SUPPLEMENTARY METHODS
Mouse cell sorting for RNA extraction
Bone marrow cell collection and staining for sorting leukocytes, HSPC, endothelial cells, MSC and
osteoblasts are described in supplementary methods. Following euthanasia, femurs were collected and
flushed into ice-cold PBS + 2% newborn calf serum (NCS) for cBM cells for RNA preparation. The
empty femurs were then flushed twice more in PBS then with 1mL Trizol (Invitrogen, Carlsbad, CA)
for endosteal RNA preparation as previously described1. RNA was also extracted from the following
FACS sorted BM hematopoietic cell populations: HSPC (LKS), myeloid progenitors (LKS-),
neutrophils (Gr1+F4/80-CD11b+), B-cells (B220+CD11b-) and T-cells (CD3+CD11b-). For stromal and
endothelial cells, partially crushed bones were incubated with type I collagenase (3 mg/mL from
Clostridium histolyticum, Worthington, Lakewood, NJ) for 40 min at 37°C then released cells
depleted for hematopoietic lineage-positive cells (using biotinylated lineage antibodies CD3, CD5,
B220, Ter119, CD11b, Gr1 with SAV-MACS beads and MACS depletion method) before staining
with SAV-FITC, CD45-APCCy7, CD31-APC, CD51-PE and anti-Sca-1-PECY7. The following cell
populations were sorted from viable (7AADneg) cells: BM endothelial cells (CD45-Lin-CD31+), MSC
(CD45-Lin-CD31-CD51+Sca-1+) and osteoblastic cells (CD45-Lin-CD31-CD51+Sca-1-) and RNA
extracted in Trizol.
Viral transduction of cell lines and HSCs
To generate transduced FDCP1 cell lines, full length cDNA encoding wild-type human HIF-2α was
subcloned by PCR from a plasmid vector2 into MND-X-IRES-eGFP (MXIE) bi-cistronic retroviral
vector3, 4 to generate MXIE-HIF2α vector expressing gene of interest together with green fluorescent
protein (GFP). Empty MXIE vector expressing GFP only was used as control. Retroviral particles
were generated after stable transfection and selection of the packaging cell line GP+E86 as previously
described4. FDCP1 cells were co-cultured for 24 hours at a concentration of 100,000/mL on a 30%
confluent layer of 25Gy irradiated GP+E86 cells stably transfected with MXIE-HIF2α or empty
MXIE. Transduced FDCP1 cells were selected by flow cytometry activated cell sorting gating on the
10% brightest green fluorescent protein (GFP)+ cells.
To generate HIF-2α over-expressing HSCs, mice were treated with a single dose of 150mg/kg
5-fluorouracil by retro-orbital injection 5 days prior to harvest. Following treatment, mice were
euthanized by cervical dislocation and BM extracted from femurs, hips, tibias and spine were
harvested in PBS with 10% FCS. BM cells were infected by overnight co-culture at 106/mL on 25Gy
irradiated transfected GP+E86 packaging cells as previously described4.
To generate stable knock-down HL60 cell line, RNA duplexes targeting human HIF-2α (5’GGGGGCTGTGTCTGAGAAGAGT-3’)
or
1
a
scrambled
control
(5’-
CCAAGGAGTAAGAGATAAAGGTC-3’) were cloned into the pFIV-H1-copGFP lentiviral vector
(System Biosciences, CA, USA) as previously described5. Transduced cell lines derived from HL60
cells were generated by 2 successive sorts of the brightest 10% of GFP-expressing cells at a 2 week
interval using a FCS Aria cell sorter (BD Biosciences).
Transplantations of virally transduced cells
All experiments were approved by the animal experimentation ethics committee of the University of
Queensland. C57BL/6 mice, DBA/2 and NOD/SCID IL2Rγ-/- (NSG) mice were purchased from the
Australian Resource Centre, Perth Australia. VavBcl2 transgenic mice in C57BL/6 background were
originally donated by Prof. J Adams (WEHI, Melbourne)6. These mice were produced and maintained
by breeding with wild-type C56BL/6 females and pups genotyped by using the PCR primers forward:
5’-GCCGCAGACATGATAAGATACATTGATG
and
reverse:
5’-
AAAACCTCCCACACCTCCCCCTGAA. 106 retrovirally transduced FDCP1 cells were transplanted
into non-irradiated DBA/2 female mice. In other experiments, 5x106 retrovirally transduced BM cells
from 5-fluorouracil treated vavBCl2 transgenic mice were transplanted retro-orbitally into C57BL/6
females after prior irradiation (two 5.5Gy doses at 4 hours apart prior to transplantation) as previously
described4. In other experiments, 2x106 lentivirally transduced HL60 cells were transplanted into NSG
female mice. Following transplantation, mouse health was scored weekly. At the emergence of first
clinical signs of disease, 50µL of blood was drawn from the tail viin at weekly intervals to measure
tumor burden by flow cytometry based on GFP fluorescence.
Transplantation of human B-ALL patient derived xenografts
Three B-ALL patient-derived xenografts ALL#3, ALL#7 and ALL#19 were generously provided by
Dr Richard Lock (Children’s Cancer Institute Australia for Medical Research, Randwick, Australia).
These 3 B-ALL patient-derived xenografts have been exclusively maintained and passaged in vivo in
NOD/SCID mouse recipients and have preserved many of the characteristics of the original patient
leukemia cells such as sensitivity or resistance to particular drugs 7. B-ALL cells were transplanted
exactly as described7. When mice exhibited clinical signs of disease, they were euthanized by cervical
dislocation, femurs harvested and flushed with cell urea cell lysis buffer for western-blot, or with PBS
for RNA extraction and qRT-PCR.
