Supplemental Materials and Methods
Cell lines and Tissue culture
Most of the experiments were carried in Bcr-Abl transformed imatinib mesylate (IM) sensitive mouse cell lines
[Bcr-Abl+ 32D cells (32Dp210) and BaF3p210]. For examination in IM-resistance we used Abl point mutant cell
lines 32D/BaF3-T315I and BaF3-E255K and Lyn up-regulated human CML line K562-R. All the cell lines were
cultured in RPMI 1640 supplemented with penicillin and streptomycin and 10% fetal bovine serum. K562-R
cells were cultured in 5 μM IM.
CML patient cells
At UT MD Anderson Cancer Center, the patients’ blood, and bone marrow are obtained with IRB approved
protocols where the patients’ consent is mandatory before collection of any samples from patients. We do
research in our lab using an IRB approved protocol (LAB05-0410) for analyzing the patient’s samples provided
by the clinicians. Cord blood samples were provided by Prof. Ravi Bhatia, Director of CML and Stem cell
Research from City of Hope. Cord Blood (CB) samples were provided by StemCyte (Arcadia, CA). All donors
signed an informed consent form. Sample acquisition was approved by the Institutional Review Board at the
City of Hope National Medical Center (COHNMC) and met all requirements of the Declaration of Helsinki.
Peripheral blood mononuclear cells (PBMC) and bone marrow cells were separated from whole blood or
bone marrow by centrifugation through Histopaque- 1077 (Sigma) and the cells from white buffy coat were
washed two times with PBS and the pellets were suspended in RPMI medium with 10% FBS. Using these
cells Western blotting and apoptosis experiments were carried out using TG101209 and WP1193.
CD34+ progenitor cells
For separation of CD34+progenitor cells from bone marrow or PBMC we followed the protocols provided by
Stem Cell Technologies (www.stemcell.com). The cells were suspended in the recommended medium (Ca++/
Mg++ free PBS containing 2% FBS and 1mM EDTA). The amount of medium added to the cells depends on
the cell number as recommended in product information sheet of the kit provided by the company. For positive
selection of CD34+ cells, EasysepR positive selection cocktail was added (100l/ ml) and incubated for 15 min
and then magnetic nanoparticles were added (50l/ml) and mixed well and incubated for 10 min. The volume
was made 2.5 ml with the recommended medium, mixed well and kept in the magnet for 5 min. The tubes
were rinsed 5 times with medium, each time kept for 5 min and finally the positive selected cells were collected
from the tubes after removal of the magnetic tube holder. The purity of the cells were examined in flow
cytometry using anti-CD34+ antibody coupled to PE. The purity of the CD34+ cells was 87-90%. Apoptosis
and Western blotting of these cells were carried out after treating these cells with Jak2 inhibitors.
Knockdown Jak2
Short interfering RNA (siRNA) duplexes targeting mouse Jak2 was designed and synthesized by Dharmacon
(Thermo Scientific, Lafayette, CO) and used as described. The human and mouse siRNA were specific to Jak2
targeting mouse mRNAs and did not have sequence overlap with Jak1, Jak3 and Tyk2. For transfection of
siRNA by electroporation, we followed the Nucleofection protocol of the manufacturer program- # E032 (for
Nucleofactor II, Lonza Walkersville Inc., Walkersville, MD). To examine efficiency of transfection, GFPmax
transfected cells were examined under a fluorescent microscope to ensure 60-70% transfection efficiency. For
knockdown experiments, 200pmole of siRNA (either targeted or non-targeted) was used. The pre- warmed
cells (2x106 cells) were centrifuged at 700 rpm. The cells were suspended in 100 l.
containing the siRNA in a cuvette used in the electroporation machine.
The appropriate program for
electroporation of a particular cell line was selected. After transfection, the cells were incubated for 48-72h.
Then Western blotting was performed as above. No effects on levels of Jak1 and Jak3 were detected (not
shown). Rescue of Jak2 knockdown by siRNA was performed by transfection of Jak2 cDNA. For expression
of protein, Western blotting was carried out.
Jak2 shRNA
For lentivirus transfection, 293T cells were transfected using Fugene-6 (Roche, Indianapolis, IN,) with Jak2
shRNA and lentivirus packaging vectors PMDG and 
After 48hrs
of expression, the supernatant containing viral particles was collected and used for infection to K562R cell line
for over night period. Polybrene (1:1000) is added to the media. Virus-transfected cells were selected with the
addition of 2g/ml of puromycin. The marker gene GFP and shRNA are induced with the addition of 2g/ml of
doxycyclin for 48hrs. Top 10% RFP positive cells were collected by FACS analysis and sorted cells were used
for western blotting following the standard protocol. Reduction of Jak2 signals was detected using anti-Jak2
antibody.
