Leukemia (2008) 22, 1354–1360
`& 2008 Macmillan Publishers Limited All rights reserved 0887-6924/08 $30.00
`
`www.nature.com/leu
`
`ORIGINAL ARTICLE
`
`Bruton’s tyrosine kinase is not essential for Bcr-Abl-mediated transformation of
`lymphoid or myeloid cells
`
`M MacPartlin1, AM Smith2, BJ Druker1,3, LA Honigberg2 and MW Deininger1
`
`1Center for Hematologic Malignancies, Oregon Health & Science University Cancer Institute, Portland, OR, USA;
`2Pharmacyclics, Sunnyvale, CA, USA and 3Howard Hughes Medical Institute, Portland, OR, USA
`
`Bcr-Abl, a constitutively active tyrosine kinase, is the cause of
`chronic myeloid leukemia (CML) and a subset of acute
`lymphoblastic leukemias (ALL). Bruton’s tyrosine kinase
`(BTK), a member of the Tec family of tyrosine kinases with a
`crucial role in B-cell development,
`is consistently tyrosine
`phosphorylated in Bcr-Abl expressing murine pre B cells. BTK
`has been implicated in Bcr-Abl-mediated B-cell transformation
`and resistance to imatinib, implying that inhibiting BTK may be
`therapeutically beneficial. We decided to test whether BTK is a
`critical node in Bcr-Abl transformation and potential drug target
`in imatinib-resistant Bcr-Abl-positive cells. We depleted BTK in
`Ba/F3 and 32D cells expressing native and kinase domain (KD)
`mutant (E255K and T315I) Bcr-Abl, using shRNA. BTK levels
`were reduced to o10% of controls. However, no differences in
`viability and cell proliferation were observed and the response
`to imatinib was not altered. Consistent with this, proliferation
`and viability were unaffected by inhibition of BTK with
`reversible (PC-005) and irreversible (PCI-31523) small molecule
`inhibitors. Lastly, BTK inhibition did not affect the ability of Bcr-
`Abl to transform primary murine hematopoietic cells in colony
`forming and B-cell transformation assays. Collectively this data
`argues against a critical role for BTK in Bcr-Abl-mediated
`leukemogenesis.
`Leukemia (2008) 22, 1354–1360; doi:10.1038/leu.2008.126;
`published online 12 June 2008
`Keywords: Bruton’s tyrosine kinase (BTK); Bcr-Abl; transformation
`
`Introduction
`
`Bcr-Abl is a chimeric tyrosine kinase whose constitutive activity
`is critical to the pathogenesis of chronic myeloid leukemia and
`Philadelphia chromosome (Ph)-positive B-cell acute lympho-
`blastic leukemia (B-ALL).1–3 Infection of murine hematopoietic
`cells with BCR-ABL retrovirus
`induces myeloproliferative
`disease or B-ALL, depending on the experimental conditions.4
`Bcr-Abl is known to activate multiple signaling pathways, whose
`relevance to leukemogenesis is incompletely understood. Using
`phosphoproteomics we have profiled tyrosine phosphorylated
`peptides in Ba/F3 cells expressing native and kinase domain
`mutant Bcr-Abl.5 Among others we observed consistent
`phosphorylation of Bruton’s tyrosine kinase (BTK) on tyrosine
`223 (Y223). BTK is critical to normal B-cell development.6,7
`Upon B-cell receptor stimulation, Src family tyrosine kinases are
`activated, which rapidly phosphorylate tyrosine 551 (Y551) in
`
`Correspondence: Dr MW Deininger, Center for Hematologic Malig-
`nancies, Oregon Health and Science University Cancer Institute, 3181
`SW Sam Jackson Park Road, Mailcode L592, Portland, OR 97239,
`USA.
`E-mail: deininge@ohsu.edu
`Received 23 April 2008; accepted 24 April 2008; published online 12
`June 2008
`
`followed by the subsequent
`the activation loop of BTK,
`autophosphorylation of Y223 in the SH3 domain.8,9 It is thought
`that phosphorylation of Y223 may play a role in modulating key
`protein–protein interactions required for B-cell development.10–13
`Consistent with our findings, another study found that ABL
`phosphorylates BTK at Y223, but not Y551.14 Furthermore, a
`recent study postulated a critical role for BTK in Bcr-Abl-
`mediated transformation of B-cells15 and a gene expression
`study found an association between high levels of BTK and
`imatinib resistance.16 This raises the question whether BTK may
`be a critical signaling node and thus potential drug target in
`Bcr-Abl-positive leukemia, particularly in patients with imatinib
`resistance. To validate this assumption, we decided to verify the
`relevance of BTK for Bcr-Abl-mediated transformation.
