Molecular Targets for Therapy

Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts

Abstract

We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.

Introduction

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease driven by the bcr-abl oncogene with constitutive tyrosine kinase activity.1 One of the characteristic features of CML is abnormal release of the expanded malignant stem cell clone with altered homing function from the bone marrow (BM) into the circulation.2 Tyrosine kinase inhibitors (TKIs) have revolutionized the natural history of CML, with overall survival and event-free survival rates of 88% and 83%, respectively for imatinib-treated patients, and low frequency of transformation into blastic phase.3 Quiescent primitive CML CD34+ cells are insensitive to TKIs, however, and these cells persist even when complete responses are achieved,4 leading to disease recurrence following discontinuation of TKIs in a fraction of CML patients.5 The quiescent CML cells are in general resistant to a wide variety of proapoptotic stimuli,6 implying unique mechanisms of protection from imatinib and other insults that induce apoptosis. In particular, BM stromal cells are known to mediate protection of CML progenitor cells from TKI-induced apoptosis.4 Elucidation of the mechanisms involved in the interactions between CML cells and BM microenvironment might provide novel molecular targets in CML therapy.

CXC chemokine ligand 12 (CXCL12), a member of the CXC subfamily previously called stromal cell-derived factor-1α, is a chemokine produced by stromal cells that acts through its cognate receptor CXCR4.7 CXCL12 functions both as a chemoattractant and a modulator of cellular growth/survival.8 G protein-coupled CXCR4, which is expressed on the membrane of normal and malignant hematopoietic cells, mediates chemotaxis and has a key role in the homing of these cells to the BM microenvironment.9 Defective leukemia–stroma interactions are inherent to CML cells, whereby p210 Bcr-Abl oncoprotein suppresses CXCR4-mediated interaction of CML cells with BM stromal cells.10 We have previously reported that blockade of bcr-abl activity with TKI imatinib induces cell-surface expression of CXCR4.11 This in turn results in enhanced migration of CML cells towards BM stromal cells and paradoxically promotes non-pharmacological resistance to Bcr-Abl inhibitors.12 These in vitro observations were recently substantiated by the in vivo studies, whereby disruption of the CXCL12/CXCR4 axis by CXCR4 antagonists restored the sensitivity of Bcr-Abl-expressing cells to TKIs and prolonged the survival of mice co-treated with CXCR4 inhibitor and TKI.4, 11, 13 However, the molecular mechanisms of interplay between CXCR4 and Bcr-Abl signaling have not been understood.

Src-family tyrosine kinase Lyn is one of the key components of CXCL12/CXCR4-mediated migration of normal hematopoietic cells.14, 15 In CML, Lyn can be additionally activated through p210 Bcr-Abl tyrosine kinase. It was shown that p210 Bcr-Abl binds and constitutively activates Lyn.16 Hence, oncogenic p210 Bcr-Abl ‘hijacks’ Lyn, which in turn becomes unresponsive to CXCL12-induced chemotaxis and increases the ability of immature cells to escape from the marrow.16 Lyn has two major sites of tyrosine phosphorylation that regulate its activity. Phosphorylated Lyn (p-Lyn)Tyr396 increases the specific activity of Lyn,17, 18 whereas p-LynTyr507 reduces Lyn activity.19 Both CXCR4 and Lyn can reside in lipid rafts, plasma membrane microdomains highly enriched in cholesterol, sphingolipids and signaling molecules. Lipid rafts act as signal transduction platforms for a variety of intracellular processes.20 However, the precise location and molecular interactions between Lyn and CXCR4 in CML are unknown.

In this study, we investigated the molecular and cellular mechanisms of leukemia–stroma interactions upon inhibition of Bcr-Abl by TKIs. Our data indicate that stroma interferes with the ability of Bcr-Abl inhibitors to block Lyn activation. Further, stromal signals promote clustering of CXCR4 in lipid rafts where it co-localizes with p-LynTyr396, which in turn facilitates migration of CML cells to the cells of BM microenvironment. Lipid raft disruption by cholesterol depletion inhibits CML cell migration, suggesting that lipid rafts represent key signaling modules responsible for CML cell homing and persistence of dormant CML stem cells in the BM niche.

