Down-regulation of microRNAs 222/221 in acute myelogenous leukemia with deranged core-binding factor subunits.
ABSTRACT Core-binding factor leukemia (CBFL) is a subgroup of acute myeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most common mutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3' untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133(+) stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.
- [Show abstract] [Hide abstract]
ABSTRACT: Improvements in technology and resources are helping to advance our understanding of cancer-initiating events as well as factors involved with tumor progression, adaptation, and evasion of therapy. Tumors are well known to contain diverse cell populations and intratumor heterogeneity affords neoplasms with a diverse set of biologic characteristics that can be used to evolve and adapt. Intratumor heterogeneity has emerged as a major hindrance to improving cancer patient care. Polygenic cancer drug resistance necessitates reconsidering drug designs to include polypharmacology in pursuit of novel combinatorial agents having multitarget activity to overcome the diverse and compensatory signaling pathways in which cancer cells use to survive and evade therapy. Advances will require integration of different biomarkers such as genomics and imaging to provide for more adequate elucidation of the spatially varying location, type, and extent of diverse intratumor signaling molecules to provide for a rationale-based personalized cancer medicine strategy.Neoplasia (New York, N.Y.) 12/2012; 14(12):1278-89. · 5.48 Impact Factor
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ABSTRACT: The elegance of fundamental and applied research activities have begun to reveal a myriad of spatial and temporal alterations in downstream signaling networks affected by cell surface receptor stimulation including G protein-coupled receptors and receptor tyrosine kinases. Interconnected biochemical pathways serve to integrate and distribute the signaling information throughout the cell by orchestration of complex biochemical circuits consisting of protein interactions and covalent modification processes. It is clear that scientific literature summarizing results from both fundamental and applied scientific research activities has served to provide a broad foundational biologic database that has been instrumental in advancing our continued understanding of underlying cancer biology. This article reflects on historical advances and the role of innovation in the competitive world of grant-sponsored research.Neoplasia (New York, N.Y.) 12/2011; 13(12):1183-93. · 5.48 Impact Factor
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ABSTRACT: Valproic acid (VPA) exhibits important pharmacological properties but has been reported to trigger side effects, notably on the hematological system. We previously reported that VPA affects hematopoietic homeostasis by inhibiting erythroid differentiation and promoting myeloid and megakaryocyte differentiation. Here, we analyzed the effect of VPA on regulatory factors involved in erythro-megakaryocytic differentiation pathways, including transcription factors and microRNAs (miRs). We demonstrate that VPA inhibited erythroid differentiation in erythropoietin (Epo)-stimulated TF1 leukemia cells and CD34(+)/hematopoietic stem cells (HSCs) and in aclacinomycin-(Acla)-treated K562 cells. miR-144/451 gene expression was decreased in all erythroid and megakaryocyte models in correlation with GATA-1 inhibition. In Epo-stimulated CD34(+)/HSCs, VPA induced the expression of the ETS family transcription factors PU.1, ETS-1, GABP-α, Fli-1 and GATA-2, which are all known to be negative regulators of erythropoiesis, while it promoted the megakaryocytic pathway. PU.1 and ETS-1 expression were induced in correlation with miR-155 inhibition; however, the GATA-1/PU.1 interaction was promoted. Using megakaryoblastic Meg-01 cells, we demonstrated that VPA induced megakaryocyte morphological features and CD61 expression. GATA-2 and miR-27a expression were increased in correlation with a decrease in RUNX1 mRNA expression, suggesting megakaryocyte differentiation. Finally, by using valpromide and the Class I HDACi MS-275, we validated that the well-described HDACi activity of VPA is not required in the inhibitory effect on erythropoiesis. Overall, this report shows that VPA modulates the erythro-megakaryocytic differentiation program through regulatory micro-networks involving GATA, ETS family transcription factors and miRNAs, notably the GATA-1/miR-144/451 axis.Biochemical pharmacology. 09/2014;
Down-regulation of MicroRNAs
222/221 in Acute Myelogenous
Leukemia with Deranged
Core-Binding Factor Subunits1,2
Matteo Brioschi*,3, John Fischer†,3,
Roberto Cairoli‡, Stefano Rossetti†, Laura Pezzetti§,
Michele Nichelatti‡, Mauro Turrini‡,
Francesca Corlazzoli*, Barbara Scarpati‡,
Enrica Morra‡, Nicoletta Sacchi†and
*Dipartimento di Biologia e Genetica per le Scienze
Mediche, Facoltà di Medicina, Università degli Studi di
Milano, Milan, Italy;†Cancer Genetics Program, Roswell
Park Cancer Institute, Buffalo, NY, USA;‡Department of
Oncology, Niguarda Hospital, Milan, Italy;§Department
of Hematology, Niguarda Hospital, Milan, Italy
involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more,
gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for
221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that
MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133+stem progenitor
significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21)
AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/
221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/
This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by
down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and over-
expression of wild type or mutant KIT in AML.
