AML1/ETO Cooperates with HIF1α in Boosting Leukemogenesis through ... - Nature … ·...

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SUPPLEMENTARY INFORMATION

Supplementary Materials and Methods

Supplementary References

Supplementary Tables

Supplementary Figures

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SUPPLEMENTARY MATERIALS AND METHODS

Patient samples

Bone marrow (BM) diagnosis of AML was performed according to the French-American-British

criteria and the WHO classification.1,2

Detection of t(8;21) was routinely accomplished by

standard cytogenetic techniques and (or) by FISH using commercially available AML1/ETO

(A/E) probe (Vysis Inc.). Molecular genetic analysis for A/E transcripts and KIT mutation was

carried out by using RT-PCR. Patients were treated with cytarabine 100 mgm2day continuous

infusion on days 1 to 7 plus daunorubicin (60 mgm2day, DA) or idarubicin (10 mgm

2day, IA)

or mitoxantrone (10 mgm2day, MA) on days 1 to 3 for induction therapy, followed by

intermediate/high dose cytarabine (1.5-2 gm2, every 12 hours on days 1 to 3) or standard-dose

cytarabine-based chemotherapy (DA/IA/MA) for consolidation. Allogeneic and autologous

hematopoietic stem-cell transplantations were performed in a risk-adapted and priority-based

manner. Mononuclear cells from BM samples of patients and 15 healthy donors were prepared

by Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO) gradient centrifugation. Fresh primary blasts

were obtained from BM samples of 3 newly diagnosed AML patients with >80% blasts and

cultured as previously described3 for subsequent clonogenic assays.

Cell lines

Human AML cell line Kasumi-1 [carrying t(8:21) (q22;q22) and mutKIT], murine myelogenous

leukemia cell line C1498 and human embryonic kidney cell line 293T were purchased from

American Type Culture Collection (Manassas, VA). SKNO-1PGK [carrying t(8:21) (q22;q22)

and mutKIT], SKNO-1siA/E, U937MT and U937A/E cell lines were kindly provided by Dr.

Clara Nervi.4 Kasumi-1, SKNO-1PGK, SKNO-1siA/E, U937MT and U937A/E cell lines were

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maintained in RPMI 1640 medium supplemented with 50 µg/ml streptomycin, 50 IU/ml

penicillin plus 20% (Kasumi-1) or 10% (others) FBS (Life Technology). C1498 and 293T cell

lines were maintained in DMEM medium supplemented with 50 µg/ml streptomycin, 50 IU/ml

penicillin and 10% FBS.

Plasmids

The following mammalian expression plasmids were purchased from Addgene (Cambridge,

USA): pCMV-AML1/ETO (Addgene ID 12428),5 HA-HIF1a-pcDNA3.0 (Addgene ID 18949).

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A 1000-2000 bp of sequence upstream of the transcription start site of the human AML1, HIF1

or DNMT3a gene was subcloned into the KpnI and HindIII sites of the luciferase reporter vector

pGL3-basic (Promega, Madison, WI). Various mutants were generated by QuikChange

Lightning Site-Directed Mutagenesis Kit according to manufacturer’s instruction (QuikChange,

Agilent Technologies). All constructs were verified by DNA sequencing. Primer sequences are

listed in Supplemental Table S5.

Cell transfection and drug treatment

For siRNA-mediated gene knockdown, 1×106 of Kasumi-1, SKNO-1PGK or C1498 cells were

seeded into 6-well plate for overnight before transfection. siRNAs used in this study were ON-

TARGETplus SMARTpool purchased from Thermal Scientific Dharmacon RNAi Technologies

(Thermo Scientific, MA): siHIF1 (L-004018-00), siRUNX1T1 (L-011824-00), siDNMT3a (L-

065433-01) and ON-TARGETplus Non-targeting Pool (D-001810-10-05). A total of 100 nM of

siRNAs or the corresponding negative control was transfected into cells using Lipofectamine™

RNAiMAX Reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s

instruction. For plasmid overexpression, 2×105 of 293T or C1498 cells were seeded into 6-well

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plate for overnight before transfection. A total of 2.5 μg of expression plasmids or the

corresponding empty vectors were transfected into cells using Lipofectamine™ 2000 reagent

(Life Technologies) according to the manufacturer’s instruction. For electroporation, 1×107 of

C1498 cells were resuspended in 380 L serum- and antibiotic-free DMEM medium. The cells

were mixed with 30 μg of plasmids in a 0.4 cm gap sterile electroporation cuvette and

electroporated using Gene Pulser Xcell electroporation system (Bio-Rad, Hercules, CA). For co-

transfection experiments, the total amount of the transfected DNA or siRNA in the individual

agent group was kept constant with the co-transfection group by adding equal amount of empty

vector or negative control. Note, in co-expression and co-knockdown experiments determining

the cooperation between A/E and HIF1, the amount of expression plasmids or siRNAs was

reduced to half of those used as single agents. Regarding the drug treatment, Kasumi-1, SKNO-

1PGK, SKNO-1siA/E or A/E-positive primary blasts isolated from patients were treated with

indicated concentrations of Echinomycin Streptomyces sp. (Calbiochem-Novabiochem, San

Diego, CA) or vehicle (DMSO, 0.1% v/v) for 24 hours.

