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`
`Regular Article
`
`LYMPHOID NEOPLASIA
`
`Egress of CD191CD51 cells into peripheral blood following treatment
`with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell
`lymphoma patients
`Betty Y. Chang,1 Michelle Francesco,1 Martin F. M. De Rooij,2 Padmaja Magadala,1 Susanne M. Steggerda,1
`Min Mei Huang,1 Annemieke Kuil,2 Sarah E. M. Herman,3 Stella Chang,1 Steven T. Pals,2 Wyndham Wilson,3
`Adrian Wiestner,3 Marcel Spaargaren,2 Joseph J. Buggy,1 and Laurence Elias1
`
`1Research Department, Pharmacyclics, Inc., Sunnyvale, CA; 2Pathology Department, Academic Medical Center, Amsterdam, The Netherlands;
`and 3Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
`
`Key Points
`
`(cid:129) MCL cells are mobilized
`into the peripheral blood of
`patients treated with the BTK
`inhibitor ibrutinib.
`Ibrutinib dose-dependently
`inhibits BCR- and chemokine-
`mediated adhesion and
`migration of MCL cells.
`
`(cid:129)
`
`Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in
`clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic
`lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells
`following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR)
`pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle
`cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized
`among patients with MCL and further investigate the mechanism of this effect. Peripheral
`blood CD191CD51 cells from MCL patients were found to have significant reduction in
`the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma
`chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment.
`Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemo-
`taxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/
`CXCL13 stimulations of pBTK, pPLCg2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in
`coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of
`malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738. (Blood. 2013;122(14):2412-2424)
`
`Introduction
`
`Mantle cell lymphoma (MCL) is an aggressive type of B-cell
`malignancy, constituting 8% of non-Hodgkin lymphomas.1-3 MCL
`is typically characterized by the t(11;14)(q13;q32) translocation,
`which drives cyclin D1 overexpression. Constitutive activation of
`the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and
`nuclear factor kB pathways contribute to the pathogenesis of MCL.1
`The majority of MCL patients present with advanced disease at
`diagnosis, and more than 90% of patients have extranodal man-
`ifestations with circulating MCL cells, bone marrow, and gastroin-
`testinal involvement. In general, MCL patients have a poor prognosis,
`with a median overall survival time of 30 to 43 months and fewer
`than 15% of them are long-term survivors.2,3 This demonstrates a
`clear need for new therapeutics for the treatment of this disease.
`The interaction of neoplastic B cells with stromal cells in the
`lymph node (LN) or bone marrow microenvironment plays a critical
`role in the survival, progression, and drug resistance of various
`B-cell malignancies,4-7 including MCL.6,8,9 Importantly, the homing
`and trafficking of B cells into the microenvironment is tightly
`controlled and regulated by the interaction of chemokine receptors
`and adhesion molecules.10-15 The contact between MCL cells and
`
`mesenchymal stromal cells (MSCs) is established and maintained
`by chemokine receptors and adhesion molecules. Stromal cells in
`lymphoid tissues constitutively express chemokines such as CXCL12
`and CXCL13, forming gradients that allow the homing of
`B lymphocytes from the periphery into tissue compartments. MCL
`cells express G protein–coupled chemokine receptors such as
`CXCR4 and CXCR5 that bind CXCL12 and CXCL13, respec-
`tively.6 Adhesion is facilitated by binding of integrins such as
`VLA-4 on B cells to VCAM-1 on stromal cells and fibronectin in the
`extracellular matrix.16 In addition to the chemokine receptor and
`integrin engagement, it has been shown that B-cell receptor (BCR)
`activation is involved in integrin (such as VLA-4) -mediated
`adhesion7,14,16-18 and is thought to contribute to the growth and
`survival of most types of B-cell malignancies.17,19-21 BCR signaling
`pathway phosphoproteins are represented abundantly in MCL cell
`lines,1,22,23 and genomic lesions or constitutive activation of sig-
`naling proteins downstream of the BCR pathways such as SYK and
`PI3KA have been reported in MCL.23-25
`Since BCR signaling is important for integrin-mediated adhesion,
`growth, and survival of B lymphocytes, Bruton tyrosine kinase
`
`Submitted February 1, 2013; accepted August 2, 2013. Prepublished online as
`Blood First Edition paper, August 12, 2013; DOI 10.1182/blood-2013-02-
`482125.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked “advertisement” in accordance with 18 USC section 1734.
