism of imatinib with other chemotherapeutic drugs. Leukemia 2002; 16: 2349–2357.

    CAS  Article  Google Scholar 

  35. 35

    Chou TC, Talalay P . Quantitative analysis of dose–effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984; 22: 27–55.

    CAS  Article  Google Scholar 

  36. 36

    Chomczynski P, Sacchi N . Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 1987; 162: 156–159.

    CAS  Article  Google Scholar 

  37. 37

    Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423–1432.

    CAS  Article  Google Scholar 

  38. 38

    Major P . The use of zoledronic acid, a novel, highly potent bisphosphonate, for the treatment of hypercalcemia of malignancy. Oncologist 2002; 7: 481–491.

    CAS  Article  Google Scholar 

  39. 39

    Ibrahim A, Scher N, Williams G, Sridhara R, Li N, Chen G et al. Approval summary for zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin Cancer Res 2003; 9: 2394–2399.

    CAS  PubMed  Google Scholar 

  40. 40

    Senaratne SG, Pirianov G, Mansi JL, Arnett TR, Colston KW . Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer 2000; 82: 1459–1468.

    CAS  Article  Google Scholar 

  41. 41

    Kerkhoff E, Rapp UR . Cell cycle targets of Ras/Raf signalling. Oncogene 1998; 17 (11 Reviews): 1457–1462.

    CAS  Article  Google Scholar 

  42. 42

    Pruitt K, Der CJ . Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Lett 2001; 171: 1–10.

    CAS  Article  Google Scholar 

  43. 43

    Deininger MW, Vieira SA, Parada Y, Banerji L, Lam EW, Peters G et al. Direct relation between BCR-ABL tyrosine kinase activity and cyclin D2 expression in lymphoblasts. Cancer Res 2001; 61: 8005–8013.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    La Rosee P, Johnson K, Corbin AS, Stoffregen EP, Moseson EM, Willis S et al. In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines. Blood 2004; 103: 208–215.

    CAS  Article  Google Scholar 

  45. 45

    Hoover RR, Mahon FX, Melo JV, Daley GQ . Overcoming STI571 resistance with the farnesyl transferase inhibitor SCH66336. Blood 2002; 100: 1068–1071.

    CAS  Article  Google Scholar 

  46. 46

    Corbin AS, La Rosee P, Stoffregen EP, Druker BJ, Deininger MW . Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood 2003; 101: 4611–4614.

    CAS  Article  Google Scholar 

  47. 47

    Chen T, Berenson J, Vescio R, Swift R, Gilchick A, Goodin S et al. Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases. J Clin Pharmacol 2002; 42: 1228–1236.

    CAS  Article  Google Scholar 

  48. 48

    Skerjanec A, Berenson J, Hsu C, Major P, Miller Jr WH, Ravera C et al. The pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with varying degrees of renal function. J Clin Pharmacol 2003; 43: 154–162.

    CAS  Article  Google Scholar 

  49. 49

    Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD et al. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 1991; 88: 2095–2105.

    CAS  Article  Google Scholar 

  50. 50

    Croucher PI, De Hendrik R, Perry MJ, Hijzen A, Shipman CM, Lippitt J et al. Zoledronic acid treatment of 5T2MM-bearing mice inhibits the development of myeloma bone disease: evidence for decreased osteolysis, tumor burden and angiogenesis, and increased survival. J Bone Miner Res 2003; 18: 482–492.

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to Dr Elisabeth Buchdunger and Dr Jonathan Green (Novartis, Switzerland) for their kind gift of IM and ZOL, respectively, and to Dr Alex Tipping, Ms Christalla Dimitriadis and Ms Kara Johnson for their advice and technical assistance. This work was supported by the Leukaemia Research Fund, United Kingdom; Singapore General Hospital Medical Fellowship and National Medical Research Council-Totalisator Board Medical Research Fellowship, Singapore.

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Correspondence to J V Melo.

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Chuah, C., Barnes, D., Kwok, M. et al. Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells. Leukemia 19, 1896–1904 (2005). https://doi.org/10.1038/sj.leu.2403949

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Keywords

  • chronic myeloid leukaemia
  • imatinib mesylate
  • zoledronic acid

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