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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton's tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)−6– or stroma–dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. Am. J. Hematol. 88:463–471, 2013. © 2013 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase of the TEC family, is preferentially expressed in hematopoietic cells [1, 2]. BTK is particularly critical for development of B-lymphocytes, as deduced from mice or humans who harbor BTK null mutations that cause X-linked agammaglobulinaemia [3, 4]. BTK is also important for effective osteoclastogenesis because its deficiency has resulted in incomplete osteoclast differentiation and mild osteopetrosis [5]. Indeed, BTK inhibitors are being developed for complications involving B-lymphocytes or myeloid cells such as cancer (e.g., lymphoma, chronic lymphocytic leukemia) [6-9] and inflammation (e.g., arthritis) [10, 11].

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of low-proliferating malignant plasma cells in the bone marrow and severe osteolytic bone disease induced by activation of osteoclasts and suppression of osteoblastogenesis [12]. Plasma cells express lower levels of BTK than most hematopoietic cells [13]. BTK activity is indispensable for B-lymphocyte migration and homing that is controlled by stromal cell-derived factor-1 (SDF-1), a chemokine that is highly expressed in bone [14]. The SDF-1/CXCR4 (C-X-C chemokine receptor type 4) signaling pathway is critically involved in metastasis, homing to bone and adhesion of myeloma cells [15, 16].

Recent studies demonstrated expression of BTK in myeloma cells and the ability of BTK inhibitor, PCI-32765 (Ibrutinib) to inhibit myeloma cell growth [17, 18] and migration towards SDF-1 [17]. Ibrutinib also shown to inhibit osteoclastogenesis and osteoclast-induced myeloma cell survival and growth [17]. In our clinical gene expression profiling (GEP) database, with samples from patients worldwide [19], we noted variable but overall higher expression of BTK in myeloma plasma cells compared to their normal, nonmyeloma counterparts. It has also been reported that cell-surface CXCR4 is expressed in a subpopulation of myeloma plasma cells and is highly variable among MM patients [15]. Based on this information, we hypothesized that BTK expression and cell-surface CXCR4 are linked and sought to further explore the role of BTK in myeloma cell migration, osteoclastogenesis and MM bone disease.

We demonstrated BTK expression in a large number of clinical myeloma samples and myeloma cell lines. We further explored the consequences of BTK inhibition by small hairpin RNA (shRNA) or LFM-A13, a BTK inhibitor [20], on myeloma cell migration, homing to bone and myeloma-induced bone disease in the SCID-rab model for MM [21-23].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

Primary myeloma cells and MM cell lines

The MM cell lines ARP-1 and CAG were established by our group at the University of Arkansas for Medical Sciences (UAMS) [24]. Other lines (H929, U266, OPM2, and JJN3) were obtained from American Type Culture Collection (ATCC; Manassas, VA). These cell lines were grown in vitro using RPMI-1640 (Mediatech, Inc., Manassas, VA) medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. The stroma-dependent BN MM line was also established at UAMS and was grown as described [25]. The myeloma cell lines ANBL6 and INA6 are interleukin (IL)−6–dependent [26]. For in vivo trafficking, the INA6 line was infected with luciferase/eGFP (enhanced green fluorescent protein) constructs containing lentivirus as previously described [25].

Primary myeloma cells were obtained from heparinized bone marrow aspirates from 28 patients with active MM during scheduled clinic visits. Purity of CD138+ primary samples was routinely >90% as assessed by flow cytometry [19]. Signed Institutional Review Board–approved informed consent forms are on record. Bone marrow samples were separated by density centrifugation using Ficoll-Paque (specific gravity, 1.077 g/mL, Amersham Biosciences Corp., Piscataway, NJ). Plasma cells were isolated using CD138 immunomagnetic bead selection and the autoMACs automated separation system (Miltenyi-Biotec, Auburn, CA). In some experiments, cell-surface expression of CXCR4 was determined by flow cytometry using isotype control and CXCR4 antibodies conjugated with phycoerythrin (PE, R&D Systems, Minneapolis, MN). CXCR4+ and CXCR4 myeloma cell populations were sorted using a fluorescence-activated cell sorter (BD Biosciences, Franklin Lakes, NJ). Mesenchymal stem cells (MSCs) from healthy donors and patients with MM were isolated and molecularly analyzed as previously described [27]. These cells were used to compare expression of BTK.

Migration assay

Chemotaxis assays for myeloma cells and osteoclast precursors were performed in triplicate using 8-µm and 5-µm pore size Transwell inserts, respectively, in 24-well plates. Osteoclast precursors were isolated from bone marrow aspirates using CD14 immunomagnetic bead selection and the autoMACs automated separation system (Miltenyi-Biotec, Auburn, CA). Myeloma cells or osteoclast precursors (150,000 and 50,000, respectively, in 0.3 mL medium) were placed in the system's upper chamber. Bottom chambers contained 0.7 mL media. As indicated, SDF-1alpha (30 nM) (R&D Systems, Minneapolis, MN) was added to the bottom chambers, and LFM-A13 (25 µM, Cayman Chemical Company, Ann Arbor, MI) was added to the upper chamber.