`
`Materials and methods
`
`Kinase inhibitors
`referred to as
`PCI-31523 is an irreversible BTK inhibitor
`compound 4 in Pan et al.17 PC-005 is a reversible inhibitor of
`BTK, Src-family kinases and Abl described in Supplementary
`Figure 4.
`
`Cell culture
`Ba/F3 cells expressing native Bcr-Abl and the kinase domain
`point mutants E255K and T315I have been described pre-
`viously.18 The lines were maintained in RPMI 1640 (Invitrogen,
`Carlsbad, CA, USA) supplemented with 2 mM L-glutamine
`(Invitrogen) 1 U/ml penicillin (Invitrogen) and 1 mg/ml strepto-
`mycin (Invitrogen) and 10% fetal bovine serum (FBS, HyClone,
`Logan, UT, USA). 293T and 293FT cells19 were maintained in
`Dulbecco’s modified Eagles medium (DMEM,
`Invitrogen)
`supplemented with 10% FBS 1 U/ml penicillin, 1 mg/ml strepto-
`mycin and 2 mM L-glutamine. All cells were grown at 37 1C in
`5% CO2. Viable cell numbers upon treatment with tyrosine
`kinase inhibitors were measured in triplicate using methlythia-
`zoletetrazolium assays as described previously.18
`
`Immunoblotting
`Cells were collected by centrifugation and lysed in phosphate-
`buffered saline containing 1% NP40, 1 mM EDTA, 1 mM
`Na3VO4, 0.5 mM PMSF and 1 mg/ml aprotinin. Lysates were
`separated by SDS–PAGE (sodium dodecyl sulphate–polyacryla-
`mide gel electrophoresis) (50–200 mg protein/lane). Blots were
`probed with specific antibodies to test for expression of Bcr-Abl
`(8E9; Pharmingen, San Jose, CA, USA), Abl phosphorylated on
`tyrosine-245 (2861, Cell Signaling Technology, Danvers, MA,
`USA), BTK (05-415 Upstate/Millipore, Billerica, MA, USA) and
`tubulin (T4026; Sigma, St Louis, MO, USA).
`
`Coalition for Affordable Drugs IV LLC - Exhibit 1011
`
`

`

`Determination of BTK active site occupancy using a
`fluorescent probe
`Cells were lysed by freeze/thaw in D-PBS and total protein was
`quantitated by BCA Protein Assay (Pierce, Rockford, IL, USA).
`Lysates were diluted in D-PBS to a final concentration of 0.9 mg/
`ml and incubated with 2.5 mM of the BTK probe PCI-33380 in a
`37 1C water bath for 1 hr. PCI-33380 is a Bodipy-FL-conjugate of
`the pure R enantiomer of PCI-31523 that binds irreversibly to
`BTK through a covalent
`interaction. When cell
`lysates are
`exposed to probe after cells are treated with the covalently
`binding inhibitor PCI-31523, probe binding is
`inhibited,
`demonstrating occupancy.20 Labeling was halted by the
`addition of LDS sample buffer and sample reducing agent
`(Invitrogen). Samples were heated at 70 1C for 10 min prior to
`gel electrophoresis. Fifteen micrograms of total cell lysate was
`run on a NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen) in the
`dark and Bodipy-FL labeled BTK was visualized by scanning the
`gel with a Molecular Dynamics Typhoon scanner (Ex¼ 532 nm
`and Em¼ 555 nm). Gels were then blotted (Invitrogen iBlot) and
`total BTK was detected using anti-BTK (BD Biosciences, San
`Jose, CA, USA) and standard immunoblotting techniques.
`
`Production of retrovirus and determination of viral titer
`The MSCV-p210BcrAbl-IRES-GFP (MIG) construct has been
`described previously.21 For the production of native, E255K
`and T315I
`retrovirus
`for use in the bone marrow (BM)
`transduction experiments, 293T cells were transiently co-
`transfected using Fugene6 (Roche, Nutley, NJ, USA) with the
`MIG construct of choice and the Ecopac vector helper virus
`DNA (a kind gift of Dr Richard Van Etten, Tuft’s University,
`Boston, MA, USA). Viral supernatants were harvested at 72 h
`post-transfection, filtered through a 0.45 mm filter, flash frozen in
`liquid nitrogen and stored for determination of viral titer or use.
