SUPPLEMENTARY MATERIALS AND METHODS
Preparation of genomic DNA
To prepare genomic DNA either 1x106 cells (for FLT3 ITD detection) or 100 μl of frozen and thawed
whole blood (to detect the JAKV617F mutation) were centrifuged at 1000 g for 10 min in 1.5 mL
microcentrifuge tubes. The supernantant was discarded. Cells were washed twice with PBS before
being re-suspended in 200 μl cold PBS containing 20 μl proteinase K. High quality genomic DNA was
prepared using the Genomic DNA preparation Kit (# 69506, Qiagen, Hilden, Germany), according to
manufacturer’s instructions.
Genotyping for FLT3-ITD
FLT3-ITD mutations were detected with the use of site specific primers to exons 14 and 15, to
amplify a 324-bp fragment in the wild-type FLT3 sequence (forward primer: GCA ATT TAG TA TGA
AAG CCA GC, reverse primer: CTT TCA GCA TTT TGA CGG CAA CC), using 10-40 ng of genomic DNA
and a PCR mastermix containing 0.5 μl of the Phusion® High Fidelity DNA polymerase (New England
Biolabs, Ipswich, MA) in 50 μl total reaction volume, according to manufacturer’s instructions. The
PCR cycle was 98°C 30”, 35x (98°C 30”, 60°C 30”, 70°C 30”), 70°C 5’). ITD mutations were detected by
the appearance of a larger band using gel electrophoresis on a 1.5% agarose gel.
Detection of JAK2V617F mutation
25 ng of extracted genomic DNA were used for Real Time PCR in the 7500 Real Time PCR system
(Applied Biosystem, Foster City, CA), using a PCR Mastermix from Applied Biosystem, (Cat# 4324018)
to determine the JAK2V617F mutation burden. The PCR cycle was as follows: (95C 10 min, 45 cycles at
95C 15 s, 62C 1 min). The TaqMan® probe used was AS-JAK2 (CTT GCT CAT CAT ACT TGC) and the
forward primer used for JAK2 wild type/mutant determination was g-JAK2F (TTA TGG ACA ACA GTC
AAA CAA CAA T). The reverse primers used for wild type/mutant JAK2 determination were G-gJAK2
(TTT ACT TAC TCT CGT CTC CAC AGT C) and T-gJAK2 (TTT ACT TAC TCT CGT CTC CAC AGT A),
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respectively. The mutation burden of the sample was calculated according to the following formula:
JAK2 V617F%=[copy number of V617/(copy number of V617F+copy number of wild type)]*100%. All
percentages above 0% indicate the presence of the JAK2V617F mutation.
pFLT3 ELISA
The pFLT3 capture ELISA (Cat#7206) from Cell Signaling Technologies was used according to
manufacturer’s protocol. Briefly, tumor lysates were thawed on ice, protein concentrations were
measured and equal amounts of protein were assayed in duplicates.
Flow cytometry
For cell cycle analysis, 5 x 105 /ml MV4-11 cells were treated for up to 72 hr with 0.1 µM TG02. Cells
were fixed using 70% ice-cold ethanol and stained with 20ng/mL propidium iodide (BD Biosciences).
For apoptosis analysis, cells were treated with 0.1 µM TG02 and stained using the Annexin V-FITC
apoptosis detection kit from BD Biosciences (Franklin Lakes, NJ), according to manufacturer’s
instructions. To characterize expanded progenitor cells or AML blasts, cells were labeled with
monoclonal antibodies against CD71 and CD235A or CD34, CD38 and CD123 respectively, and
analyzed on a FACSCalibur equipped with CellQuest Pro software (BD Systems, San Jose, CA).