To evaluate myeloma cell migration, the filters were removed after approximately 6 hr at 37°C. Cells in the bottom chamber were fixed using HistoChoice (AMRESCO, Solon, OH) for 20 min. Migrating cells were counted microscopically in four nonoverlapping areas per well under 10× magnification.

Migration of osteoclast precursors was assessed as described [28]. Briefly, the filters were removed after 4 hr at 37°C. Cells were fixed in phosphate-buffered formalin (10%) for 10 min and stained with hematoxylin. Cells on the upper surface were removed using cotton-tipped swabs, and then the filters were flipped and glued onto slides. The number of osteoclast precursors in four nonoverlapping areas (0.5-mm2) of the filter's lower surface was determined microscopically.

Infection with shRNA or cDNA lentiviral particles

For the shRNA experiments, two types of particles were purchased from Sigma-Aldrich (St. Louis, MO) for this purpose: Non-target control (scramble, SHC002V) and BTK (SHCLNV, NM_000061) shRNA particles. To infect cells with indicated shRNA lentiviral particles, cells (20,000 cells/100 µL RPMI/well) were first incubated overnight in a 96-well plate. After 24 hr, lentiviral particles were added at a multiplicity of infection of 10 in the presence of 8 μg/mL polybrene. After overnight incubation with particles, the plate was spun in an Eppendorf 5810R centrifuge at 1,200 for 1 min (Eppendorf, Hauppauge, NY), and 80 μL of the medium was replaced with fresh RPMI medium. The next day, propagation of puromycin-resistant cells was started with puromycin dihydrochloride (Sigma-Aldrich) at a final concentration of 0.75 μg/mL. Selection continued until puromycin-resistant cells were propagated (approximately 2 weeks).

For enforced expression of BTK, CAG myeloma cells (100,000 cells/500 µL) were incubated in a 24 well plate overnight with eGFP lentiviral particles (LP-EGFP-LV105–200) or BTK lentiviral particles (LP-A0534-LV103, GeneCopoeia, Rockville, MD). The next day, propagation of puromycin-resistant cells was initiated with puromycin dihydrochloride (Sigma-Aldrich) at a final concentration of 0.75 μg/mL. Cell selection continued until puromycin-resistant cells were propagated (approximately 2 weeks).

GEP analyses

GEP with the Affymetrix U133 Plus 2.0 microarray (Affymetrix, Santa Clara, CA) was performed on CD138-purified plasma cells or MM cell lines as previously described [19]. Signal intensities were preprocessed and normalized by GCOS1.1 software (Affymetrix).

The signal of probe set 205504_at, representing BTK, was used in this analysis. GEP data on the CD138-selected plasma cells used in this study, from 559 newly diagnosed patients with MM, have been deposited with the Gene Expression Omnibus at the National Center for Biotechnology Information. The plasma cell GEP data are accessible through GEO Series accession number GSE2658.

Real-Time qRT-PCR

Total RNA (1 μg) from each sample was reverse-transcribed with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen Corp., Carlsbad, CA). The qRT-PCR was performed with the TaqMan gene expression assay on an ABI Prism 7000 sequence analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's recommended protocol. Reverse-transcribed RNA (10 ng) was amplified using the TaqMan Universal PCR Master Mix and TaqMan gene expression assays (ID HS99999905_m1 for glyceraldehyde 3-phosphate dehydrogenase [GAPDH] as an endogenous control; ID HS00975865_m1 for BTK, Applied Biosystems). Each reaction was run in triplicate. The comparative threshold cycle method was used to calculate the amplification fold, as specified by the manufacturer.

Western blots

INA6 MM cell lines were cultured for 3 hr with or without serum and then stimulated with SDF-1 at 37°C. Cells were spun down at indicated times, and pellets re-suspended in 1X RIPA buffer (Cell Signaling Technology, Danvers, MA) containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA (ethylenediaminetetraacetic acid), 1 mM EGTA (ethylene glycol tetraacetic acid), 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 (sodium orthovandate) and 1 µg/mL Leupeptin. Lysates were run in 10% SDS-PAGE gels (Biorad, Hercules, CA) and immunoblotted against anti-BTK antibody (BD Biosciences, Franklin Lakes, NJ) and anti-p-Y223-BTK (Epitomics, Burlingame, CA).

For the immunoprecipitation method, freshly obtained primary myeloma cells and INA6 or H929 MM cells were serum-starved for 3 hr and then seeded on a 24-well plate (2 × 106 cells/500 µL/well) and induced with SDF-1 (30 nM) for 2 and 5 min. The cells were then spun, and reaction was stopped by adding 200 µL RIPA buffer on the pellet. The pellet was vortexed and lysate rocked at 4°C for 15 min and quantified using the Pierce BCA Kit (Thermo Scientific, Rockford, IL). The lysate (1mg/mL protein) was incubated with 30 µL anti-phosphotyrosine 4G10 beads (Millipore, Temecula, CA) overnight at 4°C. BTK phosphorylation at tyrosine sites was then evaluated by immunoblot analysis.