`
`M.F. and M.F.M.D.R. contributed equally to the manuscript.
`
`The online version of this article contains a data supplement.
`
`© 2013 by The American Society of Hematology
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`EGRESS OF MCL CELLS AFTER IBRUTINIB TREATMENT
`
`2413
`
`(BTK), a key component of the BCR pathway, is thus significant in
`B lymphocyte adhesion and survival.7,14,16,18 More recently, it has
`been shown that BTK plays a role in chemokine (such as CXCL12)
`-controlled B-cell chemotaxis and homing.14 The BTK inhibitor
`ibrutinib (PCI-32765) is an irreversible covalent inhibitor with a
`50% inhibitory concentration of 0.5 nM against BTK in biochemical
`assays, has been found to have broad antitumor activity in B-cell
`malignancies including MCL,26,27 and is currently being evaluated
`in phase 3 clinical studies. In the initial phase 1 study, we noted
`that many MCL patients had rapid increases of CD51 B lymphocytes
`in the peripheral blood (PB) following ibrutinib treatment. This
`often occurred concomitantly with rapid reductions of lymphade-
`nopathy, suggesting an egress of malignant cells from tissues into
`PB. The schedule of drug administration for most patients in the
`phase 1 study was in cycles of daily administration for 28 days,
`followed by 7 days without drug administration. We observed that
`the lymphoid flux was rapidly reversed during the 7-days-off portion
`of the treatment, with prompt reappearance at the beginning of the
`next cycle, resulting in a sawtooth pattern in PB. These patterns,
`along with immunophenotypic characterization of the cells involved,
`are presented here. We further investigated the mechanism of this
`effect by using MCL cell lines and primary cells and also in an
`MCL–stromal coculture system. We demonstrated that ibrutinib
`suppressed BCR- and CXCL12-/CXCL13-mediated adhesion and
`chemotaxis, suppressed migration of cells beneath the stromal cells
`(pseudo-emperipoiesis), and inhibited the phosphorylation of
`BTK, PLCg, and ERK in MCL cells. These observations effec-
`tively highlight the importance of BTK catalytic activity in the
`homing of MCL cells into lymphoid tissues and provide insight
`into the relevant mechanisms.
`
`Materials and methods
`
`Refer to supplemental Methods for details (available on the Blood Web site).
`
`Primary human MCL specimens from drug-treated patients
`
`Blood was drawn from MCL patients enrolled in PCYC-0475326 or PCYC-
`110427 (before April 1, 2012) in accordance with International Conference
`on Harmonisation Good Clinical Practice guidelines and principles of the
`Declaration of Helsinki, with informed consent from each patient and in
`compliance with the protocols approved by the relevant institutional review
`board. The blood samples were drawn into sodium heparin cell preparation
`tubes (BD) and shipped overnight to Pharmacyclics, Inc., within 24 hours.
`In a laminar flow hood, the peripheral blood mononuclear cells (PBMCs) were
`collected, washed with phosphate-buffered saline (PBS), and frozen in 90%
`fetal bovine serum and 10% dimethylsulfoxide in liquid nitrogen until use.
`
`Cell lines and primary material for ex vivo studies
`
`Adhesion studies were performed with PBMCs from MCL patients of the
`Academic Medical Center (Amsterdam, The Netherlands). For ex vivo
`CXCR4 and CD38 staining studies, PB and LN biopsies were collected
`from treatment-naive MCL patients enrolled in National Cancer Institute Study
`05-C-0170 (www.clinicaltrials.gov #NCT00114738) with approval from
`the National Institutes of Health Institutional Review Board and informed
`consent. Matched PB and LN samples were obtained on the same day and
`were processed and analyzed in parallel.