Materials and methods

Cells and cell culture conditions

CML cell line KBM-5 (provided by Dr M Beran),21 murine pro-B cell line (BaF3) and BaF3/wild-type p210 BCR-ABL (provided by Dr C Sawyers)22 were used.

All cell lines were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C in 5% CO2. For the culture of parental BaF3 cells, 1 ng/ml murine interleukin-3 (Wako Pure Chemical Industries, Osaka, Japan) was added to the medium. Samples procured from CML blastic crisis patients and normal BM were obtained after informed consent in accordance with institutional guidelines set forth by the MD Anderson Cancer Center and the Declaration of Helsinki. Clinical characteristics of CML patients are summarized in Table 1. BM-derived stromal cells (MSCs) obtained from a normal BM donor were cultured at a density of 5000–6000 cells/cm2 in minimum essential medium alpha supplemented with 20% FBS, 1% L-glutamine and 1% penicillin–streptomycin, as described elsewhere.23 The isolated, cultured MSCs at passage 3 comprised of a single phenotypic population, as determined by flow cytometric analysis, positive for SH2 and SH3, and negative for markers of hematopoietic lineage as described elsewhere.24 Passage 3 or 4 MSCs were used for the co-culture experiments.

Table 1 Clinical characteristics of CML patients

Treatment of cells

To study the effect of BM stroma on CML cells, KBM-5 cells were cultured, at a density of 0.5 × 106, with or without a layer of MSCs plated at a density of 0.2 × 105 cells/cm2 under serum-starved conditions (0.5% FBS). Co-cultured KBM-5 cells were separated from the MSC monolayer by careful pipetting with ice-cold phosphate-buffered saline (PBS; repeated twice). After the KBM-5 cells were collected, to rule out the possibility of contamination with MSCs, MSC monolayer was examined by microscopy ( × 100) to confirm that the monolayer was not damaged and that <10 leukemic cells per visual field remained attached. In indicated experiments, co-cultures were performed in the presence of imatinib or nilotinib (LC Laboratories, Woburn, MA, USA). The following reagents were also used: dasatinib (LC Laboratories), CXCL12/stromal cell-derived factor-1α (R&D Systems, Minneapolis, MN, USA) and PP2, a selective inhibitor for Src-family kinases (Calbiochem, La Jolla, CA, USA). Methyl-beta-cyclodextrin (Wako Pure Chemical Industries) was used to selectively extract cholesterol from the plasma membrane.

Isolation of lipid rafts

Lipid rafts were isolated as described elsewhere.25 Briefly, KBM-5 cells co-cultured with MSCs for 24 h were harvested, resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor mixture Complete mini (Roche, Indianapolis, IN, USA), 1 mM Na3VO4 and 2 mM NaF; pH 7.4) and incubated at 4 °C for 20 min. The solubilized cells were homogenized with ten strokes of a Dounce homogenizer, and 1 ml of the homogenate was added to an equal volume of 85% sucrose solution. The solubilized cells were overlaid successively with 6 ml of 35% sucrose solution and 2 ml of 5% sucrose solution. After centrifugation at 40 000 r.p.m. in a Beckman SW40Ti rotor (Beckman Coulter, Brea, CA, USA) for 16 h, 1-ml fractions were collected starting from the top of the gradient (fraction 1 (top) to fraction 10 (bottom)) and stored at −80 °C. In some experiments, collected fractions were concentrated by centrifugation at 10 000 rpm for 1 h. Protein samples from the lipid microdomain flotation experiments were subjected to western blot analysis.

Flow cytometry analysis of CXCR4 expression

For CXCR4 expression studies, cells were washed with a 20-fold volume of ice-cold buffer without FBS, stained at 4 °C with saturated concentrations of phycoerythrin-conjugated anti-CXCR4 monoclonal antibody (12G5; DAKO Cytomation, Carpinteria, CA, USA) and then analyzed by flow cytometry.