For IL-6 signaling immunoblot, INA6 infected with scramble or BTK shRNA were lysed and immunoblotted with antibodies against phospho-Y705-STAT3, STAT3, phospho-Y694-STAT5, STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Cell Signaling Technology, Danvers, MA).

Immunohistochemistry

Cytospin slides of myeloma cells (30,000/slide) were fixed with HistoChoice (AMRESCO) followed by antigen retrieval using a water bath at 80°C for 25 min. After peroxidase quenching with hydrogen peroxide (3%) for 5 min, sections were reacted with mouse anti-human BTK or control immunoglobulin G (IgG) antibody (4 µg/mL, Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min. The assay was completed using Dako's immunoperoxidase kit (Dako Corp., Carpintera, CA). Sections were lightly counterstained with hematoxylin. An Olympus BH2 microscope (Melville, NY) was used to obtain images with a SPOT 2 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Adobe Photoshop version 10 (Adobe Systems, Inc., San Jose, CA) was used to process the images.

Osteoclast differentiation and activity

Osteoclast differentiation and activity were performed as previously described [28]. Briefly, mononucleated blood cells from patients with MM were cultured (2.5 × 106 cells/mL) in α-minimal essential medium supplemented with FBS (10%), antibiotics, receptor activator of nuclear factor κ-B ligand (RANKL, 50 ng/mL, PreproTech Inc., Rocky Hill, NJ) and macrophage colony-stimulating factor (M-CSF, 25 ng/mL, Sigma-Aldrich) for 3–4 days. At that time, nonadherent cells were removed from the osteoclast medium, and the remaining adherent cells were used as osteoclast precursors. To test the effect on osteoclast differentiation, osteoclast precursors were incubated in the osteoclast medium with and without LFM-A13 for 5–7 days. At that time, the cultures were fixed and stained for tartrate-resistant acid phosphatase (TRAP); the number of multinucleated osteoclasts that were TRAP-positive was counted as described [28].

Osteoclast activity was assayed by enumerating resorption pits based on dentin resorption [28]. Mononucleated blood cells (2.5 × 106 cells/mL) were cultured on dentin slices (Immunodiagnostic Systems, Inc., Fountain Hills, AZ) in 96-well plates in osteoclast medium for approximately 10 days. By that time, mature, multinucleated osteoclasts had been formed. The cultures were then treated with LFM-A13 (10 µM) or control diluent for 5 days. Dentine slices were treated with a bleach solution (10%) for 5 min and washed in distilled water. Resorption pits were photographed with a Nikon Eclipse 450 microscope (Nikon Instruments, Inc., Melville, NY). The ratio of resorption area to total area was quantified using OsteoMeasure XP (Osteometrics, Atlanta, GA), a bone histomorphometric system [28].

In vivo study

SCID mice were obtained from Harlan Laboratories (Indianapolis, IN). SCID-rab mice were constructed as previously described [21]. SCID-rab mice were used for in vivo trafficking study and long-term treatment with LFM-A13. Animals were housed and monitored in the Division of Laboratory Animal Medicine facility at the University of Arkansas for Medical Sciences. The Institutional Animal Care and Use Committee approved all experimental procedures and protocols (Assurance Number A3063-01, File 2779).

To evaluate in vivo trafficking of myeloma cells, INA6 cells that expressed luciferase/EGFP were treated overnight with 0.01% dimethyl sulfoxide (DMSO) or LFM-A13 (50 µM) in serum-free medium overnight in the presence of IL-6. These cells were then intravenously injected into SCID-rab mice tails (5 × 106 cells/mouse) and implanted rabbit bones were subjected to ex vivo imaging after 2 hr. To determine in vivo BTK inhibition on myeloma cell growth and bone disease, LFM-A13 stock was prepared in DMSO and diluted with Phosphate buffered saline (PBS) for a final concentration of 40 mg/kg in 100 μL PBS and 1% DMSO. SCID-rab mice were engrafted with the primary cells by direct injection of myeloma cells (0.5 × 106 cells/mouse) into the implanted bones. The mice were monitored for MM progression by measuring circulating human immunoglobulins (hIg) as previously described [29]. Myelomatous SCID-rab mice were injected intraperitoneally with LFM-A13 or vehicle (10 hosts/group) twice daily for 3 weeks. At the experiment's end, implanted bones were fixed in phosphate-buffered formalin (10%) for 24 hr. Implanted bones were further decalcified with EDTA (10% wt/vol, pH 7.0) and embedded in paraffin for sectioning. Sections (5 µm) were deparafinized in xylene, rehydrated with ethanol, and rinsed in saline prior to undergoing antigen retrieval by microwave. Sections were histochemically stained for TRAP as previously described [21, 30].

