Download SUPPLEMENTARY MATERIALS AND METHODS Generation of

SUPPLEMENTARY MATERIALS AND METHODS
Generation of DNA constructs
After obtaining informed consent for all patients from whom clinical samples were collected,
RNA was isolated from bone marrow mononuclear cells from CML patients harboring BCR-ABL
fusion transcripts with e1a2 (p190BCR-ABL), e8a2 (p200BCR-ABL), or e14a2 (p210BCR-ABL)
breakpoints by guanidinium thiocyanate phenol-chloroform extraction and reverse transcribed
using random hexamer primers as previously described (1). For the patients with either e1a2 or
e14a2 fusions, the full-length BCR-ABL sequence was amplified by polymerase chain reaction
(PCR), and PCR products were subcloned into the pGEM5 vector (Promega, Madison, WI)
according to the manufacturer’s instructions. To generate the p200BCR-ABL construct, in the
patient with the e8a2 fusion (which is characterized by an inserted 55 bp fragment corresponding
to an inverted portion from ABL intron 1b (2)), BCR-ABL was amplified with primers flanking
the e8a2 BCR-ABL junction (3). The 1140 bp PCR product was digested with KpnI and NheI
restriction enzymes and subcloned into the existing pGEM5-p210BCR-ABL construct after
removing the e14a2 BCR-ABL junction region (1561 bp) from this construct using the same set
of enzymes. Each pGEM5 construct was verified through direct sequencing, and the EcoRI
fragment (including the coding sequence of the designated BCR-ABL variant) was ligated into
the retroviral vectors pSRα and MIGR1 (a kind gift of Ruibao Ren, Brandeis University).
Retroviral stock preparation
Bosc23 cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10%
fetal calf serum, 1 U/mL penicillin, and 1 g/mL streptomycin. For production of retrovirus,
Bosc23 cells were transiently transfected with one of the BCR-ABL-containing pSRα constructs
or pSRα alone (‘vector only’), using Fugene reagent (Roche, Indianapolis, IN). Viral
supernatants were harvested 48 h post-transfection. For titration of retroviral supernatants, 1x105
NIH3T3 cells were cultured in the presence of 0, 5, 10, 20, 40, 80, or 100 μL of the viral
supernatant and 8 μg/mL polybrene for 48 h. Subsequently, the percentage of GFP-positive cells
was determined using flow cytometry (FACSAria; BD Biosciences, San Jose, CA) and this
information was used to adjust the volumes of viral supernatants used in each experiment, as
previously described (4).
Cell lines
Ba/F3 and 32D cells expressing BCR-ABL variants were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum, 1 U/mL penicillin, and 1 μg/mL streptomycin. Parental
Ba/F3 and 32D cells were maintained in the same media supplemented with 15% WEHI-3Bconditioned medium as the source of murine IL-3. Bosc23 and NIH3T3 cells were maintained in
Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum, 1 U/mL penicillin, and 1
μg/mL streptomycin.
Transformation of factor-dependent hematopoietic cell lines
Ba/F3 and 32D cells were transduced with retroviral supernatant of the pSRα-p190BCR-ABL,
pSRα-p200BCR-ABL, or pSRα-p210BCR-ABL constructs and selected for neomycin resistance in
presence of G418 followed by IL-3 withdrawal to select for factor independence. Ba/F3 cells
were transduced by one round of infection, whereas transduction of 32D cells required four
rounds due to lower infection efficiency. All experiments were carried out using bulk cultures
with comparable levels of BCR-ABL protein.
Cellular proliferation assays
32D cells harboring p190BCR-ABL, p200 BCR-ABL, p210 BCR-ABL, or pSRα (vector only) were plated
(1x104 cells/well) in 96-well plates in the presence of graded concentrations of imatinib (0, 0.25,
0.5, 2 μM) for 72 h, and cellular proliferation was measured using an MTS-based viability assay
(CellTiter 96 AQueous One Solution Reagent; Promega) as previously described (5). Each
condition was performed in triplicate, and data from two independent experiments were
averaged.
Immunoprecipitation and immunoblotting
Protein lysates were prepared by resuspending 1x107 cells in 1 mL NP-40 lysis buffer (20 mM
Tris, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 10 μg/mL aprotinin, 1 mM
sodium vanadate, 1 mM phenylmethylsulfonylfluoride). Peripheral blood lysates were made by
boiling the cells in Laemmli buffer (Sigma-Aldrich, St. Louis, MO) for 10 min. BCR-ABL and
phosphotyrosine immunoblots of Ba/F3 and 32D cell lysates were prepared following 6 h serum
starvation (1% fetal calf serum) and analyzed using an anti-c-ABL antibody (8E9; BD
Biosciences) and an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Lake Placid,
NY). To analyze STAT5 and STAT6 phosphorylation, the proteins were immunoprecipitated
from Ba/F3 lysates using anti-STAT5 or -STAT6 antibodies (C-17 and M-20, respectively; Santa
Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were resolved by SDS-PAGE and
phosphorylated proteins were detected by immunoblotting with an anti-phospho-STAT5Tyr694
antibody (Cell Signaling Technology, Beverly, MA) or anti-phospho-STAT6Tyr641 antibody
(Upstate Biotechnology).