Western blot analysis

Total cell lysates were collected as described elsewhere.26 Total protein (20 μg) was separated by SDS–polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), transferred to polyvinylidene-fluoride membranes, then probed with primary and secondary antibodies according to the manufacturer's protocol. Each membrane was probed for α-tubulin (Sigma-Aldrich, St Louis, MO, USA) as loading control after being stripped with a stripping buffer (Pierce Chemical Co., Rockford, IL, USA). Proteins were visualized by the ECL Plus Western Blotting Detection System kit (GE Healthcare, Piscataway, NJ, USA), detected by a luminescent image analyzer (LAS-100 plus; Fujifilm, Tokyo, Japan) and quantified by Image Gauge (Fujifilm).

For immunoblotting, the following antibodies were used: CXCR4 (Fusin, C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), flotillin-1 (clone18; BD-Pharmingen, San Diego, CA, USA), α-tubulin (Sigma-Aldrich), p-LynTyr396 (Upstate Biotechnology, Lake Placid, NY, USA), and Lyn, p-LynTyr507, horseradish peroxidase–linked anti-mouse and anti-rabbit IgG, all from Cell Signaling Technology (Danvers, MA, USA).

Immunoprecipitation

Protein extracts were first precleared by incubation with protein A/G plus-agarose beads (Santa Cruz Biotechnology) for 30 min and incubated overnight with rabbit anti-CXCR4 antibody (Affinity BioReagents, Rockford, IL, USA). Protein A/G-agarose beads were added for 2 h, and the extracts were then washed four times with cold PBS. Sample buffer (500 mM Tris HCl (pH 6.8), 10% sodium dodecyl sulfate, 25% glycerol and 0.1% bromophenol blue) was added, and proteins were separated by SDS-polyacrylamide gel electrophoresis and then subjected to western blotting. Membranes from the CXCR4 immunoprecipitation were probed with mouse anti-Lyn antibody (H-6; Santa Cruz Biotechnology).

Confocal immunofluorescent microscopy

Primary CML cells were washed with PBS, stained with anti-B-cholera toxin antibody (incubated for 20 min in ice, 1:100, FITC Conjugate; Sigma-Aldrich), fixed with 2% paraformaldehyde in PBS for 5 min at room temperature and permeabilized with 100% methanol for 15 min at −20 °C. Next, cells were blocked with 5% chicken serum in PBS containing 2% bovine serum albumin for 60 min at room temperature and incubated with the antibodies CXCR4, Lyn, p-LynTyr396 (Abcam, Cambridge, MA, USA) or flotillin-1 overnight at 4 °C (1:50). Excess antibody was removed by washing with PBS. Cells were then incubated with secondary antibody, Alexa Flow 488-, 546-, 647-labeled anti-goat, anti-rabbit or anti-mouse IgG (H+L; 1:2000; Molecular Probes, Carlsbad, CA, USA), respectively, for 60 min at room temperature. Cells were washed with PBS, mounted on slides and analyzed under a Zeiss LSM 510 laser confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Transfection with short interfering RNA (siRNA)

KBM-5 cells were transfected with siRNA for human Lyn (siGenome SMARTpool reagent; Dharmacon, Lafayette, CO, USA) and non-targeting siRNA (control siRNA) at a final concentration of 250 nM using an Amaxa Nucleofector with a Nucleofector Kit V in accordance with the manufacturer’s protocol with slight modifications (Amaxa Biosystems, Cologne, Germany). After transfection with siRNA, cells were cultured for 24 h in RPMI 1640 medium supplemented with 10% FBS, then treated with 0.5 μM imatinib for 24 h. Cells were collected and subjected to western blot and chemotaxis assay.