Statistical analyses

All values are expressed as mean ± standard error of the mean, unless indicated otherwise. Student's t-test was used to analyze BTK expression, the effect of treatment on myeloma cell migration and growth, in vivo homing, osteoclast formation and bone mineral density (BMD).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

BTK is expressed in myeloma plasma cells

Using our GEP database [19], we examined expression of BTK in bone marrow B-lymphocytes as well as in normal and myeloma plasma cells. As expected, BTK was highly expressed in normal B-lymphocytes and reduced in normal plasma cells (NPC, Fig. 1A). BTK expression in plasma cells from all patients (MM) was moderately higher (by approximately 20%, P < 0.056) than in normal plasma cells and markedly reduced in MM cell lines independently grown in culture than in normal plasma cells (Fig. 1A). In contrast to its expression in hematopoietic cells (e.g., B-lymphocytes) and osteoclasts, BTK expression was completely absent in bone marrow MSCs (Fig. 1A). Our GEP data also revealed that TEC (a BTK family-related kinase) is not expressed in myeloma cells (data not shown). BTK expression was validated by qRT-PCR in certain primary myeloma samples and MM cell lines (Fig. 1B). Among the MM cell lines that were examined by qRT-PCR, BTK expression was detected in the two IL-6–dependent lines (ANBL6 and INA6); in our BN stroma–dependent line [25]; and, at lower but detectable levels, in H929, CAG, ARP1, and JJN3 myeloma cells. Expression of BTK was undetectable in OPM2 cells (Fig. 1B). BTK expression was demonstrated at the protein level in primary myeloma cells and cell lines using Western Blot analysis (Fig. 1C). Taken together, these data show variable expression of BTK in primary myeloma cells and MM cell lines.

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Figure 1. Primary myeloma cells and IL-6 or stroma-dependent myeloma cell lines expressed BTK. A: GEP analysis demonstrated expression of BTK in bone marrow B-lymphocytes (BC, n = 6), plasma cells from healthy donors (NPC, n = 25), patients with MM (MM, n = 559), myeloma cell lines (MMCL, n = 42), cultured osteoclasts (OC, n = 8), and mesenchymal stem cells (MSCs, n = 15). Note that BTK expression is absent in MSCs, high in B-lymphocytes and variably expressed in primary myeloma plasma cells, while expressed at very low levels in most MM lines. B: Validation of BTK expression by qRT-PCR is shown for MM cells freshly obtained from nine patients, independent MM lines (OPM2, JJN3, ARP1, CAG, H929), the stroma–dependent BN line, IL-6–dependent INA6 and ANBL6 lines and osteoclast precursors (pOC). C: Western blot analyses: upper panel demonstrated variable expression of BTK in cell lines H929 and INA6, two freshly obtained primary MM plasma cell samples and osteoclast precursors (pOC). The Namwala (a human Burkitt's Lymphoma line) cell lysate was used as positive control. Lower panel showed BTK expression in four freshly obtained myeloma plasma cell samples. The MEG-01 (Chronic Myelogenous Leukemia whole cell lysate) was used as positive control.

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BTK mediates myeloma cell migration toward SDF-1 and homing to bone

The SDF-1/CXCR4 axis has a major role in homing of myeloma cells to bone [15, 16], and BTK has been shown to mediate migration of B-lymphocytes toward SDF-1 [14]. For these reasons, we analyzed the effect of BTK inhibition or overexpression on myeloma cell migration as induced by SDF-1. For BTK gene expression and protein knockdown, we used the INA6 cell line and found that BTK shRNA containing lentivirus resulted in significant knockdown of BTK in these cells compared to scramble (SCR) control (Fig. 2A), which inhibited the ability of the cells to migrate toward SDF-1 (Fig. 2B). BTK knockdown had no effect on myeloma cell-surface CXCR4 expression (data not shown). We also examined the effect of the BTK inhibitor LFM-A13 on migration of INA6 cells (Fig. 2C) and on primary myeloma plasma cells from eight patients, which all expressed BTK (Fig. 2D, Supporting Information Fig. 1). We found that SDF-1 (30 nM) consistently, although variably, promoted migration of myeloma cells, but this effect was significantly inhibited by LFM-A13 (25 µM) in 5 of the eight experiments (Fig. 2D, Supporting Information Figure 1A). LFM-A13 had no effect on SDF-1-induced migration of ARP-1 cells which express very low BTK (data not shown).

image

Figure 2. Inhibition of BTK by shRNA or LFM-A13 impeded migration of primary myeloma cells toward SDF-1. A. Knockdown of BTK in INA6 cells using lentiviral containing shRNA. Upper panel; relative expression of BTK as assessed by qRT-PCR in the INA6 cell line infected with lentiviral-containing non-target scramble (SCR) or BTK shRNA. Lower panel: Western Blot analysis demonstrating marked reduction in BTK protein in BTK knockdown INA6 cells. B: Introduction of BTK shRNA inhibited migration of INA6 MM cells toward SDF-1 (30 nM). C: Migration assay of the INA6 MM cell line in the presence of SDF-1 and its inhibition by pharmacological BTK inhibitor LFM-A13 (25 μM). D: Average effect of LFM-A13 (25 μM) on SDF-1-induced migration of primary myeloma cells from 8 patients. Cell viability in all experiments was >90%. Note significant inhibition of SDF-1-induced migration by LFM-A13. Individual migration assays for each of the primary myeloma cell samples are shown in Supporting Information Fig. 1A.