`Relative viral titer was determined by infection of NIH3T3 cells.
`Briefly, 1 105 cells were plated in 30 mm tissue culture plates.
`Twenty-four hours later cells were exposed to 100, 200 and
`400 ml of viral supernatant in 2 ml total volume of media with
`4 mg/ml polybrene (Sigma). Forty-eight hours post
`infection
`NIH3T3 cells were harvested with trypsin (Invitrogen) and
`washed twice in D-PBS. GFP (green fluorescent protein)
`expression was analyzed by FACS (fluorescent-activated cell
`sorter, FACSAria, BD Biosciences) to determine relative viral
`titers, as described previously.5 In all cases viral titers were in
`the linear range.
`
`BTK is not required for Bcr-Abl transformation.
`M MacPartlin et al
`
`(10 mg/ml) were added to each well. Cells were then co-
`sedimented at 30 1C for 90 min at 2500 rpm. The cells were
`then allowed to recover overnight and the following day
`2.5 mg/ml puromycin was added for the selection of lentivirally
`transduced cells.
`
`1355
`
`Colony forming assays
`For myeloid progenitor colony formation assays, BM was
`harvested from 6–10 week old female Balb/c mice. The BM
`was subjected to 24 h of prestimulation in Iscove’s modified
`Dulbecco medium (IMDM, Invitrogen) supplemented with 15%
`heat-inactivated FBS, 5% WEHI conditioned media, 6 ng/ml rm
`IL-3 (StemCell Technologies, Vancouver, BC, USA), 10 ng/ml rm
`IL-6 (StemCell Technologies) and 50 ng/ml rm SCF (StemCell
`Technologies).22 After 24 h of prestimulation equal numbers of
`cells were transferred to 6-well plates and exposed to matched
`viral supernatants in the presence 2 mg/ml polybrene. Cells were
`then co-sedimented at 30 1C for 90 min at 2500 rpm. After 4 h
`the media was replaced with fresh prestimulation media. At 48 h
`a second round of co-sedimentation was performed. After an
`additional 4 h adsorption period the cells were washed twice in
`IMDM to remove cytokines, counted and plated in triplicate in
`MethoCult (M3234, without cytokines or M3534, with cyto-
`kines, StemCell Technologies). Inhibitors were re-dosed every 2
`days. Colony formation was scored at day 10.
`
`B-cell transformation assays
`For the analysis of the transformation of primary BM B-lymphoid
`progenitors, BM from Balb/c mice was subjected to red cell lysis
`using ammonium chloride solution (StemCell Technologies).23
`The cells were subjected to a single round of transduction and
`co-sedimentation with matched retroviral stock for native
`Bcr-Abl and vector control in DMEM with 10% FBS and 2 mg/ml
`polybrene and incubated overnight in the presence of viral
`supernatant. Inocula of 105, 5 104 and 104 cells were plated in
`triplicate in Whitlock/Witte media (RPMI 1640 supplemented
`with 5% FBS, 200 mM L-glutamine, 50 mM 2-mercaptoethanol
`in 24-well plates with 106 un-
`and penicillin/streptomycin)
`transduced BM cells/well as feeders.5 Cells were cultured for 4
`weeks and fed twice weekly by replacing 0.5 ml of supernatant
`with fresh media and fresh inhibitor. Cultures were scored as
`positive for transformation when the number of nonadherent
`cells exceeded 1 106 per milliter of culture medium.