Reporter assays

293T cells were seeded into 24-well plates at a concentration of 5×104 per well and grown

overnight. The transfection mixtures, contained 400 ng of firefly luciferase reporter plasmid, 2

ng of pRL-TK Renilla luciferase plasmid (Promega) and 0 to 100 ng of indicated expression

vectors were transfected into 293T using Lipofectamine™ 2000 reagent (Life Technologies)

according to the manufacturer’s instructions. For co-transfection experiments, the total amount

of the transfected DNA was kept constant by adding empty vector. Firefly luciferase activity was

measured 48 hours after transfection by using a dual luciferase reporter assay system (Promega).

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Clonogenic assays

The Methylcellulose colony-forming assays were performed in triplicate using human or mouse

methylcellulose media (R&D Systems, Minneapolis, MN) according to the manufacturer's

instructions. Briefly, 6 hours after transfection with siRNAs or exposure to drugs when the cells

did not undergo apoptosis, the cells were harvested, diluted with cell resuspension solution and

mixed with human or mouse methylcellulose complete media. For the colony-forming assays

determining the biologic significance of DNMT3a in A/E and HIF1 cooperation, C1498 cells

were transfected with A/E or HIF1 expression vector using Lipofectamine™ 2000 reagent (Life

Technologies) for 24 hours, and then DNMT3a siRNA or scramble using Lipofectamine™

RNAiMAX Reagent (Life Technologies) for another 24 hours before subjected to

methylcellulose media. Colonies were scored in 7 to 10 days.

Dot blot

Genomic DNA was purified from the treated cells using DNeasy Blood & Tissue Kit (QIAGEN,

Valencia, CA). A total of 0.5 to 1 μg of DNA was denatured in 1buffer (0.4 M NaOH, 20 mM

EDTA) at 100°C for 10 minutes and mixed with an equal volume of cold 2 M ammonium acetate

(pH 7.0). Pre-wet Nylon membrane (Hybond-N, Amersham, Buckinghamshire, UK) with

6Nucleic acid transfer buffer and then assembled the membrane onto a Bio-Dot apparatus (Bio-

Rad) connected to the vacuum. After the membrane was rehydrated with 500 μl H2O, the

denatured DNA in a 200 μl solution was loaded onto the membrane. When the sample has been

filtered through, the membrane was washed by 2Nucleic acid transfer buffer, baked at 80°C for

2 hours and blocked with 5% nonfat milk for 1 hour. The DNA spotted membrane was incubated

with mouse anti-5m

C antibody listed in Supplemental Table S6 and the signal was detected by

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horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. Equal

DNA loading was verified by staining the membranes with 0.2% methylene blue (Sigma-Aldrich)

(Supplementary Figure S5e). Densitometric analysis was performed using ImageJ (NIH Image,

Bethesda, MD) for quantification.

RNA isolation, cDNA preparation, and qPCR

RNA was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer's

instructions. RT for obtaining cDNA was performed SuperScript® III First-Strand Synthesis

System (Invitrogen) according to the manufacturer’s instructions. The expression of A/E, HIF1a,

DNMT1, DNMT3a or DNMT3b was detected by qPCR using TaqMan® Gene Expression Assay

(Applied Biosystems, Foster City, CA). Expression of the target genes was determined by the

comparative Ct method using ABL1 levels for normalization.7 The expression of p15

INK4b gene

was measured by qPCR with Power SYBR® Green PCR Master Mix (Applied Biosystems) using

GAPDH levels for normalization. The sequences for the primers and probes are listed in

Supplemental Table S5.

Bisulfite sequencing analysis

About 1 g of genomic DNA was modified with sodium bisulfite using an EpiTect Bisulfite Kit

(QIAGEN). The 5' upstream flanking sequence from p15INK4b

gene was amplified by PCR using

the bisulfite-treated DNA as template. Primer sequences are shown in Supplemental Table S5.

PCR products were subcloned and then sequenced.

Chromatin immunoprecipitation (ChIP)

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ChIP assays were performed as described previously using EZ-ChIP Assay Kit (Millipore,

Billerica, MA).8 Briefly, about 210

6 cells/assay were cross-linked with 1% formaldehyde

(Sigma-Aldrich), washed and resuspended in 1% SDS lysis buffer for sonication, in order to

yield DNA fragments with an average size of 300 to 500 bp. After being precleared with protein

G agarose, the lysates were immunoprecipitated by 5 µg of anti-HIF1 or anti-ETO antibody.