Neoplasia (2010) 12, 866–876
Abbreviations: AML, acute myeloid leukemia; BFU-E, erythroid burst-forming units; BM-MNC, bone marrow mononuclear cells; CBFL, core-binding factor leukemia; CFU-GM,
granulocyte/monocyte colony-forming unit; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor;
HSPCs, hematopoietic stem/progenitor cells; non-CBFL, non–core-binding factor leukemia
Address all correspondence to: Alessandro Beghini, PhD, Dipartimento di Biologia e Genetica per le Scienze Mediche, Facoltà di Medicina, Università degli Studi di Milano, via Viotti
3/5, 20133 Milano, Italy. E-mail: firstname.lastname@example.org
1This study was supported by FIRST 2007 and PUR 2008 (to A.B.), Progetto Integrato Oncologia 2006 (RO 4/2007) and Associazione Malattie del Sangue Onlus (A.M.S.),
Piano Regionale Sangue-Regione Lombardia 2006 (DDG 7917, to E.M.), and Roswell Park Cancer Institute’s startup funds (to N.S.).
2This article refers to supplementary materials, which are designated by Tables W1 and W2 and Figures W1 and W2 and are available online at www.neoplasia.com.
3These authors contributed equally to this work.
Received 26 March 2010; Revised 12 July 2010; Accepted 15 July 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00
Volume 12 Number 11 November 2010pp. 866–876
The multistep model of acute myeloid leukemia (AML) pathogenesis
postulates the cooperation between class I mutations, which confer a
proliferative and antiapoptotic advantage to leukemic cells, and class
II mutations, which impair cell differentiation . Core-binding factor
leukemia (CBFL) defines a subgroup of AML characterized by class II
cytogenetic mutations involving the master hematopoietic transcrip-
tion factor CBF . CBF consists of two subunits, CBFα and CBFβ,
both critical for proper transcriptional activation of CBF target genes.
Whereas the CBFα (AML1/RUNX1) is the actual DNA-binding sub-
unit, CBFβ is necessary to strengthen AML1 DNA binding . The
two most common leukemia-associated CBF rearrangements are the
t(8;21)(q22;q22) and inv(16)(p13;q22), which affect the CBFα and
CBFβ subunit, respectively. Knock-in mice models harboring either
consequent to the t(8;21)(q22;q22), or the CBFβ-MYH11 fusion pro-
other mutations are necessary, in addition to the mutant CBF fusion
tions, so far identified, that would cooperate with CBF fusion proteins
FLT3, N-Ras and K-Ras genes [6–10]. Specifically, we and others found
that the frequency of mutations involving the KIT gene, which encodes
the receptor for the steel factor or stem cell factor (SCF) receptor, is sig-
nificantly higher inboth adultandchildhoodCBFL than innon-CBFL
[11–13]. Furthermore, the expression level of both KITmRNA and
proteins is much higher in t(8;21) AML, with either wild type or mu-
tant KIT, than in leukemia cells negative for t(8;21) . Despite these
observations, it is not yet clear whether there is a mechanistic link be-
tween CBF fusion proteins and overexpression of wild type and mutant
MicroRNAs (MIRs) have been recently found to play an important
role in the circuits that regulate the lineage differentiation fate of hema-
topoietic cells by modulating the expression of known oncogenes or tu-
mor suppressors [15–20]. Human MIR-222/221, on chromosome X,
has been predicted to target the 3′ untranslated region (3′UTR)
of KIT mRNA . By performing in silico analysis of the promoter
region of the MIR-222/221 gene, we identified a few conserved AML1
consensus sequences. By chromatin immunoprecipitation (ChIP), we
found that AML1 indeed binds these AML1-binding sites.
