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Keywords:

  • CML;
  • Bcr-Abl;
  • T315I mutant;
  • NF kappaB

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The Bcr-Abl inhibitor imatinib is the current first-line therapy for all newly diagnosed chronic myeloid leukemia (CML). Nevertheless, resistance to imatinib emerges as CML progresses to an acute deadly phase implying that physiopathologically relevant cellular targets should be validated to develop alternative therapeutic strategies. The NF-κB transcription factor that exerts pro-survival actions is found abnormally active in numerous hematologic malignancies. In the present study, using Bcr-Abl-transfected BaF murine cells, LAMA84 human CML cell line and primary CML, we show that NF-κB is active downstream of Bcr-Abl. Pharmacological blockade of NF-κB by the IKK2 inhibitor AS602868 prevented survival of BaF cells expressing either wild-type, M351T or T315I imatinib-resistant mutant forms of Bcr-Abl both in vitro and in vivo using a mouse xenograft model. AS602868 also affected the survival of LAMA84 cells and of an imatinib-resistant variant. Importantly, the IKK2 inhibitor strongly decreased in vitro survival and ability to form hematopoietic colonies of primary imatinib resistant CML cells including T315I cells. Our data strongly support the targeting of NF-κB as a promising new therapeutic opportunity for the treatment of imatinib resistant CML patients in particular in the case of T315I patients. The T315I mutation escapes all currently used Bcr-Abl inhibitors and is likely to become a major clinical problem as it is associated with a poor clinical outcome. © 2009 UICC

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) chromosomal translocation, resulting in expression of the fusion oncoprotein Bcr-Abl with constitutive tyrosine kinase activity.1, 2 Bcr-Abl triggers several cellular signaling pathways, such as the mitogen activated protein kinase (MAPK) cascade, signal transducers and activators of transcription (STAT), PI3K/Akt and NF-κB.3 The deregulated kinase activity is responsible for the transforming and leukemogenic properties of Bcr-Abl.4, 5 Targeting Bcr-Abl has therefore been an attractive therapeutic strategy in CML. Imatinib (Sti571/Gleevec®) was isolated as a small molecule inhibitor of the Abl tyrosine kinase that specifically inhibited proliferation of Bcr-Abl expressing cell lines,6 growth of Bcr-Abl tumors in mouse xenograft models7 and in vitro colony formation from CML primary cells. Imatinib induced a complete cytogenetic response (CCR) in 80% of newly diagnosed chronic phase (CP) patients and is now the first-line therapy for CML.8 Unfortunately, the efficiency of imatinib decreases in both magnitude and duration in more advanced stages of the disease, dropping to only 10% in the acute phase of the leukemia.9, 10 Clinical resistance to imatinib is primarily mediated by mutations within the kinase domain of Bcr-Abl, to a lesser extent by amplification of the BCR-ABL genomic locus and by other yet unknown mechanisms. Mutations within the kinase domain of Bcr-Abl not only interfere with drug binding but can also lock the kinase in a highly active state. In particular, the T315I mutation, also called the “hell mutation” induces resistance to imatinib and to second generation of Bcr-Abl inhibitors (dasatinib, nilotinib). The threonine 315 residue is located in the kinase domain and acts as a gatekeeper. Substitution by the larger and more hydrophobic isoleucine interferes with imatinib but not ATP binding.11 Approximately 75% of patients in CP achieve a CCR upon imatinib treatment but remain positive for bcr-abl transcripts by RT-PCR (molecular persistence)12 and imatinib appears unable to eradicate leukemic progenitors.13 To improve response rates and to circumvent resistance additional drugs are needed and mathematical modeling suggests that combination of at least 3–4 drugs will be required to eliminate CML cells.14