Chemotaxis assay

Cells were pretreated with the indicated reagents and placed in a volume of 200 μl in the upper compartment of 5-μm-pore transwell filters (Costar, Cambridge, MA, USA). Inserts were placed in the 24-well lower chambers containing 800 μl 10% FBS RPMI with a MSC monolayer (1 × 105 cells) or CXCL12 (final concentration of 100 ng/ml). Chemotaxis assays for MSCs were performed at 37 °C for 4 h, and cells that migrated were counted in triplicate. For CXCL12 chemotaxis assay, migrated cells were stained with 2 μM Hoechst 33342 (Dojindo, Kumamoto, Japan) and counted using an IN Cell Analyzer 1000 in 40 random image fields (GE Healthcare). CytoSelect cell migration assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was also utilized for CXCL12 chemotaxis assay. The chemotactic index was determined as follows: (number of cells migrating to the MSC monolayer or CXCL12)/(number of cells migrating to medium alone).

Statistical analysis

Statistical significance was determined by applying the two-tailed Student's paired t-test; P-values <0.05 were considered significant. Average values are shown as the mean±s.d.

Results

Effects of imatinib on total cellular expression of CXCR4 and Lyn in KBM-5 cells co-cultured with MSCs

We and others have demonstrated that the inhibition of Bcr-Abl by TKI imatinib, nilotinib or dasatinib significantly increases cell-surface expression of CXCR4.11, 12, 27 We have further shown that this results in enhanced migration of CML cells towards stromal cell layers and promotes nonpharmacological resistance to imatinib.12 To investigate the modulation of Lyn and CXCR4 expression and/or activity in CML cells co-cultured with MSCs,28 we first examined cellular levels and surface expression of these proteins after treatment with a TKI (imatinib or nilotinib). Both imatinib and nilotinib significantly increased cell-surface expression of CXCR4 in CML KBM-5 cells, which was further induced under MSC co-culture conditions (Figure 1a). In turn, immunoblotting from the lysates of KBM-5 cells exposed to imatinib showed no change in the total cellular levels of CXCR4 and Lyn (Figure 1b), suggesting that CXCR4 was redistributed to the cell surface from intracellular stores. Although imatinib effectively inhibited phosphorylation of Lyn-Tyr396, signals from stroma co-cultures preserved p-LynTyr396 expression known to correlate with enzyme activation.29 The level of p-LynTyr507 that negatively affects kinase activity29 was less affected by imatinib irrespective of culture conditions (Figure 1b).

Figure 1
figure1

(a) CXCR4-surface expression in KBM-5 cells cultured with MSCs and imatinib or nilotinib. Percentage of CXCR4-expressing KBM-5 cells after 72 h of indicated treatment (0.5 μM imatinib or 0.5 nM nilotinib) was analyzed by flow cytometry, as described in Materials and methods. Cells were cultured under serum-starved conditions (0.5% FBS) with an MSC monolayer. Values are mean±s.d. (n=2). *P<0.05, **P<0.01. (b) CXCR4 and Lyn expression on KBM-5 cells cultured with MSCs and imatinib. KBM-5 cells were cultured alone or co-cultured with MSCs at the indicated conditions (with or without 1 μM imatinib) for 24 h. Clarified cell lysates were probed with antibodies: CXCR4, Lyn, p-LynTyr396, p-LynTyr507 and α-tubulin in western blots. Results shown are representative of three experiments. The intensity of the p-LynTyr396 and p-LynTyr507 signals was quantified by densitometry using Image Gauge software, and the relative intensity compared with α-tubulin was calculated from the three experiments.

Effects of imatinib on CXCR4 and Lyn distribution in lipid rafts of KBM-5 cells co-cultured with MSCs

Because several reports demonstrated that activated CXCR4 interacts with signaling factors in lipid rafts,30, 31 we investigated the localization of CXCR4 and Lyn in KBM-5 cells by immunoblot analysis of the individual cellular fractions separated by sucrose gradient (Figure 2). As the marker of lipid rafts, we used the lipid raft-associated protein flotillin-1.32 CXCR4 was present only in the higher-density detergent-soluble fractions 8–10 in control KBM-5 cells, but exposure to 1 μM imatinib in the presence of MSCs induced CXCR4 localization in the flotillin-1-containing low-density raft fraction 2 (Figure 2a). At this dose, imatinib induced cell cycle arrest without apoptosis induction; hence, CXCR4 redistribution was not related to change in cell viability. Similar findings were observed in BaF3 cells stably transfected with wild-type Bcr-Abl, in which imatinib caused CXCR4 localization in raft-containing fractions 2 and 3 (Supplementary Figures 1A and B).