`
`Production of lentivirus expressing shRNA and cell line
`viral transduction
`(TRCN0000023692 (BTK1),
`The five pLKO.1. plasmids
`TRCN0000023691
`(BTK3),
`TRCN0000023690
`(BTK2),
`TRCN0000023689 (BTK4), TRCN0000023693 (BTK5) expres-
`sing short hairpin RNA (shRNA) against BTK were purchased
`from Open Biosystems, Huntsville, AL, USA. For the production
`of lentivirus expressing shRNA against BTK, 293FT cells were
`transiently co-transfected using Fugene6 (Roche) with the
`pLKO.1 constructs and the ViraPower Packaging Mix (Invitro-
`gen). As a control, scrambled shRNA virus was included
`(MISSION Non-Target shRNA Control pLKO1 Vector, Sigma-
`Aldrich). Viral supernatants were harvested at 72 h post-
`transfection, filtered through a 0.45 mm filter, flash frozen in
`liquid nitrogen and stored at 80 1C. Ba/F3 and 32D cells
`were infected with lentivirus using 106 cells in 3 ml of media,
`seeded in a single well of a 6-well plate. One milliliter of
`viral supernatant, 30 ml of 1 M HEPES and 2 ml of polybrene
`
`Results
`
`Recently, we reported global phosphotyrosine profiles of Ba/F3
`cells expressing native and kinase domain mutant Bcr-Abl in
`comparison with parental Ba/F3 cells.5 A BTK-derived peptide
`corresponding to KVVALyDYMPMNANDLQLR (amino acid
`218–236) was consistently phosphorylated on tyrosine 223 in
`Bcr-Abl expressing cells, but not
`in parental Ba/F3 cells,
`implicating BTK as a potential downstream mediator of Bcr-
`Abl. To test whether BTK is relevant to growth and viability of
`Bcr-Abl expressing cells we downregulated BTK expression
`using lentivirally delivered shRNA. Initial screening experiments
`were carried out
`to select
`the optimal shRNA sequence.
`Immunoblot analysis of Ba/F3 and 32D cells expressing empty
`vector, native and kinase domain mutant (E255K and T315I)
`Bcr-Abl revealed a 490% reduction of BTK expression with
`BTK1 and BTK5, as shown for Ba/F3 p210 cells (Figure 1a).
`Control experiments with scrambled shRNA confirmed the
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`

`1356
`
`BTK is not required for Bcr-Abl transformation.
`M MacPartlin et al
`
`Figure 1 Lentivirally-mediated downregulation of BTK and its effect
`on Bcr-Abl dependent proliferation.
`(a) Lentiviruses BTK1-5 were
`produced expressing short hairpin RNA (shRNA) against BTK, as well
`as a control scramble non-specific shRNA lentivirus (MISSION Non-
`Target shRNA Control Vector). These viruses were used to infect a
`panel of Ba/F3 and 32D cell lines. Stably infected cells (Ba/F3 p210
`shown) were lysed and examined by immunoblot for knockdown of
`BTK. (b and c) Cells infected with BTK1, BTK 5 and scramble shRNA
`were treated with imatinib (0, 7.8, 15.6, 31.2, 62.5, 125, 250 and
`500 nM) and viable cells were measured by methlythiazoletetrazolium
`(MTT) assay after 72 h.
`
`specificity of the effects for BTK (Figure 1a, lane 7). Cell lines
`with practically complete knockdown of BTK (shRNAs 1 and 5)
`were used in subsequent experiments.
`In our first set of experiments we compared the growth rates of
`Ba/F3 and 32D cells expressing p210 Bcr-Abl, with and without
`simultaneous expression of BTK1, BTK5 or scrambled shRNA.
`No significant differences in growth rates were observed (Figures
`1b and c), suggesting that BTK is not critical for cell growth of
`Bcr-Abl
`transformed lymphoid or myeloid cell
`lines. To
`determine whether BTK expression modulates sensitivity to
`ABL inhibitors, the same cell lines were exposed to graded
`concentrations of
`imatinib. Downregulation of BTK did not
`affect cell proliferation or viability in the presence of imatinib
`compared to cells infected with scrambled shRNA (Figures 1b
`and c). Identical experiments were performed on Ba/F3 cells
`expressing E255K and T315I mutants of Bcr-Abl, with similar
`results (Supplementary Figure 1). These data suggest that BTK is
`
`Leukemia
`
`neither required for proliferation of Bcr-Abl-positive cells nor
`does it modulate the response to imatinib.