`
`and fixed in PBS and 1.6% paraformaldehyde. Cells to be analyzed for
`proliferation with Ki67 were permeabilized with 70% ethanol at 220°C
`overnight, rehydrated with PBS, and stained with Ki67 antibody.
`
`Flow cytometry
`
`BD FACS (fluorescence-activated cell sorter) Canto II was used for all flow
`cytometry collections. Phosphoflow assays were stained and performed as
`described.28 At least 10 000 CD191 cells were collected from each staining
`sample. The data were analyzed and quantified by using FlowJo v7.6, and
`the geometric mean was derived and presented for the mean fluorescence
`intensities (MFIs) of histograms for the specifically stained cell populations.
`
`Coculture assays
`
`Cocultures of M2-10B4 stromal cells and the MCL cell line Mino were
`established according to Burger et al.10 Mino cells were treated with
`vehicle, pertussis toxin, or ibrutinib for 1 hour at 37°C and then added to
`confluent monolayers of stromal cells. The cocultures were incubated at
`37°C for 5 hours to overnight to allow migration of Mino cells beneath the
`stromal cell layer, after which they were washed extensively to remove
`unmigrated cells. For cocultures using live-cell tracer dyes, M2-10B4 and
`Mino cells were first loaded with CellTracker Green 5-chloromethylfluorescein
`diacetate (CMFDA) and CellTracker Orange 5-(and-6)-(((4-chloromethyl)
`benzoyl)amino)tetramethylrhodamine (CMTMR), respectively. For micros-
`copy, cells were fixed with paraformaldehyde and mounted on slides with
`4,6 diamidino-2-phenylindole mounting medium (Vectashield). For quantifica-
`tion of migration of Mino cells in cocultures by flow cytometry, cells were
`trypsinized and stained with APC-Cy7–labeled anti-CD19 antibody. Cells were
`counted by using CountBright absolute counting beads (Life Technologies).
`
`Actin polymerization in Mino cells
`
`Mino cells were adhered to coverslips in serum-free media for 30 minutes at
`37°C and treated with dimethylsulfoxide, pertussis toxin, or ibrutinib for 1 hour.
`Cells were fixed with paraformaldehyde, permeabilized with Triton X-100,
`and stained with Alexa Fluor 594–labeled phalloidin (Molecular Probes).
`Coverslips were mounted on glass slides by using Vectashield mounting
`medium containing 4,6 diamidino-2-phenylindole. Microscopy was performed
`on a Zeiss Axioplan 2 microscope using a 633/1.40 oil immersion Plan-
`Apochromat objective, and images were acquired with a Zeiss AxioCam
`MRm charge coupled device camera and AxioVision v.4.8 software. For
`densitometry, at least 30 cells were imaged for each condition.
`
`Adhesion and migration assays
`
`Cell adhesion7,14 and migration assays were performed as described.14,29
`
`Immunoblotting
`
`Western blots were performed as previously described.7,29
`
`Statistical analysis
`
`Analyses were performed by using GraphPad Prism 4.0. Statistically
`significant differences were determined by using either analysis of variance
`with Bonferroni’s post hoc comparison, or unpaired two-tailed Student t test
`was used to determine the significance of differences between two means.
`The one-sample t test was used to determine the significance of differences
`between means and normalized values (100%).