Figure 2
figure2

Association of CXCR4 and Lyn with lipid rafts. KBM-5 cells were cultured alone or with MSCs at the indicated conditions (with or without 1 μM imatinib) for 24 h. The cells were subjected to lysis and fractionated in a sucrose density gradient, as described in Materials and methods. An aliquot of each fraction was resolved by SDS-polyacrylamide gel electrophoresis, and flotillin-1 (a), CXCR4 (b), Lyn (c), p-LynTyr507 (d) and p-LynTyr396 (e) were visualized by western blotting. Results shown are representative of three experiments.

The tyrosine kinase Lyn is known to localize frequently in lipid raft fractions33 and has been reported to be closely associated with CXCR4.15, 16 As shown in Figure 2c, Lyn was widely distributed both in the low-density raft fractions and the high-density nonraft fractions in control KBM-5 cells. In contrast, Lyn clustered in selective lipid raft fractions (fraction 2 and 3) and nonraft fractions (fraction 8–10) in CML cells co-cultured with MSCs. We next examined the locations of two phosphorylated forms of the Lyn kinase (p-LynTyr507 and p-LynTyr396). p-LynTyr507 localized primarily in the high-density nonraft fractions and minimally in the low-density lipid raft fractions (Figure 2d). In turn, active p-LynTyr396 clustered in membrane raft fractions of KBM-5 cells, and was increased significantly by MSC co-culture (Figure 2e). Although imatinib treatment depleted p-LynTyr396 from the lipid raft fractions of KBM-5 cells cultured alone, co-culture with MSCs maintained p-LynTyr396 within lipid raft fractions. Similar findings were observed in murine BaF3/Bcr-Abl (Supplementary Figures 1A–E).

We next examined whether CXCR4 directly interacts with Lyn in CML cells, and whether CXCR4 co-localizes with active Lyn in lipid rafts. Co-immunoprecipitation experiments using total cellular extracts confirmed the direct binding of CXCR4 to Lyn in KBM-5 cells in all conditions tested (Figure 3a). To characterize spatial localization of CXCR4 and Lyn, we next utilized confocal microscopy (Figure 3b). CXCR4 localized mainly at the cell surface, and imatinib treatment of cells co-cultured with MSCs induced moderate increase in cell-surface CXCR4 that co-localized with Lyn. Visualization of the lipid rafts with flotillin-1 showed that Lyn partially co-localized with flotillin-1 in all tested conditions. Imatinib treatment under MSC co-culture conditions induced the partial clustering of CXCR4 and Lyn (Figure 3b), and of CXCR4, p-LynTyr396 and flotillin-1 into lipid rafts (Figure 3c). This has been confirmed by the CML cells from three primary CML patient samples (Figure 3d). These data indicate that CXCR4 and active Lyn interact in lipid raft domains.

Figure 3
figure3figure3

Association of CXCR4, Lyn and flotillin-1 in KBM-5 cells. KBM-5 cells were cultured alone or with MSCs at the indicated conditions (with or without 1 μM imatinib) for 24 h. (a) CXCR4 was immunoprecipitated from total cell lysates and its association with Lyn was determined by immunoblotting. Western blot images are representative of three independent experiments. (bd) The KBM-5 cells (b, c) or primary cells of CML patients in blast crisis (d) were fixed with paraformaldehyde and subjected to confocal microscopic analysis using anti-CXCR4 (green; b, c, blue; d), anti-Lyn (red; b, d), anti p-LynTyr396 (red; c), anti-flotillin-1 (blue; b, c) and anti-B-cholera toxin (green; d) antibodies, as described under Materials and methods. CXCR4 co-localized with Lyn and p-LynTyr396, and partially co-localized with lipid raft marker flotillin-1 or B-cholera toxin upon imatinib treatment in MSC co-cultured cells. Microscopic images are representative of three independent experiments (b, c), and one of the three CML blast crisis cases (d).