NF-κB transcription factors play a central role as mediators of immune and inflammatory responses as well as in control of cell proliferation and apoptosis.15 In physiological situations, NF-κB is constitutively present in a latent, inactive form in the cytosol, retained through interaction with its specific inhibitor IκB. Classical activation of NF-κB involves phosphorylation and degradation of IκB, followed by nuclear translocation of NF-κB and subsequent activation of NF-κB target genes. IκB phosphorylation is orchestrated by a multi-subunit kinase complex composed of 2 catalytic subunits, IKK1/α and IKK2/β, and the scaffold essential protein NEMO/IKKγ.16 As NF-κB controls transcription of genes that are central to cell growth and survival, dysregulation of the pathway often leads to aberrant gene expression patterns supporting oncogenesis. Indeed, abnormal constitutive NF-κB activation has been detected in several types of solid tumors17 as well as in hematopoietic malignancies, such as Hodgkin's disease,18 acute lymphoblastic leukemia (ALL),19 acute myeloid leukemia (AML)20 and CML.21 Strategies to inhibit the survival events regulated by NF-κB could represent a very interesting approach for innovative cancer therapies. Several natural as well as synthetic compounds have been described to block NF-κB (aspirin, thalidomide, PS341, BMS-345541, parthenolide, curcumin). We previously showed that a small molecule inhibitor of the IKK2 kinase (AS602868) could reveal the apoptotic potential of TNF in Jurkat leukemic cells22 and induced apoptosis of human AML cells.23 We have evaluated the potential of this IKK2 inhibitor in a preclinical study in CML. We show that NF-κB exerts survival signals downstream of Bcr-Abl, and that pharmacological targeting of NF-κB can affect CML cell viability and bypass imatinib resistance, even in the context of the T315I mutation.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients

Heparin-treated blood samples were obtained from patients with CML (14) or Ph+ ALL (1), 12 of which were resistant to imatinib therapy. Informed consent was obtained according to institutional guidelines. Clinical characteristics of patients are depicted in Supporting Information Table 1. Peripheral blood cells from patients were isolated within 24 hr after collection by Ficoll-Paque-Plus density gradient (Amersham Biosciences, Buckinghamshire, UK).

Cell lines

The BaF3 cell line is a murine IL-3 dependent preB cell line. Expression of Bcr-Abl in these cells abrogates their IL-3 dependence. Stable BaF cell lines expressing full length wt Bcr-Abl or Bcr-Abl with the T315I and M351T kinase domain point mutations have been previously described.24 The LAMA84 cell line was isolated from the peripheral blood of a 29-year-old woman with CML in blast crisis. It is sensitive to sub-micromolar concentrations of imatinib and thus designated LAMA84-s. The LAMA84-r variant is resistant to 0.6 μM imatinib and was previously described.25 Cell lines were maintained in RPMI 1640 growth media supplemented with 200 μM L-glutamine, 10% FCS, penicillin (200 U/ml) and streptomycin (200 mg/ml) (Invitrogen, Cergy-Pontoise, France).

Drugs and reagents

AS602868 is an anilinopyrimidine derivative and adenosine triphosphate (ATP) competitor, which inhibits IKK2 (patent application PCTWO 02/46171 and Ref.23).

The IL-3, IL-6, stem cell factor (SCF), erythropoietin, GM-CSF and G-CSF were purchased from Peprotech (London, UK). Antibodies used in the study were from BD Biosciences (anti-procaspase 3), MBL (Woburn, MA) (anti-procaspase 8 and 9), Cell Signaling Technology (Beverly, MA) (anti-PARP, anti-PhosphoIKK2, anti-PhosphoBcr, anti-Bcr) and Santa Cruz Biotechnology (Santa Cruz, CA) (anti-hsp60).

Measurement of cell viability

Cell viability was measured using a colorimetric assay (Cell Proliferation Kit II XTT, Roche Applied Biosciences, Meylan, France). Cells were plated in quintuplets at 2 × 104 cells/well and incubated for 3 (cell lines) or 4 days (primary cells) with effectors. Fifty microliters of the XTT labeling mixture was then added to each well and plates were placed at 37°C in a humidified atmosphere. The absorbance was measured at 450 nm on MRX II ELISA reader (Dynex Technologies, Berlin, Germany).

Methylcellulose colony assays of human primary cells

Peripheral blood cells from patients were plated in methylcellulose at 2 × 105 cells/ml (H4230, Stem Cell Technologies, Grenoble, France) containing growth factors (IL-3, IL-6, SCF, Erythropoietin, GM-CSF, G-CSF) in presence of effectors. After 10 days, colonies were counted on triplicate wells on an inverted microscope (Leica Microsystems, Rueil-Malmaison, France).

Western blotting

Whole cell lysates were prepared as previously described.26 Proteins were separated by SDS/PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA) in Tris (20 mM), glycine (150 mM), ethanol (20%) at 500 mA for 4 hr at 4°C. Antibodies were incubated in saturation buffer (50 mM Tris pH 7.5, 50 mM NaCl, 0.15% Tween, 5% BSA) and revealed with a secondary-peroxidase-conjugated antibody followed by ECL detection (Amersham Pharmacia, Saclay, France).