Aliquots (1%) were reserved for the negative control (input DNA). ChIP DNA was quantified by

qPCR with Power SYBR® Green PCR Master Mix. Fold change in binding was compared using

the corresponding input DNA. The primers specific for HIF1, A/E or DNMT3a gene promoter

and the antibodies used are respectively listed in Supplemental Table S5 and Supplemental Table

S6.

Western blot

The whole cellular lysates were prepared by harvesting the cells in 1cell lysis buffer [20 mM

HEPES (pH 7.6), 150 mM NaCl and 0.1% NP40] supplemented with 1Phosphatase Inhibitor

Cocktail 2 (Sigma-Aldrich), 1Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich), 1 mM PMSF

(Sigma-Aldrich) and 1Protease Inhibitor Cocktail Set III (Calbiochem-Novabiochem). The

Western blot was performed with whole cell lysates as previously described.8 The antibodies

used are listed in Supplemental Table S6. Equivalent gel loading was confirmed by probing with

-actin antibody. Densitometric analysis was performed using ImageJ for quantification.

H&E and IHC staining

Tumor tissues were fixed in 10% neutral-buffered formalin, deparaffinized, hydrated and stained

with H&E (Thermo-Scientific) or subjected to IHC Staining with the primary antibodies listed in

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Supplemental Table S6. A horseradish peroxidase-conjugated goat anti-rabbit antibody was used

as the secondary antibody. After developing with 3, 3’-diaminobenzidine, the sections were

counterstained with hematoxylin. Images were acquired using Nikon Eclipse Ti microscope and

the immunohistochemical signal was quantified by using Image-Pro Plus software (v.4) program

(Media Cybernetics).

FACS analysis

Cells were fixed in ethanol and stained with propidium iodide and Annexin V-FITC (BD

Biosciences PharMingen, San Diego) before analyzed by FACS. FlowJo software (Version 7.6.1,

Treestar, Ashland, OR) was used for subsequent analysis.

Tumorigenesis assays

Athymic nude mice (female, 6-8 weeks old) and C57BL/6J mice (female, 6-8 weeks old) were

purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained under conditions

based on the guidelines established by Research Animal Resources, University of Minnesota. For

xenograft mouse model, about 3106

of SKNO-1PGK cells were injected subcutaneously into the

bilateral flanks of the nude mice. Tumor diameters were measured with digital caliper and tumor

volume was calculated using the formula: tumor volume (mm3)=(lengthwidthheight /6).

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Treatment was initiated on days 10 after the first injection when mean tumor volume was ~50

mm3. The mice were divided into 2 groups of 3 animals each with an average body weight of 30

g. Echinomycin (10 µg/kg) in PEG400 and saline (ratio 15:38:47) or vehicle was administered

by i.p. injection, three doses in week 1 and two doses in weeks 2 for five dosages in total. Mice

were sacrificed on days 21 after the first leukemia cell inoculation and the tumors were harvested.

The samples were immediately fixed in 10% neutral-buffered formalin and processed for H&E

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and IHC staining. For experimental A/E and HIF1 co-transfection C1498 leukemia mouse

model, a total of 1×107

of C1498 cells, a murine myelogenous leukemia, were mixed with 12.5

g of A/E or (and) HIF1 expression plasmid and electroparated in a 0.4 cm cuvette (Bio-Rad)

using a Bio-Rad Gene Pulser II™ set to 250V, 950 µF. The total amount of the transfected DNA

was kept constant by adding empty vector. After 48 hours, about 0.1×106 of viable cells were

injected into the lateral tail vein of each C57BL/6J mouse. Mice were sacrificed on days 20 after

injection. For experimental HIF1 overexpression or knock-down in A/E9a transgenic leukemia

mouse model,10

1×106 of A/E9a primary cells were transfected with 2.5 μg of HIF1 expression

vector or 100 nM of HIF1 siRNA using Lipofectamine™ RNAiMAX Reagent (Life

Technologies). Then about 0.1×106 (HIF1α expression or empty vector) or 0.5×10

6 (HIF1α

siRNA or scramble) of viable cells were injected into the lateral tail vein of each C57BL/6J

mouse. Mice were sacrificed 7 weeks after injection. Cytospin preparations of BM cells were

processed for Giemsa staining. Lungs, spleens and livers were harvested and immediately fixed

in 10% neutral-buffered formalin and stained with H&E (Thermo-Scientific).