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To test gain of function, we selected the CAG MM line which expresses very low level of native BTK and overexpressed BTK in these cells (Fig. 3A). BTK overexpression had no effect on myeloma cell-surface CXCR4 expression (data not shown). Spontaneous and SDF-1 induced migration was insignificantly different between untreated CAG and eGFP-overexpressing CAG cells. BTK-overexpressing CAG cells had 2.2-folds increased spontaneous migration than untreated CAG cells (P < 0.0004) and 2.4-folds higher spontaneous migration than eGFP-overexpressing CAG cells (P < 0.0002). SDF-1 induced migration in all cells; however, the number of migrating BTK-overexpressing CAG cells was 10-folds higher than those of untreated CAG cells (P < 0.0001) and 3.9-folds higher than those of eGFP-overexpressing CAG cells (P < 0.001, Fig. 3B).

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Figure 3. BTK overexpression had stimulatory effect on in vitro migration of MM cell line. CAG MM line was stably infected with BTK or control eGFP constructs as described in “Materials and Methods”. A: Expression of BTK in untreated CAG cells and in eGFP- and BTK-overexpressing CAG cells. Upper panel: relative expression of BTK as assessed by qRT-PCR. Lower panel: Western blot validation of BTK overexpression. β-actin is used as a loading control. B: Enforced expression of BTK in CAG cells increased spontaneous migration and migration toward SDF-1 compared to untreated cells or cells enforced to express eGFP.

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In vivo, luciferase-expressing INA6 MM cells that were pretreated with LFM-A13 or control DMSO for overnight were injected intravenously into SCID-rab mice (Fig. 4A). Two hours after cell injection, ex vivo bioluminescence imaging of the implanted rabbit bone detected untreated INA6 MM cells in the implanted bone, but detection of LFM-A13-treated INA6 MM cells in the implanted bone was reduced by nearly sixfold in comparison with the control group (P < 0.03, Fig. 4B–D). These data suggest that BTK has a role in myeloma cell chemotaxis and homing to bone.

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Figure 4. Inhibition of BTK impeded migration and homing of myeloma cells to bone in vivo. A: X-ray radiograph of a SCID-rab mouse demonstrating the model used for the study. B: Luciferase-expressing INA6 MM cells were pretreated with LFM-A13 (50 μM) or 0.01% DMSO overnight and then intravenously (IV) injected in SCID-rab mice (5 × 106 cells/mouse, 3 mice/group). Live-animal imaging taken 2 hr after cell injection demonstrated localization of control INA6 cells (CONT) but not localization of LFM-A13–treated INA6 cells in implanted bones. C: Individual implanted bones were crunched and subjected to ex vivo bioluminescence imaging. Note reduced bioluminescence of bones from mice injected with LFM-A13–pretreated INA6 MM cells. Numbers on the side of each well represent bioluminescence intensity (p/sec/cm2/sr). D: Quantification of bioluminescence intensity showed an approximately six fold decrease (P < 0.03) in homing of INA6 MM cells treated with LFM-A13 compared to the control cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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BTK inhibition or overexpression has no effect on short term growth of myeloma cells

LFM-A13 had no effect on viability of primary myeloma cells or cell lines examined by Annexin V flow analysis (data not shown) and 3-days MTT assay up to 50 µM concentration (Supporting Information Fig. 2A). At these concentrations, LFM-A13 had no effect on normal peripheral blood mononuclear cells (PBMCs) or CD138-depleted bone marrow cells from a MM patient as assessed by MTT assay (Supporting Information Fig. 2B,C). BTK knockdown by shRNA had no effect on the growth and viability of INA6 cells (Supporting Information Fig. 2D). Enforced BTK expression in CAG cells had no significant effect on their growth assessed by MTT assay over a period of 7 days (Supporting Information Fig. 2E). Moreover, LFM-A13 had no inhibitory effect on growth of eGFP- or BTK-overexpressing CAG cells (Supporting Information Fig. 2F). These data suggest that BTK inhibition by LFM-A13 or shRNA, or enforced expression of BTK has no effect on survival and growth of myeloma cells.

BTK expression is correlated with cell-surface CXCR4 and is activated by SDF-1

Cell-surface CXCR4 is expressed on a subpopulation of myeloma cells (4–95%, Fig. 5A), which prompted us to analyze the relationship between BTK expression and cell-surface CXCR4. We found that the percent of cell-surface CXCR4 significantly correlated with BTK gene expression using a panel of 28 primary CD138-sorted myeloma plasma cell samples and 5 MM cell lines (R = 0.81, P < 0.0001, Fig. 5B).