`
`Results
`
`MCL phenotyping
`
`PBMCs were washed, pelleted, and resuspended in PBS and 2% fetal bovine
`serum containing phenotyping surface antibodies. All staining cocktails were
`run in duplicate tubes. Cells were stained for 30 minutes, washed, pelleted,
`
`Transient increase in absolute lymphocyte count following
`ibrutinib administration to MCL patients
`
`In a phase 1 study that enrolled patients with various non-Hodgkin
`lymphomas, MCL patients were treated with ibrutinib in 35-day
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`cycles during which the drug was administered once a day for
`28 days with a 7-day drug holiday between cycles.26 Under these
`conditions, a cyclical pattern of increasing and decreasing absolute-
`lymphocyte count (ALC) was observed. This was demonstrated
`by an increase in ALC following the first few weeks of treatment
`followed by a return to baseline after the 7-day drug holiday
`(Figure 1A). This cyclic ALC pattern continued for the duration of
`the treatment (data not shown). During the course of ibrutinib treat-
`ments, tumor volumes (quantified by sum of perpendicular diameters
`measurements from scans) decreased by 75% on average (Figure 1A)
`during 2 to 6 treatment cycles. Thus, during the first 6 cycles of
`treatment, the cyclic changes of PB ALC occurred concomitantly
`with nodal responses in these patients. The increase in ALC was
`observed in a subsequent (phase 2) study in which MCL patients
`were treated with a fixed continuous daily dose of 560 mg without
`interruption. In this trial, the ALC increased by 100% to 200%
`following the first 2 to 4 weeks of treatment, followed by notable
`gradual reductions in ALC commencing by the eighth week of
`treatment and continuing over the following months (Figure 1B).
`
`Elevated ALC is due to an increase of light chain–restricted
`CD191CD51 cells
`
`In order to define the population of lymphocytes increased by
`ibrutinib, the PBMCs of patients isolated before (day 1 [D1]) and
`after 1 week of treatment (day 8 [D8]) were stained with CD3, CD19,
`and CD5 and analyzed by flow cytometry. The increased lymphocytes
`were characterized as CD3–CD191CD51; both the absolute count
`and the percentage of CD191CD51 cells in the lymphocyte popu-
`lation were significantly increased after 1 week of ibrutinib treat-
`ment (P , .05), whereas the absolute count and the percentage of
`CD191CD5– cells was not (Figure 1C). An illustrative patient is
`shown in Figure 1D, in whom the CD191CD3– and CD191CD51
`populations before drug treatments were 9.29% and 84.4%, respec-
`tively, and they increased to 63% and 98.8% after 1 week of treat-
`ment. The CD191CD3–CD51 cells were light chain–restricted (data
`not shown), likely reflecting increased circulating MCL cells in the
`periphery following 1 week of drug treatment. In some cases, the
`mobilized cells made up a distinct subset of CD45dim small cells,
`which is also consistent with MCL (data not shown).
`To confirm that full inhibition of the target BTK was achieved in
`these patients, occupancy of the BTK active site by ibrutinib was as-
`sessed in PBMCs from MCL patients by using a competitive binding
`fluorescent probe assay.30 On average, more than 90% target occupancy
`was observed in patients following 1 week of treatment (Figure 1E-F).
`
`The peripheral CD191CD51 population has decreased Ki67,
`pERK, and surface CD38 and CXCR4 expression following
`drug treatment
`Peripheral CD191CD51 cells were analyzed for markers commonly
`associated with cell proliferation/activation states such as CD38,31,32
`Ki67,33 and pERK. CD381 surface expression was higher in the
`CD191CD51 cells than in the normal CD191CD5– cells prior to
`drug treatment and became even higher in some patients immediately
`after treatment (Figure 2A), likely because of the higher CD38 ex-
`pression in tissues (Figure 2D), but it decreased significantly
`following 1 week of treatment (P , .05) (Figure 2B) and decreased
`even further with longer treatment, while the CD38 expression in
`the normal B cells did not change (Figure 2A). In vitro ibrutinib
`treatment did not affect CD38 expression in MCL cells (supplemental
`Figure 1). Furthermore, Ki67 expression, commonly used as a marker
`of proliferation, was significantly reduced after treatment (P , .01)
`
`(Figure 2B; supplemental Figure 7). pERK expression was generally
`higher in the CD201CD51 cells of MCL patients compared with
`healthy volunteers and was significantly reduced by ibrutinib treatment
`(Figure 2B, lower panel; P , .05).