Statistical analysis

SPSS 20.0 software was used to process the data. Wilcoxon signed-rank test was selected to

determine the difference of AE, HIF1, DNMT1, DNMT3a or DNMT3b expression levels among

clinical samples. Spearman’s correlation coefficient (r) was used to access the correlation of

mRNA levels between genes. To compare clinical outcome of patients with different HIF1

expression levels, the cohort was stratified using the quartile grouping method described

previously.11

Patients were grouped into quartiles according to HIF1 expression levels (Q1-Q4,

each quartile containing 25% of patients) and divided into high HIF1 (Q4; n=33) and low

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HIF1 (Q1-Q3; n=99) based on the trend observed in clinical outcome after performing a Cox

regression analysis for EFS with HIF1 quartile grouping as the independent variable. HIF1

expression ranged between 0.1965 and 10.7182 with the following median expression for each

quartile: 0.3942 (Q1), 0.8725 (Q2), 1.3020 (Q3), and 3.0218 (Q4). In this model, AE-patients in

the highest HIF1 quartile showed a significantly different EFS compared with the remaining

patients with lower HIF1 expression levels. The differences in regression coefficients with SE

for each quartile were as follows: Q1 versus Q4, -1.571 (SE 0.744), P=0.035; Q2 versus Q4, -

0.867 (SE 0.404), P=0.032; Q3 versus Q4, -0.912 (SE 0.400), P=0.023. Survival curves were

generated using the Kaplan-Meier method and the log-rank test was used to compare survival

between groups. Clinical features across groups were compared using the 2-sided Fisher exact

test for categorical data and the nonparametric Mann-Whitney U test for continuous variables.

The Cox proportional hazards model with stepwise forward selection were constructed to

determine whether HIF1 expression was associated with outcome when adjusting for other

prognostic variables. The full multivariate model used the variables significant at a 10% level in

univariate analysis, including HIF1 expression (low vs. high), KIT mutation status (mutation vs.

wild-type), WBC count (10109/L increase), BM blasts (10% increase), Age (10-year increase),

Cytarabine-based chemotherapy (high- vs. standard-dose), HSCT (allo- vs. no, auto- vs. no) and

complete remission achievement (1 vs. 2 courses). The possible influence of sample bias on the

results and the stability of the model were examined by bootstrap resampling method.12

A total

of 1000 bootstrap samples were generated for each analysis. Cox regression was run separately

on these 1000 samples to obtain robust estimates of the standard errors of coefficients, and hence

the P values and 95% confidence intervals of the model coefficients. Student’s t test was applied

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to compare gene overexpression- or knockdown-induced changes to respective controls. A P-

value of less than 0.05 was chosen as a threshold for statistical significance.

Complete remission was defined by recovery of morphologically normal BM, normal blood

counts and no circulating leukemic blasts or evidence of extramedullary leukemia. Relapse was

defined by >5% BM blasts, circulating leukemic blasts or development of extramedullary

leukemia. OS was measured from the beginning of therapy until date of death or last follow-up.

EFS was defined as the time from study entry to first event. An event was defined as failure to

achieve a complete remission, relapse after achieving a complete remission, or death.

SUPPLEMENTARY REFERENCES

1. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposals

for the classification of the acute leukaemias. French-American-British (FAB) co-operative

group. Br J Haematol 1976; 33: 451-458.

2. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J et al. World

Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid

tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November

1997. J Clin Oncol 1999; 17: 3835-3849.

3. Garzon R, Liu S, Fabbri M, Liu Z, Heaphy CE, Callegari E et al. MicroRNA-29b induces

global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid

leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood 2009; 113:

6411-6418.

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4. Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, Travaglini L et al. Epigenetic

silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Cancer

cell 2007; 12: 457-466.

5. Meyers S, Lenny N, Hiebert SW. The t(8; 21) fusion protein interferes with AML-1B-

dependent transcriptional activation. Mol Cell Biol 1995; 15: 1974-1982.

6. Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG, Jr. Inhibition of HIF is necessary

for tumor suppression by the von Hippel-Lindau protein. Cancer cell 2002; 1: 237-246.

7. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408.

8. Liu S, Wu LC, Pang J, Santhanam R, Schwind S, Wu YZ et al. Sp1/NFkappaB/HDAC/miR-

29b regulatory network in KIT-driven myeloid leukemia. Cancer cell 2010; 17: 333-347.

9. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude)

mice. Cancer Chemother Pharmacol 1989; 24: 148-154.

10. Yan M, Kanbe E, Peterson LF, Boyapati A, Miao Y, Wang Y et al. A previously unidentified

alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis. Nat Med 2006; 12:

945-949.

11. Kühnl A, Gökbuget N, Kaiser M, Schlee C, Stroux A, Burmeister T et al. Overexpression of

LEF1 predicts unfavorable outcome in adult patients with B-precursor acute lymphoblastic

leukemia. Blood 2011; 118: 6362-6367.

12. Sauerbrei W, Schumacher M. A bootstrap resampling procedure for model building:

application to the Cox regression model. Stat Med 1992; 11: 2093-2109.

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SUPPLEMENTARY TABLES

Supplementary Table S1.

Clinical characteristics of AML patients in GEO database GSE6891.