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Figure 5. BTK expression was associated with cell-surface CXCR4, and BTK was phosphorylated (activated) by SDF-1 in myeloma cells. A: Representative histograms of myeloma primary (n = 3) and cell line (n = 3) samples demonstrating expression of cell-surface CXCR4 (% of cell-surface expression). B: Correlation coefficients indicate the strength of the linear relationship between the percent of cells expressing cell-surface CXCR4 (assessed by flow cytometry) and BTK expression (assessed by qRT-PCR) in myeloma cells (n = 33) including 28 freshly obtained primary myeloma cells (•) and the 5 cell lines CAG, U266, INA6, H929, and ANBL6 (▴). C: Immunoblot showing increase in phosphorylation of BTK (P-BTK) at Tyrosine 223 residue (activation state) over time by SDF-1 stimulation of INA6 cultured with no serum (starvation) for 3 hr. Note high baseline phosphorylation of BTK and no further phosphorylation of BTK by SDF-1 in serum condition. D: Anti-BTK immunoblot representing total phosphorylated BTK in INA6 cell lysates immunoprecipitated using 4G10 phosphotyrosine immunobeads. Increased BTK phosphorylation is observed at 5 min of SDF-1 stimulation only in serum-starved INA6 cells. E: Anti-BTK immunoblot representing total phosphorylated BTK in serum-starved H929 cell lysates immunoprecipitated using 4G10 phosphotyrosine immunobeads. Rapid phosphorylation of BTK at 2 minutes and decrease within 5 min of SDF-1 stimulation is observed. F: Anti-BTK immunoblot representing total phosphorylated BTK in serum-starved primary myeloma plasma cell lysates immunoprecipitated using 4G10 phosphotyrosine immunobeads. Increased BTK phosphorylation is observed at 5 min of SDF-1 stimulation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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We stimulated MM cells with SDF-1 to examine whether BTK is activated by this chemokine in myeloma cells using two different immunoblotting assays. First, INA6 MM cells were cultured for 3 hr with or without serum, then stimulated with SDF-1 (30 nM) for 2 and 5 min, followed by Western Blot analysis for phosphorylated (p-Y223) and total BTK. In serum-starved INA6 cells SDF1 induced BTK phosphorylation (Fig. 5C). However, baseline phosphorylated BTK was high in serum condition and SDF-1 did not induce further BTK phosphorylation in these cells (Fig. 5C). To confirm our findings, induction of BTK phosphorylation was demonstrated in serum-starved INA6, H929, and primary myeloma cells using pTyrosines pull-down assay (Fig. 5D-F). These assays indicate SDF-1 rapidly stimulates BTK activation in myeloma cells.

To further show the link between cell-surface CXCR4 and BTK expression, we used myeloma cells that express high (e.g., INA6 line) or low levels of BTK (e.g., H929 and CAG). We sorted the myeloma cells based on cell-surface CXCR4 expression and looked for BTK expression in the two MM cell sub-populations (Fig. 6A). Analysis by qRT-PCR showed that CXCR4+ myeloma cells from the cell lines H929 and CAG or a primary MM sample had higher BTK expression than CXCR4 myeloma cells (Fig. 6B). We observed similar results using immunohistochemistry for BTK on cytospin slides (Fig. 6C). Together, these data demonstrate the association between cell-surface CXCR4 expression and BTK expression in myeloma cells.

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Figure 6. BTK expression was higher in cell-surface CXCR4-sorted myeloma cells. Three MM cell lines (INA6, H929 and CAG) and a patient's myeloma cells were sorted based on cell-surface CXCR4. A: Representative fluorescence-activated cell sorting (FACS) analysis of myeloma cells sorted by CXCR4+ and CXCR4 cell-surface expression. The top panels demonstrate flow cytometry staining with control IgG (left) and CXCR4 antibody (right). The bottom panels demonstrate CXCR4 (left) and CXCR4+ (right) myeloma cells after sorting. B: Expression of BTK in one primary cell sample and indicated MM cell lines sorted by CXCR+ and CXCR4 as assessed by qRT-PCR. Data are expressed as fold change between CXCR4+ and CXCR4- sorted MM cells from each sample. C: Immunohistochemistry demonstrated greater BTK staining in primary myeloma cells sorted with CXCR4+ than with CXCR4 (left panels ×200 original magnification; right panels ×400 original magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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IL-6 does not mediate BTK expression and BTK has no effect on STAT3 activation in myeloma cells

Since BTK expression was observed to be high in IL-6 dependent cell lines than IL-6 independent cell lines we sought to test whether IL-6 affects expression of BTK in myeloma cells and whether BTK is involved in IL-6 signaling. When IL-6-indepndent MM cell lines as H929, ARP1, and OPM2 were cultured in the presence of IL-6 for 24 hr, BTK expression remained unchanged. In contrast, in these cells IL-6 upregulated expression of SOCS3, a known IL-6 target gene in myeloma cells [31] (Supporting Information Fig. 3A).

IL-6 is known to induce downstream signaling in myeloma cells through STAT activation [32]. To address the question if BTK is involved in IL-6 signaling, we performed immunoblot assay against STAT3 and STAT5 phosphorylation state in both control scramble and BTK knockdown INA6 lysates. We detected similar level of STAT3 phosphorylation in both scramble and BTK knockdown INA6 cells (Supporting Information Fig. 3B). STAT5 activation was absent in both groups (Supporting Information Fig. 3B). These data suggest that IL-6 and BTK signaling pathways in myeloma cells are not directly linked.