`To confirm these observations we treated the identical panel
`of cell lines with two BTK inhibitors: PCI-31523, a selective
`irreversible inhibitor and PC-005, a reversible inhibitor of BTK
`and Abl (Pharmacyclics, Sunnyvale, CA, USA).17,20 Due to the
`lack of suitable and good quality antibodies we were unable to
`directly test BTK Y223 phosphorylation inhibition. Instead we
`used an active site probe consisting of a BTK inhibitor, PCI-
`33380, a Bodipy-FL-conjugate of the pure R enantiomer of PCI-
`31523 that selectively labels BTK. This probe binds BTK at the
`same site as PCI-31523 and PC-005 and can be visualized by
`denaturing gel electrophoresis of lysates, followed by fluores-
`cent scanning. To assay occupancy of the active site, cell lysates
`were prepared from Ba/F3 cells expressing either native or T315I
`Bcr-Abl and BV173 cells (human Ph-positive pre-B-cell line)
`treated with graded concentrations of PCI-31523. The lysates
`were then incubated with the conjugated probe and analyzed as
`described in the Materials and methods. There was complete
`occupancy of the active site in cells exposed to PCI-31523, the
`irreversible inhibitor (Figure 2a). Unsurprisingly, treatment with
`PC-005 did not affect probe binding, presumably due to
`displacement of the reversible inhibitor by the irreversible probe
`(Figure 2b).
`Despite potently inhibiting BTK, PCI-31523, did not affect the
`proliferation of any of
`the cell
`lines, with IC50 values
`consistently above 5 mM (Figures 3a and c and Supplementary
`Figure 2a). However, upon treatment of
`the cells with the
`reversible inhibitor, PC-005, cells expressing Bcr-Abl showed a
`significant decrease in proliferation. Specifically, the IC50 was
`827 nM in Ba/F3 cells expressing Bcr-Abl, 613 nM in 32D cells
`expressing Bcr-Abl and 718 nM in BV173 cells (Figures 3b and d
`and Supplementary Figure 2b). In contrast, no effect was seen in
`cells expressing the T315I mutant of Bcr-Abl (Figures 3b and d).
`The resistance of T315I expressing cells against PC-005 was
`reminiscent of combined ABL/SRC inhibitors such as dasatinib
`and suggested that the effects of PC-005 may be mediated by
`inhibition of Bcr-Abl rather than BTK.24 Indeed a PC-005 dose-
`dependent decrease in Bcr-Abl tyrosine phosphorylation was
`observed by immunoblot analysis of BV173 cell
`lysates
`(Figure 4a) and Ba/F3 cell lysates expressing native Bcr-Abl
`(Figure 4b) but not T315I mutant Bcr-Abl
`(Figure 4c). This
`supports the conclusion that the effects of PC-005 are the result
`of
`inhibiting Bcr-Abl activity.
`In vitro inhibition of selected
`kinases by PC-005 was examined and IC50 values were
`determined by the Reaction Biology Hotspot assay, with
`staurosporine as a control
`(Supplementary Figure 4). This
`demonstrated an IC50 value of 3.49 nM for inhibition of Abl,
`further supporting our observations. Upon treatment of the cells
`with PCI-31523, the irreversible inhibitor, no such effects on
`Bcr-Abl phosphorylation were observed (Figures 4a and b).
`Exogenous cytokines are required for normal murine BM to
`form myeloid colonies in semi-solid media, whereas Bcr-Abl
`expression induces cytokine-independent colony formation.
`Therefore, to assess whether BTK is required for transformation
`of primary myeloid cells, we examined colony formation of
`murine BM infected with a Bcr-Abl retrovirus in the presence of
`a concentration gradient of PCI-31523 and PC-005.
`In the
`presence of cytokines comparable numbers of colonies
`were observed from the empty vector and Bcr-Abl transduced
`cells (Figures 5a and b). As expected, only the Bcr-Abl
`transduced cells formed significant numbers of colonies in the
`absence of cytokines (Figures 5c and d). PCI-31523 did not
`affect colony formation. However, upon titration of PC-005 on
`the plates, a significant decrease in colony formation was seen
`
`

`

`BTK is not required for Bcr-Abl transformation.
`M MacPartlin et al
`
`1357
`
`Figure 2 Probe labeling of cell lysates and demonstration of PCI-31523 binding to BTK. (a) Ba/F3 p210, 32D p210 and BV173 cells lines were
`exposed to PCI-31523 for 4 h and cell lysates were then subjected to probe labeling with PCI-33380, the fluorescent analog of PCI-31523 followed
`by denaturing gel electrophoresis. Fluorescent gel scan shows inhibition of probe labeling by pre-treatment with PCI-31523 and western blot
`confirms the presence of BTK in all lanes. (b) The experiment was repeated with the reversible inhibitor PC-005.