`Since the chemokine receptor CXCR4 is important for B-cell
`homing to lymphoid tissues,34,35 we determined the surface CXCR4
`expression in the MCL cells and found that CXCR4 was signif-
`icantly reduced (P , .05) in the CD191CD51 population following
`1 week of treatment (Figure 2C). We then analyzed CXCR4 ex-
`pression on patient-matched LN- and PB-resident MCL cells from
`3 untreated patients and found that CXCR4 expression was lower
`in MCL cells isolated from LNs compared with PB in all 3 patients
`examined (Figure 2D), similar in trend to those noted in chronic
`lymphocytic leukemia (CLL) patients in whom CXCR4 surface
`expression is lower in LN compared with PB36,37 because of receptor
`endocytosis from high tissue concentrations of CXCL12.4,10,36,38
`Therefore, the newly circulating CXCR4lo MCL cell population
`is consistent with the mobilized cells originating from tissues such
`as LNs. This interpretation is further supported by the notable
`reduction in lymphadenopathy observed during the same period
`(Figure 1A). Importantly, ibrutinib treatment in vitro did not have a
`direct effect on CXCR4 surface expression in primary MCL cells at
`concentrations of 1 to 1000 nM (supplemental Figure 1).
`We also examined changes to plasma chemokines in the treated
`patients and found that chemokines important in B-cell trafficking/
`homing (CXCL13) and T-cell/accessory-cell attractions (CCL22,
`CCL4, CCL17)6,39,40 were reduced on average by more than 50%
`following 1 week of treatment. By the end of the first cycle of
`treatment, in addition to the decrease of CCL4 and CCL22, cytokines
`important for MCL proliferation—interleukin 10 (IL-10) and
`tumor necrosis factor a (TNF-a)41,42—were also reduced by 50%
`(Figure 2E).
`
`Ibrutinib inhibits pseudo-emperipoiesis in
`MCL–stromal coculture
`
`The transient increase of ALC in MCL patients treated with ibrutinib
`may be due to a disruption in cellular adhesion and migration within
`the LN or tissue compartment. To investigate this, we established
`the in vitro effect of ibrutinib on the pseudo-emperipoiesis of MCL
`in stromal cell cocultures. Primary MCL cells or the Mino cell line
`were grown in coculture with M2-10B4 murine bone marrow
`stromal cells. We found that primary MCL cells or Mino cells both
`adhered to and migrated beneath the M2-10B4 cells. Significant
`inhibition of pseudo-emperipoiesis by ibrutinib was observed, as
`demonstrated by light microscopy (Figure 3A-B), and the number
`of Mino cells or primary MCL cells remaining in the coculture were
`quantified by flow cytometry of hCD191 cells harvested by gentle
`washing following 4 hours of coculture (Figure 3D-E, left panels).
`Ibrutinib dose-dependently inhibited migration of Mino cells beneath
`the stromal cells, and the inhibition was significant at 100 nM
`(P , .01) and 1000 nM (P , .001). Pertussin toxin, a well-studied
`G protein coupled receptor inhibitor used as a positive control for
`inhibition of Mino cell migration significantly inhibited migra-
`tion at 200 ng/mL (P , .001). In addition, CXCL12, an important
`chemokine for B-cell homing produced by stromal cells, increased
`cortical actin of Mino cells, as assessed by phalloidin fluorescence
`microscopy, and this response was also dose-dependently and
`significantly inhibited by ibrutinib treatments at 10 and 100 nM
`(P , .001) (Figure 3C-D, right panel). Ibrutinib also suppressed
`actin polymerization of primary MCL in coculture at 100 nM
`(P , .001) (Figure 3E, right panel).