Measurement of apoptosis by Annexin V analysis

After stimulation, cells (0.5 × 106/ml) were harvested by centrifugation and washed once with PBS. Cells were then centrifuged 5 min at 1,200 rpm and resuspended in 400 μL of commercial binding buffer containing Annexin V-FITC and propidium iodide (Roche Applied Biosciences) for 15 min in the dark. Apoptosis was analyzed on the FL-1 channel of a FACScan (Becton Dickinson, Cowley, UK). Apoptosis was quantified as the percentage of Annexin V positive cells.

DNA fragmentation

After stimulation, cells (106/ml) were collected and resuspended in 200 μl of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA and 0.2% Triton X-100), incubated for 10 min at room temperature. Lysates were treated for 30 min at 37°C with 100 μg/ml RNase A and then for 30 min with 100 μg/ml proteinase K. Cellular DNA was ethanol-precipitated, dried and resuspended in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH 7.5). DNA was analysed by electrophoresis on 1.2% agarose gel after staining with ethidium bromide.

NF-κB activation assay

The activity of p65 NF-κB was examined in nuclear extracts by a TransAM kit (Active Motif, Carlsbad, CA), in accordance with the manufacturer's protocols. Nuclear pellets were extracted from cells using a hypotonic buffer and then resuspended in a lysis buffer to obtain nuclear proteins. The primary antibody used to detect NF-κB recognizes an epitope on p65 that is accessible only when NF-κB is activated and bound to its target DNA, here immobilized oligonucleotides containing a -κB consensus site (5′-GGGACTTTCC-3′) in a 96-well plate. A horseradish peroxidase (HRP)-conjugated secondary antibody provides a sensitive colorimetric readout that is quantified by a spectrophotometer at 450 nm with a reference wavelength of 655 nm (Dynex Technologies).

Luciferase assays

Cells were transiently transfected using TransFast (Promega, Madison, WI) according to the manufacturer's instructions with 10 μg of a luciferase reporter gene controlled by a minimal thymidine kinase promoter and 6 reiterated κB sites (κBx6 TK-luc). Twenty-four hours after transfection, cells were stimulated for 18 hr and lysed with reporter lysis buffer (Promega). Soluble extracts were assayed for luciferase activity using the Luciferase Assay Reagent (Promega) on a LB Centro luminometer (Berthold, Germany).

Immunofluorescence assay

Cells (106) were fixed with 4% paraformaldehyde, permeabilised with 0.1% triton ×100 and blocked with PBS containing 10% BSA for 45 min. Cells were then incubated with anti-p65 (SC-372) goat polyclonal antibodies (Santa Cruz Biotechnology). Secondary antibodies were Alexa-Fluor488-conjugated anti-goat IgG (1:1,000, Molecular Probes, Invitrogen) generating green fluorescence. Nuclei were stained with the fluorescent dye 4-,6-diamidino-2-phenylindole (DAPI). Slides were prepared with a cytospin centrifuge and mounted with Fluoromount-G solution (Southern Biotechnology Associates, Inc., Birmingham, AL). Analyses were performed using a LSM 510 confocal laser scanning microscope (Carl Zeiss, AG, Jena, Germany).

Xenograft growth assay

BaF/Bcr-Abl wt or T315I cells (106 in 0.2 ml PBS) were subcutaneously injected to NMRI female nude mice (4–6 weeks old; Janvier, France). Mice were then randomly assigned to either control (vehicle alone, i.e. 0.5% Carboxymethylcellulose/0.25% Tween80) or AS602868 treatment groups. Treatments started when tumors reached 150–200 mm3, after 10 days. AS602868 was dissolved in vehicle and administered by oral gavage (0.2 ml) at a dose of 20mg/kg twice a day for 5 days a week. Controls were given 0.2 ml vehicle and showed no signs of impaired health or toxicity. Mice were observed every day for signs of clinical deterioration. Tumors were measured once a week with a digital caliper and volumes were calculated by the formula: (a × b2)/2, where “a” and “b” are, respectively, the larger and smaller diameters. Treatment with AS602868 did not induce visible toxicity as analyzed by animal posture and behavior or functional hematopoiesis.23, 27