Pharmacokinetic analysis
TG02 was extracted from 50 μL of plasma or from tissue homogenized in 0.5 mL PBS. High
performance liquid chromatography mass spectrometry analysis was done on a Waters Alliance
HT2795/QuattroMicro (Waters Corp., Milford, MA) using a C8 column (2 X 5 mm, 5 µm Zorbax,
Quantum Analytics, Foster City, CA). Samples were resolved using an isocratic run with a mobile
phase consisting of 70% methanol and 30% of 0.1% formic acid in water, at a flow rate of 0.3
mL/min. The tandem mass spectrometry conditions for multiple reaction monitoring for TG02 were
transitions of mass to charge ratio (m/z) of 473 to m/z 96.8 and m/z 237 to 194 for the internal
standard carbamazepine respectively, both in electrospray ionization-positive mode. The cone
voltage (CV) was 40 V with collision energy (CE) of 35 eV for TG02 and 20 V / 35 eV for
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carbamazepine. The assay was linear between 1 and 1000 ng/mL and the lower limit of quantitation
was 1 ng/mL. Non-compartmental pharmacokinetic analysis was performed using WinNonlin
(version 5.1, Pharsight Corp., CA). The AUC was estimated by using the linear trapezoidal method.
Dose proportionality, which was assessed by linear regression of day 12 AUC0-∞ and Cmax with dose,
as well as statistical analyses were performed using Prism 5.
Cell proliferation assay
Cells were seeded in 96-well plates at a predetermined optimal density, in the log phase and rested
for 2 h, prior to drug treatment for 48 h. All experiments were performed in triplicates in 9 serial
dilution steps, and cell viability was measured using CellTiter 96 ® Aqueous One Solution Cell
Proliferation MTS Assay (Promega, Madison, WI).
Primary erythroid progenitor cells
Blood was drawn from consented donors at Tan Tock Seng Hospital, Singapore (IRB approval number
DSRB-B/08/159) and PBMCs were purified using Becton Dickinson Vacutainer® CPT™ tubes (Franklin
Lakes, NJ). Progenitors were expanded from PBMCs using a published protocol.9 In brief, 3 x 105
cells/ml were cultured in the StemSpan® Serum-Free Expansion Medium in the presence of 100
ng/ml of FLT3 ligand, 100 ng/ml of thrombopoietin and 100 ng/ml of stem cell factor, diluted from
the cytokine cocktail CC110 (StemCell Technologies). On day 4, the remaining viable cells were
purified using Ficoll histopaque (GE LifeScience, Uppsala, Sweden) and re-plated in the same
medium. On day 8, cells were seeded at 5 x 105 cells/ml in a second-step medium (SFEM containing
50 ng/ml of stem cell factor, 50 ng/ml of insulin-like growth factor-1, and 3 U/ml of erythropoietin).
Expanded progenitors were re-plated at 3 x 104 cells /ml in the same medium on day 12 for
treatment. On day 10 to 13, expanded cells were harvested, counted on the Z1 Particle Counter and
aliquoted as follows : ~ 1 x 105 cells for JAK2V617F analysis, ~ 5 x 105 cells for FACS, ~ 3 x 106 cells for
proliferation assay.
Western blot assays
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Cell lysis, protein quantification and Western blots were performed as described previously.12
Western blotting was performed using antibodies against pRb S807/811 (#9308), Rb (#9309), pFLT3
Y591 (#3461), pSTAT3 Y705 (#9145), pJAK2 Y1007/1008 (#3776), Mcl-1 (#4572), Cyclin E (#4129), Bcl2 (#3498), cdc6 (#3387), cleaved PARP N214 (#9541) and secondary antibodies, all purchased from
Cell Signaling Technology (Danvers, MA). The antibody against pSTAT5 (Y694, Cat #611965) was from
BD Biosciences (San Jose, CA) and anti-phospho-RNA polymerase II Ser2 (ab70324) was from Abcam
(Cambridge, UK). Antibodies against -actin and α-tubulin were from Sigma Aldrich (Cat# 2066; St.
Louis, MO) and Millipore (Cat# 05-829; Billerica, MA) respectively. Signals were detected using the
LAS-3000 (Fujifilm, Tokyo, Japan) at normal sensitivity using either ECL substrate (GE Life, Little
Chalfont, U.K.) or SuperSignal Western Dura Chemiluminescent substrate (Pierce, Rockford, IL).
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