SUPPLEMENTARY METHODS

Drug sensitivity and resistance testing (DSRT)

DSRT was performed with MOLM-13 and SHI-1 parental and the

respective cytarabine resistant cells. 250 active chemical

compounds, covering approved and investigational compounds and

probes, were dispensed on 384 well plates with the Echo acoustic

dispenser (Labcyte, Sunnyvale, CA, USA). Each compound was

plated at 5 different concentrations covering a 10 000-fold range to

generate quantitative dose response curves. Cells were added to

pre-drugged plates and incubated for 72 h at 37 o C and 5% CO2. Cell

viability was measured with the CellTiter-Glo assay (Promega,

Madison, WI, USA). Curve fitting and IC 50 values were calculated

with Dotmatics software (Dotmatics, Herts, UK). Drug efficacy was

quantified with a drug sensitivity score (DSS), which is a modified

area-under-the-curve (AUC) measurement.

In addition, same DSRT assay was performed with patient samples

and healthy bone marrow samples. Briefly, mononuclear cells were

isolated using Ficoll-Hypaque Premium density gradient method (GE

Healthcare, Little Chalfont, UK). Cells were counted and suspended

in Mononuclear Cell Medium (PromoCell, Heidelberg, Germany) for

drug testing or processed for molecular profiling. Selective DSS was

calculated by subtracting from the patient DSS, the average DSS

obtained from 15 healthy bone marrow controls.

Gene expression profiling

Total RNA was isolated from parental, 160 Ara-C, 320 Ara-C, 640

Ara-C and 1280 Ara-C of MOLM-13 and SHI-1 cells using the total

RNA purification kit (Norgen Biotek, Thorold, Canada). RNA quantity

and quality were assessed by Qubit (Thermo Scientific, Waltham,

MA, USA) and Bioanalyzer (Agilent Technologies, Santa Clara, CA,

USA) instruments, respectively. 600 ng of total RNA from each

cytarabine resistant variant and corresponding parental cells was

amplified and hybridized to HumanRef-12 version4 BeadChip

(Illumina, San Diego, CA, USA). The raw gene expression data were

quintile normalized using the beadarray R-package (Bioconductor).

Mutation and copy number variation analysis

Genomic DNA was isolated from MOLM-13 and SHI-1 parental and

1280 Ara-C cells using DNA/RNA/protein purification kit (Norgen

Biotek, Thorold, Canada) and from AML patients’ mononuclear cells

using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).

Whole exome sequencing was performed with 3 μg of genomic DNA

using NimbleGen SeqCap EZ Human Exome v2.0 kit (Roche

NimbleGen, Madison, WI, USA) and paired-end sequencing was

performed with Illumina HiSeq instruments. Parental cells in case of

cytarabine resistant variants and skin sample in case of AML

patient samples were used as controls. Sequencing reads were

aligned and processed through a variant calling pipeline. False

positive variants resulted from sequence alignment error and minor

allele frequencies were detected and removed.

Western blot analysis

Cells were washed in cold PBS and lysed with a buffer consisting of

50mM Tris-HCl, pH7.5, 150mM NaCl, 10% glycerol, 1% Triton X-100

and protease inhibitor cocktail (Roche Applied Systems, Penzberg,

Germany). Protein quantification was performed using Pierce BCA

protein assay kit (Thermo Scientific, Waltham, MA, USA). 80 μg of

protein lysates were separated on a 4-20% precast Mini-PROTEN

TGX gel (Bio-Rad, Hercules, CA, USA) and transferred to PVDF

membrane. The membrane was blocked with Odyssey blocking

buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h and

subsequently incubated with primary antibody NR3C1 (1:200

dilution, sc-1003, Santa Cruz Biotechnologies, Dallas, TX, USA)

overnight and with anti-GAPDH (1:10 000 dilution, clone GAPDH-

71.1, Sigma Aldrich, St. Louise, MO, USA) for 2 h. Secondary anti-

mouse and anti-rabbit fluorescence antibodies from Odyssey (LI-

COR Biosciences, Lincoln, NE, USA) were used to detect the

specific protein bands after 1 h incubation. The protein band

intensities were quantified with the Odyssey software.