The promoter of the myelopoiesis-regulator MIR-223, a MIR on
chromosome X, contains an AML1-consensus sequence, and its ex-
pression is epigenetically silenced by the t(8;21) CBFL-specific fusion
protein AML1-MTG8 . Thus, we hypothesized that MIR-222/
221 is another direct transcriptional target of AML1 and that down-
regulation of MIR-222/221 expression by AML1 fusion proteins is a
potential mechanism leading to KIToverexpression. Reporter gene ex-
periments showing that the expression of exogenous AML1-MTG8
can repress MIR-222/221-luciferase expression supported this hypoth-
esis. To further tackle our hypothesis, we analyzed the expression of
MIR-222/221, along with the expression of the myeloid-specific
MIR-223, in different contexts: 1) normal bone marrow mononuclear
epitope (AC133), a hallmark of primitive progenitors and stem cell
populations ; 2) AML samples characterized for the presence or ab-
sence of the most common CBF chromosome rearrangements, namely,
32D mouse model of a rare CBFL characterized by the t(16;21) rear-
lower in AC133-positive (AC133+) cells relative to AC133-negative
(AC133−) cells but are sharply upregulated in the course of AC133+
granulocyte/monocyte differentiation. Significantly, we detected lower
levels of MIR-222/221 and MIR-223 expression in CBFL, in correla-
levels of mouse MIR-222/221 and mouse MIR-223 as well as a higher
levelofmouse KIT(CD117) expression were also detectedinthe32D/
WT1 cell model of AML1-MTG16, the CBF fusion protein resulting
from the t(16;21) .
The overall findings suggest that CBFL-related fusion proteins are
capable of inducing the concerted down-regulation of both MIR-223
and MIR-222/221, thus leading to the concerted block of myeloid
differentiation and KIT overexpression.
Materials and Methods
In Silico Analysis of the MIR-222/221 Gene Cluster
Human MIRs sequences were obtained from the miRBase Sequence
Griffiths-Jones). The ENSEMBL Database (http://www. ensembl.org/
index.html) provided full-length DNA sequences of the MIRs genes
on chromosome X and the sequence of the 3′UTR of the KIT gene.
To identify the transcription start site, potential control elements, and
consensus sites of MIR-222/221 cluster gene sequence, the upstream
pri-MIRs sequence was analyzed by MAPPER (http://tftargetmapper.
erasmusmc.nl/), which is a platform for the computational identifica-
tion of transcription factor–binding sites in multiple genomes. It uses
an innovative technique that combines TRANSFAC and JASPAR data
with the search power of profile hidden Markov models. A “good”
match usually has a score greater than 0.8 and an E value less than
20. The greater the score, the better the match between the hit and
the model is. A more stringent set of parameters was used for the query
by setting the score greater than 1. The E value, computed with respect
to the number of the sequences in the database queried, is a measure of
the expected number of false-positives that will have scores equal to or
larger than the score of the hit. The smaller the E value, the more sig-
nificant the hit is .
Cell Lines and Culture Conditions
The human leukemic monocyte lymphoma cell line U937 was cul-
tured in RPMI 1640 supplemented with 10% heat-inactivated fetal
bovine serum (HyClone, Thermo Fisher Scientific, Waltham, MA).
by Dr. Shujun Liu, Ohio State University) was cultured in RPMI 1640
and 10 ng/ml human granulocyte/macrophage colony-stimulating
factor (GM-CSF; PeproTech, Rocky Hill, NJ). Clones derived from
the mouse myeloid 32D/WT1 cell line, ectopically expressing human
granulocyte colony-stimulating factor receptor (G-CSFR)  and in-
fected with either AML1-MTG16 (RUNX1-CBFA2T3) (A16 clones),
or the empty vector pLNCX2 (PL clones) were previously described
10% heat-inactivated fetal bovine serum (HyClone), and 10 ng/ml
of mouse interleukin 3 (IL-3; PeproTech), adjusting the cell density to
ing IL-3 with 10 ng/ml human G-CSF (Amgen, Thousand Oaks, CA).
Granulocytic differentiation was microscopically evaluated after Giemsa
staining of cytospin preparations.