`
`transduced cells grown in the absence of
`in the Bcr-Abl
`cytokines (Figure 5d). Thus, as with cell lines, the BTK/Abl
`inhibitor PC-005 is anti-proliferative, whereas the BTK selective
`inhibitor is not.
`To assess the role of BTK in the transformation of primary
`B-lymphoid progenitor cells by Bcr-Abl, murine BM was retro-
`virally infected with native Bcr-Abl or the empty MIGR1 (Mig)
`retroviral vector as a control.21 Cells were plated in triplicate in
`Whitlock–Witte cultures at various dilutions. The day of initial
`outgrowth and lowest dilution supporting outgrowth were used
`as a semi-quantitative measure of
`transformation potency
`(Figure 6).5,25 Wells were scored as positive when the number
`of non-adherent cells reached 106 per well. As expected the
`empty vector failed to transform primary B cells. In contrast,
`expression of native Bcr-Abl induced rapid outgrowth of B cells
`(Figure 6a and b). Outgrowth was unaffected by 50 nM of
`PCI-31523 (Figure 6c), an inhibitor concentration that resulted
`in nearly complete occupancy of BTK (Figure 4c). Higher
`concentrations of PCI-31523 (500 nM) led to a reduction of
`B-cell outgrowth and a complete block was observed at 5 mM
`(Supplemental Figures 3c and d). With PC-005 we found a
`marginal reduction of outgrowth at 50 nM (Figure 6d), with
`complete inhibition at concentrations of 500 nM and above,
`concentrations where significant inhibition of Bcr-Abl is seen
`(Supplemental Figure 3a and b).
`
`Discussion
`
`Imatinib is a highly effective therapy for the treatment of early
`chronic myeloid leukemia.26–28 However, drug resistance due
`to kinase domain mutations or other mechanisms is frequent in
`patients with accelerated or blastic phase or with Phþ ALL.29–32
`Second-line ABL inhibitors like dasatinib or nilotinib have
`promising activity in patients with imatinib resistance. Unfortu-
`nately, responses in patients with blast crisis or Phþ ALL are
`usually only transient. In many cases failure to second-line ABL
`inhibitors is caused by the T315I mutant, which is resistant to all
`
`Bcr-Abl inhibitors that are currently available for clinical use.
`Targeting essential pathways downstream of Bcr-Abl is a logical
`approach to address this clinical challenge.
`BTK, a Tec family kinase with a central role in B-cell
`development, has been implicated as a critical mediator of
`B-cell transformation by Bcr-Abl. Thus, Feldhahn et al.15 described
`Bcr-Abl as mimicking a constitutively active pre-B-cell receptor
`in pre-B lymphoblastic leukemia cells. The authors concluded
`that BTK is contributing to Bcr-Abl driven survival signaling in
`these cells,
`independent of pre-B-cell
`receptor dependent
`signals. Furthermore, a microarray study of patients with
`Phþ ALL found a correlation between high levels of BTK
`mRNA and imatinib resistance.16 Lastly, using phosphoproteo-
`mics we have detected phosphorylation of
`tyrosine 223
`of BTK in Ba/F3 cells expressing Bcr-Abl, consistent with active
`BTK.5 These data suggest that BTK may be a critical node and
`potential drug target
`in imatinib-resistant Bcr-Abl-positive
`cells.15,16,33
`In a first series of experiments, we used shRNA to down-
`regulate BTK in Ba/F3 and 32D cells expressing native Bcr-Abl
`and the kinase domain mutants E255K and T315I. However, in
`contrast to the report by Feldhahn et al.15 we saw no effect on
`the proliferation of these cells in culture (Figure 1). Moreover,
`sensitivity to imatinib was unaffected by the lack of BTK. The
`reasons for the discrepancy with the published data are not
`immediately obvious. To extend our observations, we treated
`the cell lines with two small molecule inhibitors of BTK. PCI-
`31523, a selective irreversible inhibitor that did not inhibit Abl
`phosphorylation in cells, had no effect on the proliferation of
`Bcr-Abl-positive cells (Figure 3). Using a fluorescent BTK probe,
`we confirmed that cellular BTK was fully occupied by PCI-
`31523 in these experiments (Figure 2). In contrast, PC-005, a
`less selective and reversible inhibitor,
`led to a decrease in
`proliferation of Bcr-Abl positive cells (Figure 3). However, this
`anti-proliferative effect was correlated with inhibition of Bcr-Abl
`rather than BTK, as evidenced by the complete resistance of
`cells expressing the T315I mutant (Figure 4c). We also tested
`LFM-A13, an established BTK inhibitor with a reported IC50 of
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`1358
`
`BTK is not required for Bcr-Abl transformation.