Characteristic Total t(8;21) non-t(8;21) P value

Patients number 461 35 426

Median age, y (range) 43 (15-61) 37 (16-54) 45 (15-61) 0.000

Sex (male/female) 231/230 21/14 210/216 0.224

FAB subtypes, no. (%) 0.003

M0 16 (3.5) 0 (0) 16 (3.8)

M1 95 (20.6) 2 (5.7) 93 (21.8)

M2 106 (23.0) 30 (85.7) 76 (17.8)

M3 24 (5.2) 0 (0) 24 (5.6)

M4 84 (18.2) 3 (8.6) 81 (19.0)

M5 104 (22.6) 0 (0) 104 (24.4)

M6 6 (1.3) 0 (0) 6 (1.4)

MDS (RAEB/RAEB-t) 4 (0.9)/13 (2.8) 0 (0)/0 (0) 4 (0.9)/13 (3.1)

unknown 9 (2.0) 0 (0) 9 (2.1)

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Supplementary Table S2

Comparison of clinical characteristics of AML patients according to HIF1 expression.

Characteristic

Overall cohort

(n=132) P value

A/E-positive

(n=73) P value

A/E-positive & wtKIT

(n=53) P value

HIF1high HIF1low

HIF1high HIF1low

HIF1high HIF1low

Patients no. 33 99 26 47 15 38

Age a 36 (13-60) 36 (11-84) 0.885

33.5 (13-

60) 25 (11-82) 0.500 34 (13-59)

30 (11-

62) 0.560

Sex, no. (%) 0.536

Male 22 (66.7) 60 (60.6) 15 (57.7) 28 (59.6) 9 (60) 25 (65.8)

Female 11 (33.3) 39 (39.4) 11 (42.3) 19 (40.4) 0.876 6 (40) 13 (34.2) 0.692

WBC ( 109/L)

a

13.1 (1.5-

290.0)

12.8 (0.3-

362.0) 0.898

12.9 (1.5-

53.6)

14.0 (2.1-

76.3) 0.489

12.2 (2.9-

37.4)

13.9 (2.1-

76.3) 0.447

BM blasts (%) a

65.0 (21.2-

92.0)

56.0 (7.4-

97.0) 0.194

67.8 (34.0-

92.0)

54.7 (7.4-

95.0) 0.065

70.0 (36.8-

92.0)

53.1 (7.4-

95.0) 0.058

FAB subtypes, no. (%)

0.127

M2 28 (24.3) 87 (75.7) 26 (35.6) 47 (64.4) 15 (28.3) 38 (71.7)

M3 0 (0) 2 (100)

M4 5 (55.6) 4 (44.4)

M5 0 (0) 4 (100)

M6 0 (0) 2 (100)

A/E status, no.

(%) 0.002

A/E positive 26 (78.8) 47 (47.5)

A/E negative 7 (21.2) 52 (52.5)

KIT mutation status

b, no. (%)

0.001 0.034

mutKIT 11 (42.3) 9 (12) 11 (42.3) 9 (19.1)

wtKIT 15 (57.7) 66 (88) 15 (57.7) 38 (80.9)

Chemotherapy b,

no. (%) 0.905

High-dose

cytarabine-

based

8 (32.0) 20 (33.3) 8 (42.1) 11 (50.0) 5 (38.5) 9 (50.0)

Standard-dose

cytarabine-

based

17 (68.0) 40 (66.7) 11 (57.9) 11 (50.0) 0.726 8 (61.5) 9 (50.0) 0.524

HSCT, no. (%) 0.497

Allo-HSCT 9 (27.3) 26 (26.3) 5 (19.2) 9 (19.1) 2 (13.3) 7 (18.4)

Auto-HSCT 4 (12.1) 6 (6.1) 4 (15.4) 5 (10.6) 3 (20.0) 3 (7.9)

No HSCT 20 (60.6) 67 (67.7) 17 (65.4) 33 (70.2) 0.834 10 (66.7) 28 (73.7) 0.443

CR b, no. (%) 0.211

1 course 16 (64.0) 30 (49.2) 12 (66.7) 13 (56.5) 10 (76.9) 10 (55.6)

2 courses 9 (36.0) 31 (50.8) 6 (33.3) 10 (43.5) 0.509 3 (23.1) 8 (44.4) 0.220 a Values represent median (range).

b Information is not available in some cases. WBC, white

blood cell; HSCT, hematopoietic stem cell transplantation; CR, complete remission.

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Multivariate analysis for OS and EFS.