BTK mediates migration, differentiation and activity of osteoclast precursors

Similar to myeloma cells, osteoclast precursors have been reported to express BTK and migrate in an SDF-1 gradient [33]. We first demonstrated that BTK is expressed in mature osteoclasts by GEP (Fig. 1A) and then that BTK is expressed in human osteoclast precursors using qRT-PCR and Western Blot (Fig. 1B, C). We also demonstrated that blood monocytes (used as osteoclast precursors), sorted based on cell-surface CD14 expression, expressed cell-surface CXCR4 (Fig. 7A). We anticipated that these cells may play a role in MM-induced osteoclastogenesis and that high SDF-1 level in myelomatous bone may attract these cells and induce their differentiation into osteoclasts [34]. Indeed, osteoclast precursor cells expressing CD14 migrated toward the SDF-1 gradient (30 nM), an effect that was partially but significantly inhibited by LFM-A13 (25 µM, Fig. 7B). LFM-A13 also reduced migration of the murine RAW 264.7 cell line of osteoclast precursors toward SDF-1 by 40% (data not shown). LFM-A13, in a dose-dependent manner, significantly suppressed differentiation of human osteoclast precursors into multinucleated, TRAP-expressing osteoclasts in media supplemented with RANKL and M-CSF (Fig. 7C,D). LFM-A13 (10 µM) also inhibited the bone resorption activity of mature osteoclasts on dentine slices by approximately 50% (P < 0.002, Fig. 7E).

image

Figure 7. BTK inhibition reduced migration and differentiation of osteoclast precursors and bone resorption activity of mature osteoclasts. A: Flow cytometry demonstrating expression of cell-surface CXCR4 in osteoclast precursors sorted from blood using CD14 immunomagnetic beads. B: Migration assays demonstrated migration of osteoclast precursors toward SDF-1 (30 nM), an effect that was suppressed by LFM-A13 (25 µM). C: LFM-A13 inhibited differentiation of osteoclast precursors in medium supplemented with RANKL and M-CSF in a dose-related manner (each P value indicates significant dose effect against untreated control). D: Representative photographs demonstrating TRAP staining in osteoclast precursor cultures treated with vehicle (CONT) or 10 μM LFM-A13. E: LFM-A13 (10 μM) suppressed pit formation by mature osteoclasts on dentine slices. All functional assays were performed in triplicate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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BTK inhibition prevents myeloma-induced bone disease

For the in vivo study, we exploited the SCID-rab model and engrafted mice with a patient's myeloma cells, which express high levels of BTK (Primary #2, Fig. 1B). Upon establishment of MM growth, mice were treated with LFM-A13 or vehicle (10 mice/group) for 3 weeks, as assessed by measurement of circulating human λ light chains. The Bone mineral density (BMD) of the implanted bones was reduced by 15% from pretreatment levels in control hosts, but BMD of the implanted bone was maintained close to pretreatment levels in myelomatous bones from mice treated with LFM-A13 (Fig. 8A, P < 0.04 versus control hosts). X-ray radiographs of the implanted myelomatous bone before treatment initiation and at experiment's end showed that bone loss was prevented with LFM-A13 treatment (Fig. 8B). Staining of decalcified bone sections for TRAP revealed significant reduction in the number of osteoclasts in bones treated with LFM-A13 (Fig. 8C). LFM-A13 also reduced the growth of myeloma cells in SCID-rab mice as assessed by circulating λ light chain levels, 11 and 22 days after treatment was initiated. However, the results only neared significance (P < 0.07, Fig. 8D).

image

Figure 8. In vivo, LFM-A13 markedly inhibited myeloma-induced bone disease and moderately reduced myeloma growth. SCID-rab mice engrafted with primary myeloma cells, which express high levels of BTK (see Fig. 1, Primary #2), were treated with LFM-A13 intraperitoneally (n = 10, 40 mg/kg, twice daily) or vehicle (n = 10) for 3 weeks. A: Bone Mineral Density (BMD) changes in implanted, myelomatous bones demonstrated reduced BMD in the control group but maintained BMD levels after treatment with LFM-A13. B: X-ray images of implanted, myelomatous bones prior to treatment (Pre-Rx) and at experiment's end (Final) in five representative mice from each group (Control and LFM-A13). Note bone loss in mice treated with the control compared to bone preservation in mice after treatment with LFM-A13. C: Histological sections of the implanted myelomatous bones revealed a reduced number of osteoclasts (OC) when treated with LFM-A13. D: MM burden monitored by measurement of circulating human immunoglobulins demonstrated reduced MM growth after treatment with LFM-A13, at a level close to statistical significance (P < 0.07).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

Our study revealed the expression of BTK in myeloma cells, particularly in subpopulations that express cell-surface CXCR4, and showed that BTK expression mediates the migration of myeloma cells toward SDF-1 and homing to bone in vivo. We further found that BTK activity in osteoclast precursors directly mediated their migration toward SDF-1 and their differentiation to osteoclasts. Pharmacologic inhibition of BTK effectively prevented myeloma-induced bone loss and slightly reduced tumor growth in our experimental model.