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`EGRESS OF MCL CELLS AFTER IBRUTINIB TREATMENT
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`Figure 1. Transient mobilization of lymphocytes in MCL patients treated with ibrutinib. The mean percentage change of ALC (over baseline) is graphed against
`treatment time. (A) Ibrutinib treatment was in 35-day cycles of 28 days on and 7 days off. Mean percentage ALC change and percentage of the sum of perpendicular
`diameters (SPD) change are plotted to treatment cycles (n 5 9). (B) Ibrutinib treatment was continuous with no gap in treatment. Mean percentage ALC change compared
`with baseline (plotted as mean 1 standard error [SE]; n 5 17) is plotted against treatment time. Time points plotted are D0 (day zero), D1, D8, D15, D22, and end of cycles
`1
`1
`1
`CD5– cells following 1 week of ibrutinib treatment. Note the statistically significant
`1, 2, 3, 4, and 5. (C) The absolute count (ABS) and percentage of CD19
`CD5
`vs CD19
`1
`1
`cells of MCL patients following treatment. *P , .05 (paired t test; n 5 16). (D) Flow plot of gated lymphocytes of PBMC samples
`change in the absolute count of CD19
`CD5
`1
`CD3–
`from a representative MCL patient before and after ibrutinib treatment (560 mg per day) for 7 days. PBMCs were stained with CD3, CD19, and CD5. Note increase of CD19
`1
`1
`and CD19
`CD5
`population after 7 days of drug treatment. (E) Fluorescent probe occupancy assay of BTK in PBMCs isolated from a representative MCL patient before treatment
`(predose), and after 4 hours, 24 hours after first dose, before treatment on the eighth day (Day 8, predose), and 4 hours after the eighth day dose (Day 8, 4HR). The arrows point to
`the 75 kDa BTK band on a scanned fluorescent gel (top) and a western blot (bottom). (F) An average of .90% occupancy of BTK by drug is achieved in MCL patients who were
`administered ibrutinib in the first week, determined by fluorescent probe assays (n 5 6).
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`Figure 2. CD191CD51 cells have decreased CXCR4, CD38, and Ki67 expression following ibrutinib treatment. (A) Reduction of CD38 expression (mean fluorescence
`1
`1
`1
`CD5– cells during 4 weeks of treatment in 4 patients treated with ibrutinib. (B) Surface CD38 expression (left
`intensity ratio [MFIR]) in CD19
`CD5
`cells but not CD19
`panel; *P , .05) and intracellular Ki67 (right panel; **P , .01) is significantly reduced following 1 week of treatment. MFIR of intracellular phospho-ERK (pT202/Y204/
`1
`1
`ERK1/2) of CD20
`CD5
`cells from healthy participants or MCL patients treated with ibrutinib before treatment (day 1 [D1]) and after 1 week of treatment (D8) (lower
`1
`1
`panel; *P , .05). (C) Significant reduction of surface CXCR4 expression (MFIR) in CD19
`cells following 1 week of ibrutinib treatment (n 5 14; *P , .05). The line
`CD5
`between the dot plots shows the mean. (D) CXCR4 and CD38 expression from LN biopsies and PBMCs (PB) of three MCL patients (patients A, B, and C) not treated with
`drug. (E) Plasma chemokine and cytokine concentrations on day 8 (D8; left) or day 29 (D29; right) of ibrutinib-treated MCL patients compared with pretreatment times
`100% (n 5 9).