Western blotting and immunoprecipitation
The harvested cells were washed with PBS, lysed in lysis buffer containing 1% NP-40 in 20mM HEPES pH
7.4, 150mM NaCl, 1mM EDTA, and different protease inhibitors. For immunoprecipitation, 400g (protein) cell
extract was mixed with 400l lysis buffer. The primary antibody used for immunoprecipitation was mixed with
cell lysate, and rotated for 90 min at 40C. After that 35 μl of a 50% suspension of protein A /G agarose was
added and the rotation was continued for another 90 min. After washing with lysis buffer, the beads were
treated with 30 l 2x SDS-MEO sample buffer and boiled for 5 min. For direct western blot, 30 g cell lysate
was mixed with equal amount of 2x sample buffer and boiled for 5 min and were loaded into wells and
separated in 4-20% SDS-PAGE gradient gel using protein markers. The proteins were transferred onto PVDF
membrane, blocked by either 3% BSA (for detection of pTyr) or 5% nonfat dry milk. Primary antibodies were
incubated for 1h. After washing with TBST (Tris buffered saline and Tween 20), the appropriate secondary
antibodies conjugated to horse-radish peroxidase were incubated at room temperature for 1h. After washing,
the membrane was developed using ECL/ ECL plus reagents (Amersham Biotech Company). -Actin / tubulin
/GAPDH (Sigma, St. Louis, Mo) were used as a loading control in Western blot directly and also from
supernatant for immunoprecipitation.
Kinase assay for Jak2 and Bcr-Abl
For in vitro kinase assays we used Jak2 and Bcr-Abl from the immunoprecipitates obtained from Bcr-Abl+ cell
lines using anti-Jak2/ Anti-Abl antibody (P6D) followed by immunoprecipitation with Protein A/G agarose
beads. In vitro kinase assay for Jak2 (autophosphorylation) was carried as described (35) and Sandberg et al.
(36). Cell free lysates of 32Dp210 was prepared by treating the cells with lysis buffer containing 20mM TrisHCl, 100mM NaCl, 1% NP40 and protease inhibitors. Detergent extracted lysate was aliquoted to a microfuge
tube containing 500 μg protein/500 μl lysis buffer. Then 30 μl Jak2 antibody coupled with agarose beads was
added for co- immunoprecipitation for Jak2. After washing with lysis buffer followed by washing with kinase
buffer (containing 50mM HEPES,pH7.6, 100mM NaCl, 5mM MgCl2, 5mM MnCl2), the agarose beads were
suspended in kinase buffer and different amounts of TG101209 were added and the reaction was initiated by
addition of 2.5mM ATP. The reaction was continued for 30 min at room temperature and the reaction was
stopped by addition of 2X sample buffer. The signals for the kinase reaction were detected by Western blotting
with pTyr antibody (4G10) or by using P32 gamma ATP. In vitro kinase assays for Bcr-Abl
(autophosphorylation) was performed following the method of (2) by immunoprecipitating Bcr-Abl with P6D
antibody.
Immunoprecipitates were incubated with various amount of WP1193.
Kinase reactions were
initiated with addition of cold ATP, Mg++ and 1mM DTT at 30 deg C for 30 min. Kinase activity was detected
by Western blotting with anti-pTyr antibody (4G10).
Recombinant Jak2 kinase assay
For recombinant Jak2 kinase (JH1 or JH1-JH2 domains) assay, 25ng kinase in kinase assay buffer was
preincubated for 10 min with different amount of TG101209 in an incubation mixture as described above for
cold kinase assay. After 10 min the reaction was initiated with cold ATP (5M), 10Ci /assay 32Pgamma ATP
and 5 μg of Bcr Tyr 177 peptide (Bachem Co. Torrance, CA) and continued the reaction for 10 min at room
temperature. The target peptide has the Bcr sequence H-Ala-Glu-Lys-Pro-Phe-Tyr (177)-Val-Asn-Val-GluPhe-His-His-Glu-(Lys-Lys-Lys). The bolded amino acids are the Grb2 binding site in Bcr-Abl (YVNV) and are a
Jak2 consensus phosphorylation site. The three Lys residues allow binding to Whatman 3mm filters. The
reaction was stopped with addition of 5l orthophosphoric acid (3%) in ice.
