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Supplementary Information
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Materials and Methods:
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Gene Expression Analysis
A previously published gene expression datasets (GSE88791) deposited in NCBI
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Gene Expression Omnibus (GEO) were re-analyzed in this study.
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dataset contains gene expression information on 55 diagnostic T-ALL samples, of which
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9 are early immature T-ALL and 46 are more differentiated T-ALL. Affymetrix Human
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Genome U133 Plus 2.0 Arrays were used in the microarray experiments (54675 probes
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are printed on each array).
The GSE8879
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We performed differential gene expression analysis for early immature T-ALL vs.
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more differentiated T-ALL patient samples for GSE8879 dataset,1 using GenePattern, a
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publically available open-source software package developed at the Broad Institute for
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the analysis of genomic data. First, the PreprocessDataset module, which performs a
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variety of pre-processing operations including thresholding/ceiling, variation filter and
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normalization, was used to filter the dataset.
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inputted to the ComparativeMarkerSelection module to identify differentially expressed
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genes.
Then the preprocessed dataset was
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Protein Extraction and Western Blotting Analysis
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Whole cell lysates were prepared in RIPA buffer for the following T-ALL cell lines:
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LOUCY, ALL-SIL, CCRF-CEM, HSB2, SUPT-11, SKW-3/KE-37, MOLT4, JURKAT and
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JURKAT overexpressing BCL2 and luciferase. The primary antibodies included: anti-
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BCL2 (Santa Crutz, Dallas, TX, USA), anti-BCL-XL (Cell Signaling, Boston, MA, USA),
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anti-BCLw (Abcam, Cambridge, MA, USA) and anti-ACTIN (Sigma, St. Louis, MO, USA)
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antibodies.
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mouse or anti-rabbit antibodies (Thermo, Rockford, IL, USA). Autoradiographs were
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imaged with a LAS-3000 imaging system (Fuji, Tokyo, Japan) and a CCD camera. All
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images were prepared for publication with Adobe Photoshop (Adobe, San Jose, CA,
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USA) software.
Secondary antibodies included horseradish peroxidase-conjugated anti-
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Cell Lines and Cell Viability Assay
Cell
lines
were
cultured
in
RPMI1640
(Corning,
Corning,
NY,
USA)
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supplemented with 10% fetal bovine serum (Life Technology, Carlsbad, CA, USA). For
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viability assay cells were plated at a density of 20,000 per well in a 96 well plate
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(Corning, Corning, NY, USA) and incubated for indicated times in DMSO (ATCC,
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Manassas, VA, USA), ABT-199 (Chemietek, Indianapolis, IN, USA), ABT-263
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(selleckchem), cytarabine (Sigma, St. Louis, MO, USA), doxorubicin (Pfizer, New York
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City, NY, USA), etoposide (Accord Healthcare, Durham, NC, USA), dexamethasone
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(Sigma, St. Louis, MO, USA), rapamycin (LC laboratories, Woburn, MA, USA). Cell
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Titer Blue assay (Promega, Madison, WI, USA) was used to assess cell viability and
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was reported as a relative percentage versus control cells.
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Apoptosis and Cell Cycle Analysis
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Apoptosis was assessed with Annexin V and propidium iodide (PI) staining, based on
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two populations: early apoptosis (PI-, Annexin V+) and late apoptosis (PI+, Annexin V+).
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Specifically, cells were incubated at room temperature for 20 minutes with Annexin V-
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APC (BD Bioscience, San Jose, CA, USA), PI (50ug/ml) (Sigma, St. Louis, MO, USA)
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and 1x binding buffer (BD Pharmagen, San Jose, CA, USA). For cell cycle analysis,
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cells were fixed in ice cold 100% ethanol and incubated at -20oC for 30min. Fixed cells
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were then
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incubated for 20 min at 4oC in the dark. Cells stained for apoptosis and cell cycle were
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analyzed on a FACScalibur (BD Bioscience, San Jose, CA, USA) and data were
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analyzed with either Flowjo or Modfit software.
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Combinatory Treatment of Small Molecules
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T-ALL cell lines were treated with a combination of ABT-199 and Cytarabine with a
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range of doses around the IC50 of each drug alone. Cell viability was analyzed by Cell
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Titer Blue assay (Promega, Madison, WI, USA).