Statistical analysis

The mean and SD were calculated for each experimental mice group. The non-parametric Mann-Whitney U test was performed to assess the difference of tumor volume between control and treatment group. To account for multiple comparisons, significant differences among groups were calculated at p < 0.0125 for WT and 0.017 for T315I xenografts.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Constitutive NF-κB activation in Bcr-Abl expressing cell lines

NF-κB p65 DNA binding activity was measured by an ELISA method in the various CML cellular models used in this study (Fig. 1a). Compared to unstimulated or PMA-treated Jurkat cells (Bcr-Abl negative ALL line), constitutive levels of active p65 were detected in the nucleus of BaF cells expressing wt, T315I or forms of M351T Bcr-Abl, in LAMA84-s and -r lines. Cell treatment with the IKK2 inhibitor AS602868 resulted in a significant inhibition of NF-κB activation in both imatinib-sensitive or resistant Bcr-Abl-expressing cell lines.

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Figure 1. AS602868 inhibits the constitutive NF-κB activation of CML cell lines without affecting constitutive activation of Bcr-Abl. (a)Stable BaF cell lines expressing wt or T315I, M351T Bcr-Abl forms, LAMA84-s and -r were incubated for 18 hr with 10 μM AS602868 or 1 μM imatinib. Nuclear extracts were then isolated and p65 NF-κB DNA binding activity measured by Elisa. Data are expressed as fold of activity in comparison to control (unstimulated Bcr-Abl cells). Columns : mean of duplicate samples; bars: SD. Similar results were obtained in 3 independent experiments. (b) Gene reporter analysis of NF-κB activation. wt and mutant BaF/Bcr-Abl, LAMA84-s and -r cells were transfected with a κB-luciferase vector using Transfast transfection reagent 24 hr before addition of AS602868 (10 μM) or imatinib (1 μM). After 18hr cells were lysed and luciferase activity measured on a luminometer. Columns : mean of triplicate samples; bars : SD. Similar results were obtained in 4 independent experiments. (c) BaF/Bcr-Abl and LAMA84 cells were incubated with indicated doses of AS602868 or 1 μM imatinib for 18 hr. Cell lysates were resolved by SDS-PAGE and transferred to Immobilon membranes. The autophosphorylation status of IKK2 was analyzed with antibodies against phosphorylated serines 177 and 181. Anti-phospho (Y177) Bcr antibodies were used to detect phosphorylated Bcr-Abl. Anti-IKK2 and anti-Bcr antibodies were used as loading control.

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Since DNA binding does not necessarily reflect the transcriptional power of NF-κB, experiments were performed using a κB-luciferase reporter. Twenty-four hours after transfection, BaF/Bcr-Abl and LAMA84 cell lines were incubated with inhibitors for 18 hr and harvested for luciferase assay (Fig. 1b). NF-κB was constitutively active and sensitive to inhibition by 10 μM AS602868 in all Bcr-Abl positive cell lines. In contrast, imatinib treatment was clearly less efficient than AS602868 and in particular showed no effect on BaF/Bcr-Abl T315I. Taken together, the results confirm that expression of Bcr-Abl kinase activity in CML cell lines leads to constitutive activation of NF-κB.

Activation of IKK2 and Bcr-Abl can be followed by measuring autophosphorylation on serines 177/18128 and tyrosine 17729, respectively. As shown in Figure 1c using phospho-specific antibodies, both kinases appeared constitutively phosphorylated in BaF/Bcr-Abl (lane 1) and LAMA84 cells (lane 5). Imatinib strongly interfered with both Bcr-Abl and IKK2 phosphorylation in the 2 cell lines (lanes 4 and 9). In contrast, AS602868 affected only IKK2 phosphorylation (lanes 2, 3 and 6–8). Total levels of IKK2 and Bcr-Abl were unchanged by AS602868 or imatinib treatment. These data suggest that Bcr-Abl acts upstream of IKK2 to control NF-κB activation.

Altogether our results show that NF-κB is constitutively active in CML cell lines through IKK2 activation.