Fluidigm gene expression analysis
RNA was extracted from donor and patient material using a routine phenol/chloroform method
(Trizol, Invitrogen). Purified total RNA was converted to cDNA using the QIAGEN Quantitect
Reverse Transcription Kit, and then all of the targeted genes were pre-amplified in a single 14-cycle
PCR reaction for each sample by combining 1.25ul cDNA with the pooled primers and TaqMan Pre2
Amp Mastermix (Fluidigm BioMark™) following conditions outlined in the manufacturer’s protocol.
Finally, quantitative PCR was performed for each primer pair on each sample on a 96.96 Integrated
Fluidics Circuit (IFC) using the EvaGreen detection assay on a Biomark HD system following
standard Fluidigm protocols. Primers were purchased from Sigma-Aldrich (see Supplementary table 2
for primer sequences).
Data were analyzed using Fluidigm Real-Time PCR Analysis v4.0.1 (Fluidigm), and graphed using
Prism v5.04 (GraphPad Software). The HIF-2α expression for each sample was normalized to the
geometric mean of the expressions of the three housekeeping genes (HMBS, RPLP0, HPRT1).
Samples with cycle thresholds greater than 28 were excluded from the analysis.
Mutation screening
To assess co-occurrence with other common mutations in AML we used a PCR-based fragment
analysis for FLT3-ITD screening and a multiplexed matrix-assisted laser desorption/ionization timeof- flight genotyping approach (Sequenom MassARRAY Compact System, Sequenom, Inc., San
Diego, CA, USA) for detection of the following mutations: KIT (D816V), DNMT3A (R882C/H),
FLT3 (TKD: D835H/Y//V/E, I836DEL, I836INS), IDH1 (R132C/H/P), IDH2 (R140W/ L/G,
R172W/G/K/M), JAK1 (T478S, V623A), JAK2 (V617F), KRAS (G12D/V/A, G13D/A) and WT1 as
previously8.
Statistical analyses
For cell cultures or mice, differences between treatment groups were analyzed using a two-tailed t-test
or non-parametric Mann-Whitney depending on distribution normality. Data are presented as mean ±
standard deviation. Survival curve comparisons were performed using the log-rank test. All
calculations were performed using GraphPad Prism 5 software (GraphPad Sofwares, La Jolla, CA). P
values below 0.05 were considered significant.
Differences between the clinical characteristics of AML patient groups were analysed using
the Wilcoxon rank-sum test for continuous variables and the Fisher's exact test or Chi-square test for
categorical variables as indicated. The definition of diagnosis followed recommended criteria 9. For
survival analysis eligible patients received standard induction chemotherapy as described above and
survived to first bone marrow biopsy post-treatment (28 days). Overall survival was measured from
date of diagnosis to date of death (failure), last date of contact (censored), or date of transplant
(censored) and all patients were censored at 5 years. Disease free survival was measured from the date
of diagnosis to date of relapse before transplant, date of death from AML, date of transplant
(censored), date of death from unrelated causes (censored), or last date of contact (censored). All
remaining patients were censored at 5 years. Median survival and percentage survival at 5 years were
3
estimated using the Kaplan-Meier method, and differences between survival distributions were
assessed using univariate and multivariate Cox regression analysis. All statistical analyses were
performed using IBM SPSS Statistics 19.
Refereances
1
Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, et al. Bone marrow
macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes
HSCs. Blood 2010; 116: 4815-4828.
2
Bracken CP, Fedele AO, Linke S, Balrak W, Lisy K, Whitelaw ML, et al. Cell-specific
regulation of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha stabilization and
transactivation in a graded oxygen environment. The Journal of biological chemistry 2006;
281: 22575-22585.
3
Robbins PB, Yu XJ, Skelton DM, Pepper KA, Wasserman RM, Zhu L, et al. Increased
probability of expression from modified retroviral vectors in embryonal stem cells and
embryonal carcinoma cells. Journal of virology 1997; 71: 9466-9474.
4
Shen Y, Winkler IG, Barbier V, Sims NA, Hendy J, Lévesque J-P. Tissue inhibitor of
metalloproteinase-3 (TIMP-3) regulates hematopoiesis and bone formation in vivo. PLoS
ONE 2010; 5: e13086.
5
Martin SK, Diamond P, Williams SA, To LB, Peet DJ, Fujii N, et al. Hypoxia-inducible
factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma
cells. Haematologica 2010; 95: 776-784.
6
Ogilvy S, Metcalf D, Print CG, Bath ML, Harris AW, Adams JM. Constitutive Bcl-2
expression throughout the hematopoietic compartment affects multiple lineages and enhances
progenitor cell survival. Proc Natl Acad Sci U S A 1999; 96: 14943-14948.
7
Liem NLM, Papa RA, Milross CG, Schmid MA, Tajbakhsh M, Choi S, et al. Characterization
of childhood acute lymphoblastic leukemia xenograft models for the preclinical evaluation of
new therapies. Blood 2004; 103: 3905-3914.
8
Diakiw SM, Perugini M, Kok CH, Engler GA, Cummings N, To LB, et al. Methylation of
KLF5 contributes to reduced expression in acute myeloid leukaemia and is associated with
poor overall survival. Br J Haematol 2013; 161: 884-888.
9
Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH, et al. Revised
recommendations of the International Working Group for Diagnosis, Standardization of
Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in
Acute Myeloid Leukemia. J Clin Oncol 2003; 21: 4642-4649.
4