Lipid raft disruption and Lyn inhibition abrogate imatinib-stimulated migration of KBM-5 cells

We have previously reported that inhibition of Bcr-Abl promotes migration of CML cells to recombinant CXCL12 or to CXCL12-producing MSC.12 To confirm these findings, we examined migration towards CXCL12 in paired isogenic BaF3 and BaF3–wild-type Bcr-Abl cells. Consistent with our published findings, parental BaF3 cells avidly migrated towards CXCL12, whereas the migration of Bcr-Abl-expressing cells was significantly diminished (Figure 4a). In turn, migration of BaF3–wild-type Bcr-Abl cells was fully restored upon Bcr-Abl blockade by imatinib or nilotinib. Similar findings were seen in human Bcr-Abl-expressing KBM-5 cells: although only 10% of cells migrated towards CXCL12-producing MSCs, treatment with imatinib caused 40% of cells to migrate, consistent with our published data12 (Figure 4b). To determine the role of lipid rafts and of Lyn in CML cell migration, we examined the effects of cholesterol depletion from the plasma membrane, which disturbs lipid raft integrity.33, 34 Pretreatment of KBM-5 cells with methyl-beta-cyclodextrin, which disrupts lipid rafts through sequestration of cholesterol, significantly (P=0.01) blocked MSC-induced KBM-5 cell migration. Importantly, a specific inhibitor of Src-family kinases PP2 also abrogated imatinib-stimulated migration of KBM-5 cells. Treatment with methyl-beta-cyclodextrin or PP2 did not affect CXCR4-surface expression, which was reportedly upregulated following TKI (Figure 4c). Because PP2 acts on a broad range of Src-family kinases, and MSCs produce various cytokines and chemokines that may affect KBM-5 cell migration, we utilized Lyn knockdown in KBM-5 cells to evaluate the specific involvement of Lyn. Silencing of Lyn with siRNA efficiently inhibited Lyn expression and abrogated imatinib-induced KBM-5 cell migration towards recombinant CXCL12 (Figure 4d). Further, substantiating the role of Src in CXCL12-triggered CML cell migration, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 than either imatinib or nilotinib (P=0.04) (Figure 4e). These findings collectively suggest the involvement of lipid rafts and the Lyn kinase-dependent pathway in CXCL12/CXCR4-mediated CML cell migration.

Figure 4
figure4

(a) Chemotaxis assay of parental BaF3 (i) or BaF3/wild-type p210 BCR-ABL cells were stimulated with CXCL12 (100 ng/ml) for 4 h (ii). When indicated, cells were treated with imatinib (0.5 μM) or nilotinib (0.5 nM) for 18 h. (b) Chemotaxis assay in KBM-5 cells co-cultured with MSCs. KBM-5 cells were incubated in serum-free RPMI 1640 medium. When indicated, cells were pretreated with 0.5 μM imatinib for 18 h, and 10 μM PP2 or 10 mM methyl-beta-cyclodextrin for 1 h. (c) Percentages of CXCR4-expressing KBM-5 cells incubated under the indicated conditions were determined by flow cytometry, as described in Materials and methods. Values are mean±s.d. (n=2). **P<0.01. (d) KBM-5 cells were transfected with Lyn siRNA or control siRNA as described in Materials and methods. After 48 h of transfection, cells were cultured with or without imatinib (0.5 μM) for 18 h. Clarified cell lysates were probed with antibodies Lyn, p-LynTyr396 and α-tubulin in western blots. Results shown are representative of two experiments. Chemotaxis assays of KBM-5 cells were performed 72 h after transfection with Lyn siRNA with or without imatinib treatment (0.5 μM, 18 h). Cells were stimulated with CXCL12 (100 ng/ml) for 4 h. (e) Chemotaxis assay of KBM-5 cells treated with imatinib (0.5 μM), nilotinib (0.5 nM) or dasatinib (0.5 nM) for 18 h. Dasatinib induced significantly lower cell migration towards CXCL12 (100 ng/ml) than imatinib or nilotinib. In all experiments, the chemotactic index was calculated as described in Materials and methods. Values in chemotaxis assays are mean±s.d. (n=3). *P<0.05, **P<0.01.