Patients Variable a

P value Hazard Ratio (95.0% CI)

overall cohort

OS I/HD vs. SD cytarabine-based chemotherapy 0.001 0.198 (0.076 - 0.514)

Allo- vs. No HSCT 0.001 0.270 (0.121 - 0.603)

EFS

HIF1 high vs. HIF1 low 0.015 2.181 (1.164 - 4.087)

I/HD vs. SD cytarabine-based chemotherapy 0.001 0.284 (0.130 - 0.618)

Allo- vs. No HSCT 0.008 0.410 (0.212 - 0.795)

A/E-positive patients

OS



EFS



A/E-positive & wtKIT patients

OS


EFS

HIF1 high vs. HIF1 low 0.002 3.708 (1.636 - 8.406) a Variables considered for model inclusion were: HIF1 expression (low vs. high), KIT mutation

status (mutation vs. wild-type), WBC count (10109/L increase), BM blasts (10% increase), age

(10-year increase), cytarabine-based chemotherapy (intermediate/high dose- vs. standard-dose),

HSCT (allo- vs. no, auto- vs. no) and CR achievement (1 vs. 2 courses). Only variables

significantly associated with outcomes in univariate analysis were included in the multivariate

model. SD, standard dose; I/HD, intermediate/high dose.

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Bootstrap resampling for variable in the multivariate model.

Patients Variable a

Regression

Coefficient Std. Error

Bootstrapa

P value 95.0% CI

overall cohort OS

I/HD vs. SD cytarabine-based

chemotherapy -1.621 0.867 0.001 -3.176 - -0.874

Allo- vs. No HSCT -1.310 0.457 0.003 -2.322 - -0.552

EFS

HIF1 high vs. HIF1 low 0.780 0.345 0.013 0.095 - 1.446


chemotherapy -1.260 0.401 0.001 -2.148 - -0.591

Allo- vs. No HSCT -0.891 0.344 0.005 -1.603 - -0.295

A/E-positive patients

OS



chemotherapy -1.623 1.250 0.001 -3.427 - -0.586

EFS



chemotherapy -1.475 0.732 0.002 -2.706 - -0.681

A/E-positive &

wtKIT patients

OS


EFS

HIF1 high vs. HIF1 low 1.311 0.372 0.002 0.604 - 2.106 a

bootstrap results are based on 1000 bootstrap samples. SD, standard dose; I/HD,

intermediate/high dose.

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Sequence of primers used in experiments.

Name (Genebank Accession No.) Primer Sequence (5' to 3')

AML1 promoter (NM_001754)

Forward 1 GGGGTACCCATCACACAGGTTCTCTTGACCCTATGTGTCG

Forward 2 GGGGTACCAGAGTGCTATGGCGGATGTAGGCAGAG

Forward 3 GGGGTACCACCAAGGACTTAACTCTCCCGGAGCTG

Reverse CCCAAGCTTTTGGCTGTGGGTTGGTGATGCTCACCA

AML1 promoter-1-Mut

Forward GGTTCTCTTGACCCTATGTGTCGAAATTTCATAGACGCAGAGTGCT

ATGGC

Reverse GCCATAGCACTCTGCGTCTATGAAATTTCGACACATAGGGTCAAGA

GAACC

HIF1 promoter (NM_001530)

Forward 1 GGGGTACCCATTTACTGAGTGCTTACTATGCACCAG

Forward 2 GGGGTACCACTTAGTAGACAAGGTGAGTTCCCCTG

Forward 3 GGGGTACCGAGCATTACATTACTGCACCAAGAGTA

Forward 4 GGGGTACCTGACGCTGCCTCAGCTCCTCAGT

Reverse CCCAAGCTTCGACACACTGGCCGAAGCGACGAAGA

HIF1 promoter-3-Mut Forward GCGGCGTGGGCGGGGACTTGCC

Reverse GGCAAGTCCCCGCCCACGCCGC

DNMT3a promoter

(NM_175629)

Forward 1 GGGGTACCCGGGGTTTCACCATGTTAGCCAGGCTGGTC

Forward 2 GGGGTACCGTGATGTGGGGTGATTTCAGAGCCGGATG

Forward 3 GGGGTACCGGCCGTGTTATATGTAAGCCCTTGATGAAGGC

Forward 4 GGGGTACCGGGGTGAGGGCGGTGTGTAGGCACACAC

Reverse CCCAAGCTTAGAGCCCACTGCGCTCTGCCTGCCTCAG

DNMT3a promoter-1-Mut-a Forward GCCTCCCAAAGTGCTACAGGCGTGAGCC

Reverse GGCTCACGCCTGTAGCACTTTGGGAGGC

DNMT3a promoter-1-Mut-b Forward GAGCAACTATGTTTCAAAAGGTGGGCTGGGCAC

Reverse GTGCCCAGCCCACCTTTTGAAACATAGTTGCTC

DNMT3a promoter-2-Mut Forward CCTCAGTTTCTTCATCTGTAAGGAGAGTACAAGCCA

Reverse TGGCTTGTACTCTCCTTACAGATGAAGAAACTGAGG

A/E expression (NM_001754)

Forward CAAGTCGCCACCTACCACAGA

Reverse AGCCTAGATTGCGTCTTCACATC

Probe FAM-CCATCAAAATCACAGTGGAT-NFQ-MGB

HIF1 expression

(NM_001530)