In vitro kinase assays
Immune complex assays and immunoprecipitation of ABL proteins from parental 32D cells and
from 32D cells expressing the p190BCR-ABL, p200BCR-ABL, or p210BCR-ABL variant were carried out
as described previously (6) with minor modifications. Constructs expressing the GST-Crk (120225) fusion protein (used as an exogenous substrate) and the corresponding Crk (120-212)
negative control (lacking Y221, the residue phosphorylated by c-ABL) were kindly provided by
R.A. Van Etten of Tufts University. Briefly, 32D cells (2-4x108) were lysed in NP-40 lysis
buffer supplemented with Complete protease inhibitor cocktail (Roche), 2 mM AEBSF, 2 mM
sodium vanadate, and 40 µM phenylarsine oxide. The c-ABL mouse monoclonal antibody 24-11
(25 µg; Santa Cruz Biotechnology) was added to the clarified lysate (2 mL) and the solution was
agitated at 4°C for 2 h. GammaBind G Sepharose (100 µL settled bead volume; Amersham
Biosciences, Piscataway, NJ) was added, and the mixture was agitated at 4°C overnight.
Following centrifugation and removal of supernatant, beads were washed four times with kinase
buffer (Cell Signaling Technology) and resuspended in kinase buffer (final volume ~400 µL,
including beads), then used immediately in kinase activity assays and western blot analysis. In
vitro kinase assays were carried out in kinase buffer containing GammaBind G Sepharose-bound
enzyme, GST-Crk (120-225) substrate (10 µg), and ATP/[-32P]ATP (50 µM; 2.5 µCi per assay).
Reactions were initiated by addition of ATP and terminated by addition of SDS-PAGE loading
buffer followed by heating at 100°C for 10 min. Quenched reaction mixtures were separated on
10.5-14% SDS-PAGE gels, and
32
P incorporation into the GST-Crk(120-225) substrate was
quantitated by phosphorimager analysis (Typhoon 9400; Amersham Biosciences). Kinase assay
results were corrected for relative BCR-ABL protein levels, which were determined by western
blot densitometric analysis using a Lumi-Imager fluorescence imaging system (Roche). Results
of individual experiments conducted in triplicate were expressed as the average value  standard
deviation. Statistical analysis between BCR-ABL isoforms was performed using a paired
Student’s t-test for sixteen individual experiments conducted in triplicate.
Colony formation assays
Bone marrow cells isolated from 3-5 week-old female Balb/c mice (Charles River Laboratories,
Wilmington, MA) were infected with MIGR1-p190BCR-ABL, MIGR1-p200
BCR-ABL
, or MIGR1-
p210 BCR-ABL retrovirus or MIGR1 alone (vector control) retrovirus in the presence of IL-3, IL-6,
and stem cell factor (Stem Cell Technologies, Vancouver, BC), and cells were plated (3x105
cells per 35 mm dish) in MethoCult medium with or without growth factors (M3234 or M3334,
respectively; Stem Cell Technologies), according to published protocols (7). Plates were
incubated at 37ºC for 12 days, and colonies were counted under an inverted microscope. Blymphoid colony forming assays were also performed as previously described (8) with minor
modifications. Briefly, 1-3x106 bone marrow cells from 3-5 week-old female Balb/c mice were
transduced with 2x105 retroviral infectious units for 24 h prior to plating in soft agar. Plates were
incubated at 37ºC for 15 days and colonies were scored under an inverted microscope.
Bone marrow transduction and transplantation
Balb/c mice used in the study were housed at the Oregon Health & Science University animal
care facility. Bone marrow transduction and transplantation were performed as previously
described (9), with the following modifications. Five to six week-old male donor mice were
injected retro-orbitally with 300 mg/kg 5-fluorouracil (5-FU) 4 days prior to harvesting bone
marrow. Following two rounds of viral infection over a 48 h period in the presence of IL-3, IL-6,
and stem cell factor (Stem Cell Technologies), 0.4x105 bone marrow cells from 5-FU-treated
mice or 1x106 bone marrow cells from non-5-FU-treated mice were injected into the orbital sinus
of each lethally irradiated (900-cGy, divided into 2 doses 3 hours apart) mouse. To determine the
onset of leukemia and monitor disease progression in recipient mice, 10 μL of peripheral blood
was drawn from the tibial vein of each animal every 48 h and diluted 1:1 in PBS containing 10
mM EDTA. Subsequently, the white blood cells, platelets, differential counts, and hemoglobin
levels of the peripheral samples were measured using a VET ABC blood analyzer (Heska, Fort
Collins, CO). Survival curves were compared by Kaplan-Meier analysis.
Flow cytometry
Cells from peripheral blood, bone marrow, spleen, liver, and lymph node tissues of diseased
mice were prepared for FACS analysis as previously described (8). Hematopoietic cells were
marked with PercpCy5-conjugated CD45 antibody (BD Biosciences) and screened for
expression of B-cell (B220), T-cell (Thy1.2), myeloid (Ly6-G), early myeloid (Mac-1), and
erythroid (Ter-119) markers using PE-conjugated antibodies (BD Biosciences).
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