The IKK2 inhibitor AS602868 decreases viability of Bcr-Abl expressing cell lines

Imatinib sensitive- and resistant-Bcr-Abl positive cells were cultured in the presence of increasing doses of AS602868 and imatinib for 72 hr and cell viability assessed by an XTT assay. The percentage of viable cells is presented in Figure 2. Upon expression of Bcr-Abl, BaF3 cells become dependent on the oncogenic tyrosine kinase for their growth. Proliferation of BaF/Bcr-Abl wt was therefore profoundly inhibited by 1 μM imatinib (Fig. 2a). AS602868 exhibited a dose dependent anti-proliferative effect with an IC50 of 3 μM for BaF/Bcr-Abl. The IKK2 inhibitor also reduced cell viability of parental BaF3 cells, but with a higher IC50 (8 μM). This could be due to an interference with IL3, a cytokine that activates NF-κB. This has already been observed in this cellular model with another IKK2 inhibitor.30 While proliferation of BaF cells expressing M351T or T315I mutants of Bcr-Abl was not, or only slightly affected by imatinib, AS602868 strongly inhibited their growth in a dose-dependent manner, with an IC50 of 1–2 μM.

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Figure 2. Inhibition of cell survival of CML cell lines by the IKK2 inhibitor AS602868. Parental BaF3 cells, BaF (a) and LAMA84 (b) cell lines sensitive and resistant to imatinib were incubated for 72 hr. in the presence of AS602868 or imatinib at indicated doses. Cell viability was measured by an XTT colorimetric assay. Columns, mean of quintuplets samples; bars, SD. Similar results were obtained in 3 independent experiments. (c) BaF3, BaF/Bcr-Abl wt and mutant cells as well as LAMA84-s and -r were stimulated for respectively 48 and 72 hr with AS602868 or imatinib. Cells were stained with AnnexinV-Fitc and Propidium Iodide. Histograms display the percentage of apoptotic cells (AnnexinV positive cells). Representative of 3 different experiments.

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LAMA84 cells were highly sensitive to imatinib (IC50:0.1 μM) wheras AS602868 induced a dose-dependent inhibition of LAMA84 proliferation (IC50:3 μM) (Fig. 2b). The LAMA84-r variant still proliferated with 0.6 μM imatinib (dose used for selection) but remained sensitive to higher concentrations (i.e., 1 μM imatinib), although to a lesser extent than parental cells. Incubation of LAMA84-r cells with AS602868 dose dependently inhibited cell proliferation (IC50:3.2 μM).

Altogether, these results show that the IKK2 inhibitor, AS602868, was as efficient as imatinib to block cell survival of imatinib-sensitive, Bcr-Abl-expressing cells and most interestingly was able to counteract imatinib resistance in different cellular models.

NF-κB inhibition leads to apoptosis of Bcr-Abl expressing cells

As shown in Figure 2c, neither AS602868 nor imatinib could induce cell death in BaF3 parental cells as assessed by AnnexinV/propidium iodide staining. In contrast, AS602868 increased apoptosis to 80% in all CML cell lines whereas imatinib only affected BaF/Bcr-Abl and LAMA84-s lines (70% apoptotic cells).

These results were further confirmed by analysis of DNA fragmentation (Fig. 3a). Treatment of BaF/Bcr-Abl wt cells with AS602868 (lane 3) or imatinib (lane 4) for 48 hr induced DNA fragmentation. Apoptotic responses were comparable to that obtained after Fas death receptor engagement (lane 1). Interestingly, the IKK2 inhibitor, but not imatinib, induced DNA fragmentation in BaF/Bcr-Abl T315I cells (lane 6).

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Figure 3. AS602868 induces apoptosis in BaF/Bcr-Abl cells and causes regression of BaF/Bcr-Abl xenografts in nude mice. (a) DNA fragmentation: BaF/Bcr-Abl cells were incubated for 48 hr with 10 μM AS602868 or 1 μM imatinib. Anti-Fas antibody treated Jurkat cells were used as positive control. After lysis, DNA was extracted and analysed by electrophoresis on an ethidium bromide stained 1.2% agarose gel. (b) BaF/Bcr-Abl cells were incubated with indicated doses of AS602868 or imatinib for 48 hr. Cell lysates were resolved by SDS-PAGE and transferred to PVDF membrane. Apoptosis was evaluated by processing of the zymogenic forms of caspases 3, 8 and 9 and by the cleavage of poly ADP ribose polymerase in its characteristic apoptotic 89 kDa fragment. Hsp60 levels were used as loading control. (c) BaF/Bcr-Abl wt (upper graph) and T315I (lower graph) cells were subcutaneously injected in nude mice. After 10 days, when tumors reached 150–200 mm3, AS602868 was administered p.o. at 20 mg/kg twice a day at 12-hr intervals from days 0 to 22. Tumors were measured with a digital caliper. (non-parametric Mann-Whitney U test : * p < 0.0119, **p < 0.075).