Forward GTACCCTAACTAGCCGAGGAAGAA

Reverse CTGAGGTTGGTTACTGTTGGTATCA

Probe FAM-TTGCACTGCACAGGCCACATTCAC-TAMRA

DNMT1 expression

(NM_001130823.1)

Forward CAAGTCCGATGGAGAGGCTAA

Reverse GTTTGCCTGGTGCTTTTCCTT

Probe FAM-CGTTCAAGAGACCCTCCTGCCTCAGC-TAMRA

DNMT3a expression

(NM_175629)

Forward CATGCCGAGGCTCACCTT

Reverse GTTTTCTTCCACAGCATTCATTCC

Probe FAM-ACCCCTACTACATCAGCAAGCGCAAGC-TAMRA

DNMT3b expression

(NM_175849.1)

Forward TCCGAGGTCTCTGCAGACAA

Reverse AGCTTTCTCCAGAGCATGGTACA

Probe FAM-CTGTTCAGCCAGCACTTTAATTTGGCCA-TAMRA

ABL1 expression (NM_007313)

Forward CTCCATTATCCAGCCCCAAA

Reverse CCCAGCTTGTGCTTCATGGT

Probe FAM-CGCAACAAGCCCACTG-NFQ-MGB

P15INK4b

expression

(NG_023297.1)

Forward CCAGATGAGGACAATGAG

Reverse AGCAAGACAACCATAATCA

18

GAPDH expression

(NM_002046.4)

Forward CTCTGCTCCTCCTGTTCGAC

Reverse GCCCAATACGACCAAATCC

P15INK4b

bisulfite sequencing

(NG_023297.1)

Forward GGTTGGTTTTTTATTTTGTTAGAG

Reverse ACCTAAACTCAACTTCATTACCCTC

HIF1 ChIP primer

(NM_001530)

Forward GCTAAACACAGACGAGCACGTG

Reverse TCACCTGAGGTGGAGGCGGGTT

AML1 ChIP primer

(NM_001754)

Forward CATTTACAACCCATCATCACA

Reverse CTCTGCTCTGCCTACATC

DNMT3a ChIP primer

(NM_175629) (P2)

Forward AACCTGGCTTTGAATCCT

Reverse TACTCTCCTCACGCTACA

DNMT3a ChIP negative control

primer (P1)

Forward ATTCCACTTCTGTCTCTATGA

Reverse AACCTTGAGGATACTATGCTAA

19


Antibodies used in experiments.

Antibody Application Company Catalog No. Source Dilution

DNMT3a Western blot

IHC

Santa Cruz

Biotechnology sc20703 Rabbit

Western blot:1:500

IHC: 1:250

DNMT1 Western blot Biolab M0231L Rabbit 1:500

DNMT3b Western blot Abcam ab13604 Mouse 1:500

AML1 Western blot

IHC

Cell Signaling

Technology 4334 Rabbit

Western blot: 1:500

IHC: 1:100

HIF1 Western blot

IHC

Cell Signaling


Western blot: 1:500

IHC: 1:100

Cleaved

Caspase-3

Western blot

IHC

Cell Signaling


Western blot: 1:500

IHC: 1:250

Cleaved

Caspase-8 Western blot

Cell Signaling

Technology 9496 Rabbit 1:500

Caspase-9 Western blot Cell Signaling

Technology 9502 Rabbit 1:500

-actin Western blot Santa Cruz

Biotechnology sc-1616 Goat 1:1000

Ki-67 IHC Abcam ab15580 Rabbit 1:250

5-m

C IHC

Dot blot Active Motif 39649 Mouse

IHC: 1:250

Dot blot: 1:1000

ETO ChIP Santa Cruz

Biotechnology sc-9737 Goat 5 µg

HIF-1 ChIP Novus Biologicals NB100-134 Rabbit 5 µg

20

SUPPLEMENTARY FIGURES

Supplementary Figure S1

Supplementary Figure S1. Selectively high expression of HIF1 in AML cell lines and patients

bearing t(8;21). (a) qPCR showing HIF1 mRNA expression in myeloid leukemia cell lines.

CML-BC, chronic myeloid leukemia in blast crisis. Bars indicate the mean±SEM from three

independent experiments. (b) Normalized HIF1 expression in pretreatment samples of 461

patients with de novo AML (GEO database, GSE6891). Patient characteristics are described in

Supplementary Table S1. The gene expression was determined using gene-expression arrays

(Affymetrix HGU133 Plus 2.0 GeneChips). Median values are depicted by the horizontal lines.

21


Supplementary Figure S2. Overexpression of A/E induces HIF1 upregulation. (a, b) qPCR (a)

and Western blot (b) showing A/E and HIF1 levels in A/E-inducible U937 cells and control.