`M MacPartlin et al
`
`Figure 4 Immunoblot analysis of PC-005 and PCI-31523 inhibitor
`activity. (a, b and c) BV173, Ba/F3 p210 and Ba/F3 T315I cells,
`respectively, were treated with inhibitor (4 h at the concentration
`shown) and cell lysates were analyzed by immunoblot for Abl tyrosine
`245 phosphorylation of Bcr-Abl.
`
`Figure 3 Cell proliferation in the presence of BTK inhibitors PCI-
`31523 and PC-005. (a and b) Ba/F3 cells expressing empty vector,
`native Bcr-Abl (p210) and the kinase domain (KD) mutants E255K and
`T315I were exposed to a range of inhibitor concentrations (0, 10, 50,
`100, 500, 1000 and 5000 nM) and viable cells were measured by
`methlythiazoletetrazolium (MTT) assay after 72 h. (c and d) Identical
`assays were performed with 32D cells expressing native or kinase
`domain mutant Bcr-Abl.
`
`17.2 mM.34 However, we did not see any anti-proliferative effect
`at concentrations below 100 mM (data not shown). Any effects at
`higher concentrations are more consistent with non-specific
`toxicity than an effect related to BTK inhibition. These results
`appear to be, along with our siRNA (small interfering RNA) data,
`discordant with the data published by Feldhahn et al.15
`However, no drug concentrations are given in that paper,
`making a direct comparison impossible. To address the question
`
`of a critical role of BTK for Bcr-Abl-mediated transformation of
`primary cells, we assayed Bcr-Abl-induced myeloid colony
`formation and B-cell transformation. Consistent with our cell
`line data, myeloid colony formation was unaffected by
`inhibition of only BTK (with the irreversible inhibitor PCI-
`31523), but reduced in the presence of PC-005, which inhibits
`ABL and BTK (Figure 5). Similarly, B-cell transformation was
`unaffected at concentrations of PCI-31523 (50 nM) that pro-
`duced a complete block of BTK as shown by a specific
`fluorescence assay (Figures 2a and 6c).
`In summary, our
`data provide conclusive evidence that transformation of myeloid
`as well as B-lymphoid cells by Bcr-Abl does not depend
`on BTK, suggesting that
`the therapeutic effects of a pure
`BTK inhibitor in the setting of imatinib resistance would be
`minimal. However, it remains possible that BTK would assume a
`critical role upon simultaneous inhibition of another signaling
`pathway.
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`Leukemia
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`

`BTK is not required for Bcr-Abl transformation.
`M MacPartlin et al
`
`1359
`
`Figure 5 Myeloid colony formation by Bcr-Abl transduced cells exposed to PCI-31523 and PC-005. (a and b) Bone marrow (BM) from Balb/c
`mice infected with virus expressing empty vector or Bcr-Abl (p210) was plated in triplicate in methyl-cellulose in the presence of cytokines and
`increasing concentrations of PCI-31523 and PC-005. Colony formation was scored on day 10. (c and d) The assay was repeated in the absence of
`cytokines.
`
`Figure 6 Effects of BTK inhibition on B-cell transformation by Bcr-Abl. Bone marrow (BM) from Balb/c mice was transduced with retrovirus
`expressing Bcr-Abl (p210) and plated at the indicated cell numbers per well in each of three wells. For stromal support, 1 106 non-transduced
`cells were added per well. A well was scored as positive when the viable non-adherent cell number reached 1 106/well. The x axis is the number
`of days after plating and the y axis indicates that number of wells, from 1 to 3 that are scored as positive. (a) Mig empty vector control, (b) Bcr-Abl,
`(p210), (c) Bcr-Abl (p210 ) with 50 nM of PCI-31523 and (d), Bcr-Abl (p210 ) with 50 nM of PC-005.
`
`Acknowledgements
`
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`This study was supported by NHLBI Grant HL082978-01 (MWD)
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