Neoplasia Vol. 12, No. 11, 2010Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.
Isolation and Culture of AC133+Hematopoietic
MNCs were isolated according to standard procedures using Lym-
the posterior iliac crest of a healthy donor, after obtaining informed
consent, as per the Niguarda Hospital’s institutional review board
guidelines. The AC133+cell fraction was isolated by immunomagnetic
separation after labeling with CD133/1 (AC133)–biotin antibody and
anti-biotin MicroBeads on LS columns and Midi MACS separator
(Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the
97%.BM-MNCs (2×104per 35-mm dish) andAC133+cells (1×103
per 35-mm dish) were grown in semisolid culture using ready-made
MethoCult GF H 4534 CE-IVD medium (StemCell Technologies,
Inc, Vancouver, British Columbia, Canada), which contains human
recombinant GM-CSF, IL-3, and SCF, with or without erythropoi-
etin (EPO). Erythroid burst-forming units (BFU-E) and granulocyte/
monocyte colony-forming units (CFU-GM) were identified based on
their morphology and counted after 14 days of culture.
Flow Cytometry Analysis
Unselected BM-MNC, CD133/1-positive (AC133+), and CD133/
1-negative (AC133−) cells were incubated for 20 minutes at room tem-
perature in the dark with the appropriate monoclonal antibody (mAb)
mixture,ata concentration deriving fromspecifictitration experiments.
MAbs were directly conjugated with the fluorochromes fluorescein iso-
thiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein
ysis. Each sample was incubated with the following mAbs panels:
CD34-FITC/CD133/1-PE/CD45-PerCp/CD38-APC and CD34-
FITC/CD133/2-PE/CD45-PerCp/CD38-APC. Unselected BM-
MNC from AML patients were also incubated with the mAbs panel
CD34-FITC/CD117-PE/CD45-PerCp/CD14-APC to test for KIT
ride. Cells were centrifuged at 800×g for 8 minutes, and the cell pellet
was resuspended in 500 μl of PBS for flow cytometry analysis. All mea-
surements wereperformedon a dual-laser FACSCalibur flowcytometer
(Becton Dickinson, San Jose, CA) and contained 10,000 to 50,000
cells,adjustedtothe leukocyte subpopulationsintheCD45/sidescatter
plot. Data acquisition was performed with the CellQUEST software,
whereas both CellQUESTand Paint-a-Gate (Becton Dickinson) were
used for analysis. Multiparameter analysis including logical gates on
forward scatter, side scatter, FL1, FL2, FL3, and FL4 was used to eval-
uate cell populations. To assess mouse KITexpression in the 32D cell
as per manufacturer’s instructions, fixed in 4% paraformaldehyde, and
analyzed by using a FACScan flow cytometer (Becton Dickinson) and
FCSExpress software. The results were expressed as geometrical mean
of the fluorescence intensity of the selected markers.
Leukemic MNC cells were isolated from the BM of 39 patients
affected by de novo AML (samples were obtained as per the Niguarda
sified according to the French-American-British classification. Twenty-
five AML samples showed cytogenetic evidence of involvement of
inv(16)/t(16;16). The remaining samples included 12 samples with an
apparently normal karyotype and 2 samples with a complex karyotype,
with three to five chromosome abnormalities in at least one clone but
negative for t(8;21)(q22;q22) and inv(16)/t(16;16).
All the AML samples, previously screened for the presence of KIT
mutation in the entire coding region , were screened for this study
for mutationsinthe 3′UTRof theKIT gene and inthegenomic region
region was amplified by standard polymerase chain reaction (PCR) with
3′) and 3′UTR KITreverse primer (5′-AGA TAC TGG CCC GGT
GTC C-3′), whereas a 438-bp sequence within the MIR-222/221 ge-
nomic region in the chromosome X (chrX) was amplified with chrX
forward primer (5′-TCT GGT TTA CTA GGC TGG TG-3′) and
chrX reverse primer (5′-GTT GGT AGT AGG TAA GTC CC-3′).
Direct DNA sequencing of the PCRfragments wasperformedby using
Thermo Sequence Dye Terminator sequencing reaction and an ABI
Prism3100sequencing analyzer (AppliedBiosystems,FosterCity,CA).