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We then measured activation of caspases 3, 8, 9 and cleavage of the caspase 3 substrate Poly ADP Ribosyl Polymerase protein (PARP). Processing of caspases 3, 8, 9 was induced by imatinib and AS602868 in BaF/Bcr-Abl wt cells (Fig. 3b), respectively, lane 2 and lanes 3, 4). No caspase activation was detected in BaF/Bcr-Abl T315I cells exposed to 1 μM imatinib (lane 6), whereas upon AS602868 treatment a complete processing of caspases was observed (lanes 7, 8). PARP cleavage was induced upon imatinib (lanes 2, 3) or AS602868 (lanes 4–6) treatments of BaF/Bcr-Abl wt. PARP was only cleaved in response to AS602868 in the T315I mutant clone (lanes 10–12 compared to 8, 9).

These results showed that the negative effects of IKK2 inhibitor on survival of Bcr-Abl-expressing cells are associated with an induction of apoptosis.

In vivo activity of AS602868 against BaF/Bcr-Abl xenografts

We next tested the effects of AS602868 on BaF/Bcr-Abl xenografts in nude mice. BaF/Bcr-Abl wt and T315I cells were subcutaneously injected to nude mice. Tumors were staged to 150–200 mm3 after 10 days and AS602868 was administered p.o. at 10 mg/kg twice a day (Fig. 3c). This regimen resulted in significant regression of the tumors. After 22 days of AS602868 administration, tumors size diminished by 41% in mice injected with BaF/Bcr-Abl wt and by 71% in those injected with BaF/Bcr-Abl T315I cells.

NF-κB is constitutively active in CML and Ph+ ALL primary cells

The DNA binding activity of NF-κB was quantified in blood cells from CML and Ph+ ALL patients using the TransAm p65 Elisa kit. As shown in Figure 4a, a low p65 DNA binding was detected in the Bcr-Abl negative cell line Jurkat, whereas NF-κB activation was 7-fold higher in the LAMA84 cell line. Significant levels of constitutive NF-κB DNA binding activity could be detected in nuclear extracts of primary cells from 3 CML and 1 Phi+ALL patients. The constitutive activation of NF-κB appeared to be 2- to 4-fold higher than that of Bcr-Abl negative Jurkat and 2- to 3-fold lower than that of LAMA84 cells. Indirect fluorescence experiments show that LAMA84 cells and cells from 4 patients exhibit strong cytoplasmic and nuclear staining for p65 while p65 was exclusively cytoplasmic in Jurkat cells used as negative control for constitutive NF-κB activation (Fig. 4b).

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Figure 4. Activation of NF-κB in primary CML cells. (a) Nuclear extracts were isolated from primary CML and Phi+ ALL cells and endogenous p65 NF-κB DNA binding activity was quantified using a TransAM NF-κB kit. Patients #5, 13, 15 are resistant to imatinib therapy. Columns: mean of duplicate samples; bars: SD. Similar results were obtained in 2 independent experiments. (b) Indirect immunofluorescence staining of p65. Green indicates p65 staining, blue color DAPI, nuclear translocation is visible as overlapping green-blue staining (merge). Single cell photographs correspond to a magnification of ×63 whereas pictures of group of cells correspond to ×40.

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Blockade of NF-κB in CML primary cells affects their growth and their capacity to form hematopoietic colonies

We next tested the efficiency of the IKK2 inhibitor on primary CML and Ph+ ALL cells (see Supporting Information Table 1 for patients' characteristics). Peripheral blood cells from 10 imatinib-resistant patients were cultured for 96 hr in the presence of imatinib or AS602868 before assessment of cell viability (Fig. 5). Cells from 9 patients were completely refractory to imatinib whereas cells from patient 5 were only partially resistant to the drug (25% inhibition of viability). By sharp contrast, AS602868 inhibited cell viability of leukemic cells in a dose-dependent manner (IC50s from 0.2 to 4.5 μM). There was variability among patient samples, as some appeared good responders (2, 4, 9, 10, 11, 12) compared to others (1, 3, 5, 13). In all cases, 10 μM AS602868 induced a strong decrease (70–95%) in cell viability.