From the reaction mixture 10l aliquot was dropped into the three separate disks (2.5cm disk, grade p81,
Whatman filter paper) and the disks were washed three times for 5 min with orthophosphoric acid (180 M),
and finally the disks were washed with methanol (100%) (2min). The disks were put in the tubes containing 3ml
scintillation fluid and the tubes were used for counting 32Pgamma ATP in a liquid scintillation counter.
Recombinant Abl kinase assay
For the Abl kinase assay 20ng full length recombinant Abl kinase was mixed with the same kinase buffer used
for Jak2 kinase assay. Different amount of IM was added to the incubation mixture and preincubated for 10
min. The reaction was initiated by adding the substrate for Abl kinase (5M Abltide), 5M unlabeled ATP and
radio labeled P32 ATP (10Ci). The reaction was stopped by addition 5l of 3% phosphoric acid following the
same protocol mentioned for Jak2 kinase assay.
Apoptosis assay
For apoptosis measurement of the Bcr-Abl+ cell lines, CD34+ progenitor cells, bone marrow cells, we used the
apoptosis kit of BD Bioscience and followed the manufacturer protocols. Briefly, Bcr-Abl+ leukemic cells were
incubated with different doses of IM or Jak2 inhibitors-TG101209/ WP1193 for 48h. After incubation the
washed cells are suspended in a total volume of 50 l binding buffer (1x) containing 2.5l Annexin V/FITC, 4l
PI and incubated for 15 min in dark. After the incubation the cells are suspended to a total volume of 500l.
Then the apoptosis level of the cells was quantitatively measured (in %) in a flow cytometer (Becton Dickinson,
CA). For all the graphical presentation of apoptosis data, the sum of percentage of early and late cell death
was included. Usually, for apoptosis experiments duplicate samples and an average of data with std deviation
is presented but for patients samples in some cases single point analysis is carried out when supply of
patient’s sample or yield of cells was limited.
Solid tumor model
All animal protocols used for our studies were approved by the institutional animal care review committee at
UT MD Anderson Cancer Center and also in accordance with the National Institute of Health Guidelines for
animal care. In vivo efficacy of TG101209 was examined in nude immuno deficient SCID mouse model of
Jak2V617F-induced hematopoietic disease (34). To test the efficacy of WP1130 in vivo against the CML cells,
we used Swiss Nu/Nu mice in a tumor model. For this study we followed the protocols of Bartholomeusz, et al.
(35).
Briefly, the mice (5 weeks old) were purchased from the breeding facility of the Department of
Experimental Radiation Oncology, UT MD Anderson Cancer Center. IM resistant K562-R cells known to have
high expression of Lyn kinase (50 x 10 6/ml) were mixed with equal volume of matrigel and 200 l of this
suspension was injected subcutaneously into the mid-line dorsal region and randomly divided into two groups
(5 mice per group)-one for control (untreated) and the other for drug-treated group. On day 14, we injected one
group with 30mg/kg body weight WP1193 in 100l solvent (1:1 ratio of DMSO and polyethylene glycol 300) for
5 days (every 48 h) intraperitoneally (i.p.) into tumor bearing mice. In control group 100l only solvent was
injected following the same schedule. We measured the volume of tumor growth of the WP1193-treated and
untreated groups and the tumor volumes were plotted graphically. One day after the 6 th injection all the mice
were sacrificed (at day 22) and the weight of the tumors, livers, kidneys were measured.
Leukemia model
To assess the anti-leukemic potential of WP1193, BaF3-T315I Bcr-Abl mutant cells were injected i.v. into
female Swiss nude / nude mice (5-6 weeks old). Cells were suspended to 1-5 x 10e7 cells/ml in RPMI medium
without serum. The 0.1 ml of this suspension is injected i.v. into each mouse. One day after cell injection, the
WP1193 group (5 mice) (30 mg/Kg in DMSO mixed with PEG 3001:1) and the control group (5 mice) (50 µl
DMSO and PEG 300 1:1 ratio) were injected i.p. with every 48h for the entire period (7 injections) (14 days).
Mice were sacrificed one day after the last injection; and liver and spleens were collected and their weights
were measured to compare untreated and treated mice.
A second control set of mice experiments were carried out to examine the toxicity and side effects, if any
of WP1193, at this dosage (30mg/kg body weight) for this short treatment regiment. In these WP1193-treated
mice, no leukemic cells were inoculated. WP1193 was injected five times on alternate days with 30mg/kg body
weight. In these WP1193 treated, the livers and spleens were collected and compared with the spleens and
livers of a first set of control mice where the mice were neither injected with the leukemic cells nor with
WP1193.