Stem-loop Reverse Transcription and Real-time PCR
Total RNA from leukemic blasts isolated by Ficoll-Hypaque density-
Germany), according to the manufacturer’s instructions and treated with
DNase I (Ambion, Austin, TX). Total RNA (200 ng) was reverse-
Madison, WI) and 10 μM of stem-loop reverse transcription (RT)
primer. Stem-loop RT primers for human MIR-221 (5′-GTC GTA
TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT
ACG ACG AAA CCC-3′), human MIR-222 (5′-GTC GTA TCC
AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG
ACG AGA CC-3′), and human MIR-223 (5′-GTC GTA TCC AGT
GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG
GGG TAT TT-3′) were used for multiplex RTreactions under the
following conditions: 30 minutes at 16°C, 30 minutes at 42°C, and
15 minutes at 70°C and then held at 4°C. Human glyceraldehyde-
3-phosphate dehydrogenase (GAPDH), used for normalization of the
RNA samples, was reverse-transcribed with a linear primer (5′-CAG
tion system (Promega).
tified by real-time PCR performed on an iQ5 Multicolor Real-time
PCR detection system (Bio-Rad, Hercules, CA) by using Premix Ex
Taq (Perfect Real Time; Takara, Shiga, Japan),and primers/probes were
designed using the Beacon Designer software (Bio-Rad). The reaction
was performed by using TaqMan probe 5′FAM-TTC GTC GTATCC
GTC TGC TGG-3′, and reverse primer 5′-GTA TCC AGT GCA
GGG TCC-3′ for MIR-221; TaqMan probe 5′HEX-CTC GTC GTA
TCC AGTGCG AATACC T-3′BHQ1, forward primer 5′-AGC TAC
ATC TGG CTA CTG G-3′, and reverse primer 5′-GTA TCC AGT
GCA GGG TCC-3′ for MIR-222; TaqMan probe 5′FAM-CCG
TCG TAT CCA GTG CGA ATA CCT-3′BHQ1, forward primer 5′-
GTG TCA GTT TGT CAA ATA C-3′ and reverse primer 5′-GTA
TCC AGT GCA GGG TCC-3′ for MIR-223; and TaqMan probe 5′
FAM-CCT CCG ACG CCT GCT TCA CCA-3′BHQ1, forward
Down-regulation of MIR222/221 in CBF-leukemia Brioschi et al. Neoplasia Vol. 12, No. 11, 2010
tions, run in triplicate in a 96-well plate, were incubated at 95°C for
3 minutes, followed by either 40 cycles at 95°C for 5 seconds, 56°C
for 20 seconds, and 72°C for 10 seconds (for MIR-221) or 40 cycles
at 95°C for 5 seconds and 60°C for 1 minutes (for MIR-222, MIR-
223, and GAPDH). The level of the MIR transcripts was normalized
to the level of the GAPDH transcripts and quantified by the threshold
MIRs that differ by as little as a single nucleotide was tested with syn-
thetic MIR-221 and MIR-222. Each MIR assay was examined against
synthetic MIR-222 and MIR-221. Detection specificity was calculated
assuming 100% efficiency for the perfect match between target MIR
ranging from0%to0.17%,respectively (Table W1).We testedalsothe
sensitivity of MIRs detection using synthetic MIR-221, MIR-222, and
MIR-223 at decreasing concentrations. The TaqMan MIR assay
showed a good linearity between synthetic RNA input and Ctvalue,
demonstrating that Ctvalue correlates to the MIRs copy number (data
MA) following the manufacturer’s protocol. Occupancy of endogenous
AML1 or AML1-MTG8 at the AML1-consensus sites in the MIR-222/
221 and MIR/223 promoters was assessed by ChIP with either anti-
the AML1 N-terminus, or anti-MTG8 , recognizing the MTG8
C-terminus, respectively. ChIPs without antibody were performed as
control. The immunoprecipitated DNA was amplified by real-time
no. 1 (sense: 5′-TGACCACACTAAACCCTTGCC-3′; antisense: 5′-
AGTGTGGTTAGCTCTTGGTGG-3′), MIR-222/221 region no. 2
(sense: 5-CACAGCAAAGGATTCTAAGACG-3′; antisense: 5′-CCTG-
GCATTTGAGTGGATTCC-3′), MIR-223 promoter (sense: 5′-
GGGAGAATTGAGAAGAGGGA-3′; antisense: 5′-GATAAGCAGG-
TAAAGCCCGA-3′) , and control region (sense: 5′-GGT-
GCGTGCCCAGTTGAACCA-3′; antisense: 5′-AAAGAA-
GATGCGGCTGACTGTCGAA-3′). The DNA relative enrichment
was calculated by using the ΔΔCtmethod. The PCR signals obtained
for each gene region were normalized to the PCR signal obtained from
the input DNA (total chromatin fraction). Significance was calculated
by using the Student’s t test on three independent determinations.