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`Figure 3. Ibrutinib inhibits migration of MCL cells beneath stromal cells (pseudo-emperipoiesis) and the formation of CXCL12-stimulated cortical actin. (A) Phase
`contrast (left panel) and 4,6 diamidino-2-phenylindole (DAPI) staining (right panel) of Mino and BM stromal cell M2-10B4 coculture 24 hours after Mino cells were pretreated
`with vehicle (dimethylsulfoxide [DMSO]) (panels a and b) or ibrutinib (1000 nM) (panels c and d). Highlighted yellow outlines show typical cobblestone appearance of
`migrated Mino cells beneath stromal cells. Black arrow points to a cell that is adhered on top of stromal cells but not migrated underneath. Yellow arrow points between two
`migrated Mino cells. (B) Mino cell and stromal cell coculture of Mino cells loaded with 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (red), and M2 cells
`loaded with 5-chloromethylfluorescein diacetate (green). (A) Stromal cells alone and (B) in coculture with Mino cells. (C) Mino cells pretreated with G protein coupled
`receptor inhibitor pertussis toxin at 200 ng/mL or (D) ibrutinib at 1000 nM. (C) Mino cells were stimulated with CXCL12 at 100 ng/mL and stained with rhodamine-phalloidin to
`determine actin polymerization and were counterstained with DAPI to identify nuclei of cells. Phalloidin staining of Mino cells (A) before and (B) after CXCL12 stimulation and
`after treatment with (C) pertussis toxin at 200 ng/mL or (D) ibrutinib at 100 nM. Magnification, 3200. (D) Mino cells were pretreated with ibrutinib, pertussis toxin, or vehicle
`for 30 minutes and then placed on a stromal cell–populated plate. After 4 hours, coculture was washed several times and migrated, and adhered Mino cells were counted in
`a flow cytometer with calibrated beads after staining with hCD19. Both pertussis toxin and ibrutinib dose-dependently inhibited migration and adhesion of Mino cells (left
`panel). Mino cells stimulated with CXCL12 and treated with vehicle or drug were stained with phalloidin, and its intensity was determined by using flow cytometry (right
`1
`panel). (E) Ibrutinib (100 nM) inhibited pseudo-emperipoiesis of primary MCL (hCD19
`cells) in coculture with M2-10B4 stromal cells (left panel). Actin polymerization as
`assessed by phalloidin staining was significantly reduced by ibrutinib treatment in primary MCL cells. **P , .01; ***P , .001. One-way analysis of variance (ANOVA)
`compared with vehicle control.
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`BLOOD, 3 OCTOBER 2013 x VOLUME 122, NUMBER 14
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`Ibrutinib inhibits BTK activity in MCL/stromal cell coculture
`and suppresses stromal cell–induced chemokine and
`cytokine secretion
`
`To further understand the drug effect on MCL cells in coculture
`with stromal cells, Mino cells were treated with drug and cocultured
`with M2-10B4 or stimulated with anti-immunoglobulin M (IgM).
`Ibrutinib dose-dependently inhibited pBTK, pPLCg2, pAKT, and
`pERK in Mino cells in coculture with M2-10B4 cells and in Mino
`cells with BCR stimulation (Figure 4A). Chemokine and cytokine
`concentrations of conditioned media were determined from ibrutinib-
`treated Mino cells alone or stimulated with anti-IgM or in coculture
`(Figure 4B). Mino cells increased chemokine and cytokine secretions
`following BCR stimulation or coculture with M2 cells. Similar
`results were observed with the Jeko cell line (supplemental Figure 2).
`Ibrutinib dose-dependently and potently suppressed production of
`human IL-10, CCL22, CCL3, CCL4, TNF-a, and CCL17 following
`BCR activation or in coculture, whereas the murine stromal cells
`alone did not produce human chemokines or cytokines (Figure 4B).
`Similarly, ibrutinib suppressed the production of IL-10, CCL22,
`CCL3, CCL4, and TNF-a of Jeko1 cells in coculture with M2-10B4
`(supplemental Figure 2) or human stromal cell line HS-5 (data not
`shown). Interestingly, these chemokines/cytokines were also reduced
`in ibrutinib-treated patients, but the degree of reduction in plasma
`CCL3 was far less than that in vitro (Figure 2E).
`
`Ibrutinib inhibits BCR- and chemokine-mediated adhesion and
`migration in vitro
`
`We measured the direct effect of ibrutinib on adhesion and migration
`of the MCL cell lines Jeko1, HBL2, Mino, and JVM-1. First, the
`effect of ibrutinib on BTK signaling was determined. As expected,
`ibrutinib inhibited phosphorylation of BTK and downstream signal-
`ing proteins PLCg2, MAP kinases ERK, JNK, and AKT following
`stimulation by anti-IgM and chemokines CXCL12 and CXCL13
`(Figures 5A-B and 6A; supplemental Figures 5 and 6). Cell surface
`expression of CXCR4, CXCR5, CCR7, surface IgM, and a4b1
`integrin was confirmed by flow cytometry (supplemental Figure 3),
`and subsequent in vitro adhesion and chemotaxis assays were per-
`formed with the drug. Ibrutinib significantly inhibited anti-IgM–
`stimulated adhesion of Jeko1 and HBL1 cells onto fibronectin or
`VCAM-1 at 100 nM (a clinically relevant concentration of ibrutinib)
`with more than 50% to 70% inhibition. The inhibition of adhesion
`by ibrutinib was also dose-dependent (supplemental Figure 4).