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Figure 5. Inhibition of viability of primary CML cells by the IKK2 inhibitor AS602868. Cells from peripheral blood of 9 CML and 1 Phi+ ALL imatinib-resistant patients were purified by Ficoll-Paque-Plus density gradient and incubated for 96 hr in the presence of AS602868 or imatinib at indicated doses. Cell viability was measured by a colorimetric assay. Columns: mean of quintuplets samples; bars: SD. Similar results were obtained in 2 independent experiments.

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We analysed the ability of leukemic progenitors to form colonies in a semi-solid medium in the presence of inhibitors. A 10 μM treatment with AS602868 resulted in a significant reduction (50–85%) in the number of colonies in all cases from both sensitive- (6, 7, 8) or imatinib-resistant patients (1, 4, 5, 12, 14, 15) (Fig. 6a). Interestingly, cells from patients 12, 13, 14 (who bear the T315I mutation) appeared sensitive to NF-κB inhibition in terms of proliferation and CFU formation.

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Figure 6. AS602868 decreases hematopoietic colony formation and induces apoptosis of primary CML cells. (a) Growth of CFU-GMs and BFU-Es was assessed after continuous exposure to combined treatment for 14 days. Ficoll purified CML and Phi+ ALL primary cells were grown in the presence or not of 10 μM AS602868 in Iscove's methylcellulose medium supplemented with IL-3, GM-CSF, G-CSF, IL-6, Epo, SCF. Data represent means of triplicate experiments. Patients #1, 4, 5, 12, 14 and 15 are resistant to imatinib therapy. (b) Cells from peripheral blood of 2 CML patients resistant to imatinib therapy were purified by Ficoll-Paque-Plus density gradient and incubated for 48 hr in the presence of AS602868 or imatinib at indicated doses. Upper panels: Apoptosis was measured by AnnexinV-Fitc staining. Histograms display the percentage of apoptotic cells. Lower panels: Cell lysates were resolved by SDS-PAGE and transferred to PVDF membrane. Apoptosis was evidenced by the cleavage of PARP in its characteristic 89 kDa fragment. Hsp60 levels were used as loading control.

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Apoptosis was then analysed in cells from 2 imatinib-resistant CML patients treated in vitro with AS602868 or imatinib for 48 hr (Fig. 6b, upper part). Cells displayed some spontaneous apoptosis in culture (10% and 20%, respectively, for patients 3 and 5) that increased in a dose dependent manner after incubation with 10 μM AS602868 (50–65% of AnnexinV-positive cells). Imatinib triggered a highly lower response (15%). In parallel, AS602868 treatment induced a complete cleavage of PARP (lanes 2, 3 and 7, 8) while imatinib had no effect (lanes 4, 5 and 9, 10).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the present study, we show that the Bcr-Abl oncogene-induced activation of the transcription factor NF-κB to support cell survival in various CML cellular models such as Bcr-Abl-transfected BaF cells, human LAMA84 CML line and primary cells from CML patients. NF-κB DNA binding activity and NF-κB dependent transactivation were induced by Bcr-Abl in a tyrosine kinase dependent manner. Several studies have already reported a functional link between Bcr-Abl and NF-κB.21, 30–32 While Kirchner et al.21 suggested that Bcr-Abl activates NF-κB independently of the IKK complex our study as well as Cilloni et al.32 and Duncan et al.30 implicate IKK2 downstream of Bcr-Abl. The molecular mechanisms linking Bcr-Abl to the IKK complex are still to be fully elucidated.

The Bcr-Abl inhibitor imatinib is now the treatment of choice for all newly diagnosed CML patients. However, the initial striking efficacy of the drug is overshadowed by the appearance of clinical resistance, as leukemia progresses to blast crisis.33 A combination of several targeted drugs is expected to be required in order to effectively treat CML14 and it is therefore crucial to find additional relevant targets in the Bcr-Abl pathway in order to bypass resistance.34 As Bcr-Abl activates PI3K, Ras, STAT, and NF-κB pathways, several pharmaceutical drugs have been tested in vitro for their efficiency against CML cells, such as the farnesyl transferase inhibitor, SCH66336 (Lonafarnib)35 or the mTOR inhibitor, rapamycin.36

Aberrant activation of NF-κB is associated with carcinogenesis and constitutively high IKK activity is detected in many tumor types, including various leukemias.23, 37 The IKK2 kinase represents an attractive therapeutic target in cancer. In the present study, we observed that AS602868, an inhibitor of IKK2, induced growth arrest and apoptosis of BaF cells expressing Bcr-Abl, of cells of the LAMA84 CML line and of primary CML cells. We have previously described that blocking NF-κB could sensitize primary AML cells to apoptosis and could potentiate the effect of the chemotherapeutic drugs doxorubicin, AraC and etoposide, with only minor effects on normal CD34 positive hematopoietic precursors.23