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Cambridge, UK) was used to calculate the potency of drug combination and we used
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the Chuo-Talalay method2 to calculate a combined index (CI).
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isobolograms were also produced with Calcusyn software. The CI has been interpreted
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as follows: very strong synergy (<0.1), strong synergy (0.1 to 0.3), synergism (0.3 to
Calcusyn software (Biosoft,
Normalized
3
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0.7), moderate synergism (0.7 to 0.85), slight synergism (0.85 to 0.9), nearly additive
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(0.9 to 1.1), slight antagonism (1.1 to 1.2) and moderate antagonism (1.2 to 1.45).
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Statistical Analysis
GraphPad Prism software was used to calculate significant values (p) using a
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two-tailed Student’s t test.
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statistically significant.
P-values less than or equal to 0.05 were considered
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Supplementary Figure Legends:
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Supplementary Figure 1.
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The expression of BCL-XL, MCL-1 and BFL-1 in early immature T-ALL primary
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patient samples. BCL-XL (probe ID: 215037_s_at), MCL-1 (probe ID: 200796_s_at)
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and BFL-1 (probe ID: 205681_at) were expressed in early immature T-ALL patients
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samples at levels similar to more differentiated T-ALL. Data was generated by re-
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analyzing a published gene expression dataset (GSE8879)1 that contained 9 early
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immature T-ALL and 46 more differentiated T-ALL patient samples.
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Supplementary Figure 2.
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ABT-263 and ABT-199 induce apoptosis in LOUCY cell line. The LOUCY cell line
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was treated for 48h with either ABT-263 (0nM, 30nM and 125nM) or ABT-199 (0nM,
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15nM and 2000nM) and then cells stained with Annexin V and PI. Cells were examined
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by flow cytometry to determine early apoptosis (PI-, Annexin V+) and late apoptosis (PI+,
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Annexin V+).
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Supplementary Figure 3.
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The LOUCY cell line shows resistance to T-ALL induction therapeutics. A panel of
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eight T-ALL cell lines - LOUCY, ALL-SIL, CCRF-CEM, HSB2, JURKAT, SUPT-11,
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SKW-3/KE-37 and MOLT4 were treated for 48 hours, with the following T-ALL induction
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therapeutics: (a) etoposide, (b) dexamethasone, (c) rapamycin, (d) vincristine and (e)
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doxorubicin. Relative cell viability was measured using Cell Titer Blue reagent. The
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values represented in the graphs are the means  SD, and represent three biological
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replicates.
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Supplementary Figure 4.
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Cytarabine induces S-phase cell cycle arrest in LOUCY and JURKAT cell lines. (a-
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d) Cytarabine alone and in combination with ABT-199 induces S-phase arrest. The
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histograms represent cell cycle analysis of PI stained cells showing the DNA content of
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LOUCY cells treated for 24 hours with (a) DMSO (control), (b) ABT-199 15nM, (c)
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Cytarabine 75nM and (d) both drugs in combination (ABT-199: 15nM; cytarabine:
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75nM). Values are means  SD, and represent three biological replicates. *, P<0.05; **,
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P<0.001; ***, P<0.0005.
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Supplementary Figure 5.
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Cytarabine and ABT-199 synergizes to promote apoptosis in LOUCY cells.
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Apoptosis analysis was performed on LOUCY cells stained with Annexin V and PI 24
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hours after treatment. The LOUCY cells were treated with DMSO (control) (far left
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panel), ABT-199 15nM (middle left panel), cytarabine 75nM (middle right panel) and
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both drugs in combination (ABT-199: 15nM; cytarabine: 75nM) (far right panel). Values
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are means  SD, and represent three biological replicates. Statistical significance is
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indicated by: *, P<0.05; **, P<0.001; ***, P<0.0005.
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References:
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1.
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Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic
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leukaemia. Lancet Oncol. 2009;10:147-56.
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2.
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Chou-Talalay method. Cancer Research. 2010;70:440-6.
Coustan-Smith E, Mullighan CG, Onciu M, Behm FG, Raimondi SC, Pei D, et al.:
Chou TC: Drug combination studies and their synergy quantification using the
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