Discussion

In this study, we investigated the role of chemokine receptor CXCR4 and Lyn kinase in microenvironment-mediated resistance to TKIs in CML. Our data indicate that inhibition of Bcr-Abl by imatinib under MSC co-culture conditions promoted CXCR4 clustering in lipid rafts, where CXCR4 co-localized with active p-LynTyr396. Consistent with previous studies, TKIs (imatinib and nilotinib) induced cell-surface CXCR4 expression.11, 12, 27 Yet, the immunoblot experiments show that imatinib had no effects on total cellular CXCR4 levels, indicating that increases in cell-surface CXCR4 likely represent redistribution from the abundant intracellular sources. It has recently been demonstrated that imatinib induces cell membrane sialylation in p210 BCR/ABL-expressing CML cells.34 Because sialic acids are present on glycoproteins and glycolipids, the modulation of the sialylation status may affect the molecular composition of lipid rafts highly enriched in cholesterol and glycosphingolipids. Cebo et al.34 demonstrated that, in CML cells, imatinib induces cell-surface GM1 ganglioside, one of the cellular constituents of the lipid rafts, which notably is reported to facilitate CXCL12-induced cell migration.35, 36 Taken together, these findings suggest that modulation of lipid raft composition by imatinib may contribute to recruitment of CXCR4 into lipid rafts. In turn, stromal cells abundant in BM microenvironment clearly facilitate CXCR4 relocalization to lipid rafts. Although the mechanisms of stroma-induced raft reorganization remain to be elucidated, we have shown that cytokines secreted by BM stroma, including stem cell factor, regulate surface induction of CXCR4(ref.12) and likely modulate CXCR4 membrane localization.

We have further focused on the role of Src-family kinase Lyn, a well-characterized downstream target of the oncogenic p210 Bcr-Abl tyrosine kinase. In normal hematopoietic progenitor cells, Lyn is one of the signaling effectors downstream of CXCL12/CXCR4 stimulation. It was shown that in CML cells, phosphorylated BCR/ABL binds to and constitutively activates Lyn, which in turn becomes unresponsive to CXCL12-induced chemotaxis and increases the ability of immature cells to escape from the marrow.16 Lyn, like other Src-family kinases, has two major tyrosine residues, at Tyr507 and Tyr396, which cause inhibition and stimulation of catalytic activity, respectively. Our analysis of lipid raft fractions in KBM-5 cells demonstrates that lipid raft-associated Lyn has a higher level of Tyr396 phosphorylation than that of nonraft-resident Lyn, which must lead to its higher specific activity. Inhibition of Bcr-Abl by imatinib effectively inhibited both p-LynTyr507 and p-LynTyr396 levels. In turn, BM stromal cells strikingly prevented depletion of the active, raft-associated p-LynTyr396 by imatinib. This facilitated physical association of active Lyn with CXCR4 in lipid raft microdomains and stimulation of migration in response to CXCL12 (Figure 5).

Figure 5
figure5

Mechanisms of CXCR4 localization and Lyn activation in lipid rafts. Imatinib promotes clustering of CXCR4 in lipid rafts, which co-localizes with active p-LynTyr396 and induces CML cell migration to the CXCL12-producing BM stroma cells.