Acquired clinical resistance to imatinib relies on 2 major mechanisms : mutations within the Bcr-Abl kinase domain that interfere with imatinib binding (50–90% of cases) and Bcr-Abl overexpression after gene amplification (10%).38 Interestingly, AS602868 could bypass imatinib resistance in cellular models mimicking these types of clinical resistance. The LAMA84-r variant which is characterized by overexpression of Bcr-Abl25 retains some sensitivity to AS602868. IKK2 inhibition also strongly decreased cell viability in BaF cells expressing various mutated forms (M351T, T315I, - G250E not shown) of Bcr-Abl observed in imatinib-resistant patients.

Our results further extend the study from Duncan et al.30 which corresponds to an in vitro study of 2 NF-κB inhibitors on Bcr-Abl-transfected BaF and 32D cells. In addition, we observed in xenograft experiments that BaF/Bcr-Abl-driven tumors regressed in nude mice after administration of AS602868. Interestingly, a 22-day treatment diminished by 71% the size of tumors in mice injected by BaF/Bcr-Abl T315I. AS602868 appeared more efficient on BaF/Bcr-Abl T315I than on BaF/Bcr-Abl wt (71 vs. 41% inhibition). In vitro, BaF/Bcr-Abl T315I cells were more sensitive to IKK2 inhibition than BaF/Bcr-Abl wt as shown on Figures 2a (XTT assay) and 3b (caspase activation, PARP clivage). An attractive hypothesis would be that this difference reflects a stronger dependence on active NF-κB for BaF/Bcr-Abl T315I cells.

We also evaluated the efficiency of the IKK2 inhibitor in primary cells from CML patients and observed that survival of cells from 10 different imatinib-resistant CML or Ph+ ALL patients was dramatically affected by AS602868. Moreover, the capacity of CML PBL to differentiate in vitro was also strongly diminished after NF-κB inhibition suggesting that AS602868 could affect leukemic progenitors. We have previously shown the IKK2 inhibitor did not exert major toxicity on normal hematopoiesis.27 A major problem of CML is the persistence of leukemic stem cells (LSC) which are unaffected by imatinib39 and therefore responsible for residual disease and relapse. It would be interesting to evaluate if NF-κB inhibition by AS602868 could also target these cells, as it was suggested for AML-LSC.20

Cilloni et al.32 described the in vitro apoptotic effects of the IKK2 inhibitor PS1145 on cells from patients in the different clinical phases representing CML progression. However, the patients whose cells were used in that study were not characterized at a molecular level. In our work, half of imatinib-resistant patients displayed identified Bcr-Abl mutations : M351 (1), E255 (1), T315 (4). Among these, M351T occurs in 15% of CML patients but remains sensitive to the second generation of Bcr-Abl inhibitors (dasatinib/BMS-354825 and nilotinib/AMN107).40 In contrast, the E255V mutation is insensitive to imatinib and shows an intermediate sensitivity to dasatinib and nilotinib.40 Finally, the T315I mutation (“hell mutation”) is of critical clinical importance because it represents the most frequent cause of imatinib resistance and is associated with poor outcome.41 This mutation confers complete resistance to all currently clinically available inhibitors,42, 43 and is likely to become an increasing concern at a clinical level unless specific drugs are discovered. Alternatively, targeting one, or more likely, several signaling pathways downstream of Bcr-Abl will have the advantage of being independent of the mutational status of Bcr-Abl during transformation of the leukemia.11, 34 In this context, our data strongly support IKK2 inhibition as a possible therapeutical approach for treatment of all imatinib-resistant CML cases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Samantha Sarno (EMD-Serono) for critical reading of the manuscript. They also thank Dr. F.X. Mahon (INSERM, Bordeaux, France) and Dr. P. Dubreuil (INSERM, Marseille, France) for the gift of the BaF/Bcr-Abl variants and Dr. C. Gambacorti-Passerini (University of Milan, Italy) for LAMA84 imatinib resistant cells.

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  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_24294_sm_SuppTable.doc32KSupporting Information Table 1: Clinical characteristics of CML and Ph+ ALL patients.

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