`Similarly, the adhesion of both Mino and Jeko1 cells to VCAM-1
`or fibronectin was inhibited by ibrutinib at 100 nM following
`CXCL12 or CXCL13 activation. The extent of inhibition was
`greater in Mino cells (50% to 70%) than in Jeko1 cells (20% to 30%)
`(Figure 6B-C). We found that, in addition to changes in adhesion,
`ibrutinib dose-dependently inhibited CXCL12-induced migration
`of Mino, Jeko1, and JVM-1 cells, with Mino and Jeko1 cells being
`more sensitive to drug than JVM-1 cells (Figure 6D). Ibrutinib also
`significantly inhibited CXCL13-stimulated migration of Mino cells
`dose-dependently from 1 nM to 1 mM (Figure 6D).
`Next, we examined the effect of ibrutinib on signaling and
`adhesion in primary MCL cells. In primary MCL, pY223BTK was
`increased compared with that in normal B lymphocytes, consistent
`with elevated BCR signaling in malignant B cells. Ibrutinib inhibited
`pBTK in both primary MCL and normal B cells on Y223, the auto-
`phosphorylation site of BTK, and Y551 (phosphorylated by Src
`family kinases) and reduced pPLCg2 on Y759 and Y1217 (Figure 7A)
`at concentrations of 10 nM and above. These results demonstrate
`
`that ibrutinib directly inhibits BTK activity in MCL primary cells.
`Importantly, ibrutinib also inhibited CXCL12- or CXCL13-activated
`adhesion to VCAM-1 as well as BCR-stimulated adhesion to fibro-
`nectin at 100 nM in primary MCL cells. The degree of inhibition in
`these primary cells was about 10% to 20%, and the magnitude was
`less impressive compared with that in the MCL cell lines but the
`inhibition was statistically significant (Figure 7B).
`These studies collectively demonstrate that ibrutinib inhibits
`BCR-, CXCL12-, and CXCL13-activated adhesion and migration in
`MCL cell lines as well as in primary MCL cells, which is associated
`with the BTK inhibition in these cells.
`
`Discussion
`
`Dramatic early increases of PB lymphocytes occur consistently
`among CLL patients treated with small-molecule antagonists of the
`BCR signaling pathway (Syk, PI3Kd, and BTK inhibitors).26,43,44
`Here we report and characterize similar early transient elevations
`of lymphocytes among MCL patients treated with the irreversible
`BTK inhibitor ibrutinib. The increase in ALC commenced as early
`as the first week, was maximal after 2 to 3 weeks of treatment, and
`then gradually subsided over several cycles of treatment. As with
`CLL patients,26 MCL patients dosed on a 28-days-on and 7-days-
`off schedule exhibited decreases of ALC following the 7-day-off
`period at the end of each cycle, and subsequent increases of ALC
`were observed periodically at the beginning of each subsequent
`cycle. Despite these increases in circulating cells, lymphatic masses
`were noted to decrease concomitantly, consistent with cellular
`mobilization from tissues as opposed to disease progression.
`We found that the newly appearing cells were light chain–
`restricted CD191CD51 cells, consistent with their identification as
`MCL cells. The identity of these cells as lymphomatous is not
`surprising since circulating PB tumor cells are often detectable
`among patients with MCL. Increase of circulating MCL cells fol-
`lowing treatment has not been previously reported and, moreover,
`it was highly specific, not involving CD191CD3–CD5– c