Although BTK is reportedly expressed at low levels in normal plasma cells [13], our clinical GEP data indicate that CD138-expressing myeloma plasma cells from a large subset of patients express similar or higher levels of BTK compared to normal bone marrow plasma cells. Interestingly, myeloma cell lines grown independently in vitro expressed very low levels of BTK or do not seem to express BTK (e.g., CAG, JJN3. ARP1, H929, OPM2). However, even in such cell lines (e.g., H929 and CAG, Fig. 6B), enrichment of myeloma cells expressing cell-surface CXCR4 resulted in detectable BTK expression. In our unique myeloma lines, established by passaging in SCID-hu/SCID-rab mice [25], or in IL-6–dependent lines, BTK is consistently expressed at levels similar to those of primary myeloma cells, further emphasizing their clinical relevancy. Additionally, IL-6 had no effect on BTK expression in IL-6-independent cell lines, which indicates that IL-6 dependency of other cell lines (INA6, ANBL6) is not directly related to BTK expression. BTK knockdown in INA6 cells did not change IL-6 mediated STAT3 or STAT5 phosphorylation state, negating the role of BTK upstream of STAT activation in IL-6 signaling pathway [32]. Two intriguing aspects of our findings are that BTK expression and cell-surface CXCR4 are associated in myeloma plasma cells and that BTK is activated by SDF-1 in these cells. In addition, BTK inhibition consistently reduced in vitro migration of myeloma cells toward SDF-1, a chemokine that is highly expressed in bone. Conversely, BTK overexpression in a cell line that expresses very low level of native BTK increased their spontaneous and SDF-1-induced migration capacity. Our data confirm previous report [17] and provide evidence that links BTK with CXCR4 signaling.

Although recent studies suggest that BTK inhibitors are cytotoxic to myeloma cells [17, 18] we could not observe significant differences in myeloma cell growth by BTK inhibition or overexpression in a short-term experimental setting, while in vivo LFM-A13 slightly suppressed MM growth. The differences in the results may be related to the use of different inhibitors and myeloma cells. Based on our findings, we suggest that the small subpopulations of myeloma cells that co-express BTK and cell-surface CXCR4 may be responsible for homing of myeloma cells to bone. Whether these cells are sufficient to sustain MM or they possess tumorigenic advantages, possibly through their superior survival rates within the specialized, supportive bone marrow microenvironment, is a matter of continual investigation. In agreement with our previous findings about the ability of recognizable myeloma plasma cells to produce MM in vivo [21, 30], it is reasonable to speculate that myeloma plasma cells expressing BTK and cell-surface CXCR4 are part of a subpopulation of cells responsible for transferring and sustaining MM.

In addition, a possible outcome of BTK inhibition is inhibition of bone disease induced by MM. In osteoclast precursors, BTK has been shown to integrate the RANKL/RANK and immunoreceptor tyrosine-based activation motif (ITAM) signals, resulting in phosphorylation of phospholipase C-gamma (PLCγ, a signal-transducing element), induction of the osteoclastic transcription factor NFATC1 (nuclear factor of activated T-cells, cytoplasmic 1) and subsequent osteoclast formation [5, 17]. Evidence also indicates that BTK is required for efficient osteoclast activation [35]. We confirmed that BTK inhibition reduced the osteoclast formation and activity, and for the first time also provide evidence that migration of human CD14-expressing osteoclast precursors towards SDF-1 is reduced by BTK inhibition. Previous studies have indicated that RANKL is upregulated in myelomatous bones and mediates formation of osteolytic lesions [36, 37]. Treatment with LFM-A13 of SCID-rab mice that bear MM significantly reduced the numbers of osteoclasts in myelomatous bone and prevented osteolysis. Thus, inhibition of MM growth in vivo with LFM-A13 could be mediated by direct inhibition of BTK in myeloma cells and indirectly by inhibition of osteoclast activity, as previously shown using other osteoclast-specific inhibitors [27, 37].

In summary, this study highlights the potential role of BTK activity in myeloma cell clonogenicity and metastasis and in osteoclast-mediated bone resorption. BTK inhibitors that are being developed for B-cell malignancies and inflammatory disorders [2, 38] may also benefit patients with MM [17].

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

The authors wish to thank the faculty, staff, and patients of the Myeloma Institute for Research and Therapy for their support and the Office of Grants and Scientific Publications at the University of Arkansas for Medical Sciences for editorial assistance during preparation of this manuscript.

Authorship Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

RB performed in vitro and in vivo studies, analyzed and interpreted the data and was one of the writers for the paper. WL, AP, SK, SUV, and XL performed in vitro and in vivo studies. JS performed gene expression profiling. JE helped design the experimental studies and analyzed and interpreted the data. SU, BB, and FVR provided patient materials and interpreted the data. SY designed and performed the research, conceptualized the work, analyzed and interpreted the data and was one of the writers for the paper.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Authorship Contributions
  9. Supporting Information

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