Bars indicate the mean±SEM from three independent experiments.

22


Supplementary Figure S3. HIF1 functionally cooperates with A/E in promoting leukemia

growth. (a, b) Colony-forming assays showing growth inhibition in leukemia cells with

knockdown of A/E (a) or HIF1 (b). Representative colony morphologies were shown. Scale

bars represent 1 cm (red) or 200 µm (black). (c, d) C1498 leukemia cells were transfected with

A/E or/and HIF1 expression vector. Transfection efficiency was monitored by Western blot of

A/E and HIF1 protein levels in C1498 cells after transfection (c), and the growth stimulation in

23

C1498 cells upon A/E or/and HIF1 overexpression was assessed by colony-forming assays (d).

Scale bars represent 1 cm. Error bars indicate mean±SEM of duplicate samples from two

independent experiments. (e-g) A/E9a primary leukemia cells were transfected with HIF1

expression vector or siRNA and engrafted into C57BL/6J mouse. Transfection efficiency was

monitored by qPCR analysis of HIF1 mRNA levels in A/E9a cells before injection (e) and in

BM cells of A/E9a mice in 7 weeks after injection (f). The development of leukemic disease was

monitored by WBC count (g).

24


Supplementary Figure S4. Selectively high expression of DNMT3a in A/E-positive AML. (a-c)

Normalized expression levels of DNMT3a (two probes, DNMT3a_1 and DNMT3a_2) (a),

DNMT1 (b) and DNMT3b (c) in pretreatment samples of 461 patients with de novo AML (GEO

database, GSE6891). Median values are depicted by the horizontal lines. (d, e) qPCR showing

the mRNA levels of DNMT1 (d) and DNMT3b (e) in BM samples of AML patients and healthy

donors (HD). Median values are depicted by the horizontal lines. NS: not statistically significant.

25


Supplementary Figure S5. A/E and HIF1 induces DNMT3a expression. (a, b) qPCR (a) and

Western blot (b) showing DNMT3a levels in 293T and U937 cell lines upon A/E overexpression.

(c, d) qPCR (c) and Western blot (d) showing DNMT3a levels in Kasumi-1 cell line upon A/E

knockdown. (e, f) qPCR (e) and Western blot (f) showing DNMT3a levels in 293T cells upon

HIF1 overexpression. (g, h) qPCR (g) and Western blot (h) showing DNMT3a levels in

Kasumi-1 cells upon HIF1 knockdown. Bars in a, c, e and g indicate the mean±SEM from three

independent experiments.

26


Supplementary Figure S6. A/E and HIF1 cooperatively modulate DNA methylation by

targeting DNMT3a transcription. (a) qPCR showing DNMT3a mRNA levels in A/E-negative

293T cells transfected with empty, A/E, HIF1 or both vectors. (b) qPCR showing DNMT3a

mRNA levels in Kasumi-1 cells transfected with scramble, A/E siRNA, HIF1 siRNA or both.

(c) Western blot showing DNMT3a protein levels in 293T cells upon DNMT3a overexpression

27

or knockdown. (d) Dot blot showing 5-m

C levels in 293T cells upon DNMT3a overexpression or

knockdown. Bars in a, b and d indicate the mean±SEM from three independent experiments. (e)

Top: The genomic DNA was extracted from U937MT or U937A/E cells after ZnSO4 treatment

and subjected to Dot blot using 5-m

C antibody; bottom: equal DNA loading was verified by

staining the membranes with 0.2% methylene blue. (f, g) Western blot showing the changes of

DNMT1 and DNMT3b protein levels in U937 or 293T cells upon overexpression of A/E (f) or

HIF1 (g). (h, i) Western blot showing the changes of DNMT1 and DNMT3b protein levels in

Kasumi-1 or SKNO-1 cells upon knockdown of A/E (h) or HIF1 (i).

28


Supplementary Figure S7. Pharmacological inhibition of HIF1 disrupts A/E-HIF1-DNMT3a

axis and suppresses leukemic proliferation in vitro. (a) qPCR analysis of A/E, HIF1 and

DNMT3a mRNA levels in Kasumi-1 and SKNO-1 cells treated with echinomycin or vehicle.

Bars indicate the mean±SEM from three independent experiments. (b) FACS analysis of

apoptosis in Kasumi-1, SKNO-1PGK or SKNO-1siA/E cells treated with echinomycin or vehicle.

(c, d) Colony-forming assays showing the shape and size of A/E-positive cell lines (c) or patient-

derived leukemia blasts (d) upon echinomycin treatment. Scale bars represent 1 cm (red) or 200

µm (black).

29


Supplementary Figure S8. Echinomycin induced no significant body weight loss in SKNO-1

xenograft tumor-bearing nude mice. Measurement of body weight of the mice at the indicated

time points post echinomycin treatment. Data represent mean±SEM (n=3 mice/group).

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