Although the mechanisms of MSC effects on Lyn kinase in the lipid rafts require further studies, the ligand-induced recruitment of the stem cell factor receptor c-Kit to lipid rafts is known to activate raft-localized Src-family kinases in hematopoietic cells. Because MSCs produce Kit ligand stem cell factor,37 stem cell factor and/or other cytokines may be involved in Lyn activation in lipid rafts. It has been further described that MSCs producing stromal cell-derived factor-1α stimulated Lyn activation in the BCR/ABL-negative acute myelogenous leukemia HL-60 cell line.15 It may be possible that the potential of MSCs to activate Lyn in lipid rafts is a generic phenomenon and mediates chemoresistance in other types of leukemias. In addition to stimulating migration, CXCR4 redistribution to lipid rafts and subsequent activation of Lyn in raft fractions may positively regulate pro-survival signaling events downstream of CXCL12/CXCR4 (Figure 5), which likely contribute to the survival of residual leukemic cells within the BM niches.38

Our observations of the key role of activated Lyn in restored CXCL12/CXCR4 signaling upon inhibition of Bcr-Abl suggest, furthermore, that utilization of Src kinase inhibitors ameliorates microenvironment-mediated resistance to TKIs. Consistent with this hypothesis, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 than imatinib. Recent studies have shown that dasatinib induced significantly higher rates of complete cytogenetic response (77% vs 66%, P=0.007) and major molecular response (46% vs 28%, P<0.0001) in CML compared with imatinib.39 We here, propose that one of the mechanisms responsible for the high potency and less frequent development of resistance to dasatinib might be blockade of Lyn-dependent microenvironmental resistance. This effect was incomplete, however, indicating that mechanisms other than Lyn activation may promote CXCR4 signaling, possibly through increased cell-surface expression. Recent findings by Fei et al.11 also showed that dasatinib increased cell-surface expression of CXCR4 in p210 Bcr-Abl-positive acute lymphocytic leukemia cells, and combined treatment cells with dasatinib and a CXCR4 inhibitor resulted in enhanced cell death. These data indicate the feasibility of using combinations of CXCR4 inhibitors and TKIs in CML and Ph(+) ALL.13 Although this concept has already demonstrated improved therapeutic efficacy in the preclinical models of AML and multiple myeloma,38, 40, 41 and has translated in the ongoing clinical trials, our findings indicate the alternative approach of lipid raft disruption to ameliorate microenvironmental resistance. The data presented here, indicate that lipid rafts facilitate key functional coupling between cross-linked CXCR4 and Lyn. Statins, a family of drugs that inhibit the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, may perturb the composition of cell membranes, resulting in lipid raft disruption42 as well as depletion of cellular mevalonate pools. In CML cells, it has been reported that simvastatin or a combination of lovastatin and interferon-alpha 2β led to cell growth inhibition through cell cycle arrest and caused significant reduction of phosphorylation in tyrosine, serine and threonine protein residues.43, 44 As such, inhibition of cholesterol synthesis, for example by statins, is a potential adjunct to CML therapy. It would be curious to examine whether the encouraging response rates reported in human AML trials combining standard chemotherapy with statins45 are associated with lipid raft disruption and blockade of CXCR4 signaling. This is, in particular, pertinent in light of recent findings, reporting upregulation of CXCR4 expression in AML cells exposed to cytotoxic agents.46

In summary, our data suggest the indispensable role of lipid rafts in CML cell migration to the BM niche. The molecular signaling events in the lipid rafts of TKI-treated CML cells, such as the stimulation of migration through interactions between CXCR4 and active Lyn, will provide important insight into the tumor–host interactions that contribute to CML progenitor cell survival within the BM microenvironment.

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Acknowledgements

We thank Drs Eri Hirasawa, Zhihong Zeng and Akimasa Someya for their invaluable help and discussion, and Tomomi Ikeda, Takako Shigihara-Ikegami, Akemi Koyanagi and Hiroaki Miyajima for their technical assistance. We thank Kathryn Hale for manuscript review. This work was supported by a Grant-in-Aid for Scientific Research of the Japan Science and Technology Agency, the Japan Leukemia Research Fund, the Osaka Cancer Research Fund and the Project Research Program from Juntendo University School of Medicine (to YT).

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Correspondence to M Konopleva.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Leukemia website

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Tabe, Y., Jin, L., Iwabuchi, K. et al. Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts. Leukemia 26, 883–892 (2012). https://doi.org/10.1038/leu.2011.291

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Keywords

  • chronic myeloid leukemia (CML)
  • imatinib
  • dasatinib
  • CXCR4
  • Lyn
  • lipid raft

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