U937 cells grown in a 24-well plate (2 × 105cells/well) were trans-
fectedbyusingLipofectamine LTX(Invitrogen)with 20ng of pRL-TK
and the indicated amounts of (−1600) MIR-222/221-Luc (kindly pro-
vided by C. Croce, Ohio State University), alone or in combination with
either pCMV5-AML1B (Addgene, Cambridge, MA) or pcDNA3.1-
AML1-MTG8-V5 . Luciferase activity was measured 48 hours after
transfection by using Dual Glow Luciferase Assay System (Promega) and
was normalized to Renilla Luciferase expression.
All data were analyzed with usual descriptive statistical technique,
after checking their distribution with the Shapiro-Wilk test. Quantita-
tive expression of MIR among genotypes (wt vs inv(16) vs t(8;21)) were
compared using the Kruskal-Wallis test; in case of statistical significance
(P < .05), the pairwise evaluations were carried out by means of the
Mann-Whitney U test, adjusted with the Bonferroni method for mul-
tiple comparisons. Differences in expression among CBFL versus non-
CBFL, non-CBFL versus inv(16) versus t(8;21) were checked by the
Mann-Whitney U test. Subject variations in AC133+and AC133−
were analyzed by the Wilcoxon signed rank test.
AML1 Is Implicated in the Transcriptional Control of the
MIR-222/221 Gene Cluster
The CBF transcription factor regulates the transcription of critical
hematopoietic genes by binding the consensus sequence TG(T/C)
GGT through its CBFα (AML1/RUNX1) subunit . MIR-222
and MIR-221 are clustered on chromosome X and transcribed from
the minus strand into a common precursor. MIR-222/221 transcrip-
tion is driven from the same promoter region, which spans approx-
imately 1.6-kb upstream of the transcription start site . The 2-kb
region upstream of the MIR-222/221 gene cluster transcription start
site was searched for the presence of AML1-consensus sequences by
using the MAPPER program. This program identified the most
probable combinations of bases for AML1-binding sites within the
context of the MIR-222/221 promoter (Figure 1A). Specifically, four
AML1-consensus sequences were identified: three canonical AML1-
consensus sequences (at −1012, −1102, and −1296) and one nonca-
nonical AML1-consensus sequence (at −1749; Figure 1B). One of
the canonical AML1-consensus sequences is conserved also in the
mouse MIR-222/221 promoter (at −1155; Figure 1B). To establish
whether AML1 plays a role in MIR-222/221 transcriptional regula-
tion, we tested whether endogenous AML1 can bind one, or more, of
the MIR-222/221 AML1-consensus sequences by ChIP analysis. To
this end, we chose U937 cells, in which endogenous AML1 was pre-
viously shown to bind an AML1-consensus sequence in the MIR-
223 promoter . ChIP with an anti-AML1 antibody shows that
endogenous AML1 binds two regions containing AML1-consensus
sequences in the MIR-222/221 gene (Figure 1C, left) as well as the
previously described AML1-consensus sequence in the MIR-223
promoter (included as a positive control; Figure 1C, right) but does
not bind a negative control region lacking AML1-binding sites
(Figure 1C ). Further, we tested whether AML1 affects MIR-222/
221 transcription by using a reporter construct carrying the luciferase
gene under the control of the MIR-222/221 promoter (from −1600 to
+1) . This construct could be efficiently expressed in a dose-
dependent manner when transiently transfected in U937 cells (Fig-
ure 1D, left), and its expression was significantly (P < .05) enhanced
bycotransfection with increasing amounts of AML1 (Figure 1D, right).
Altogether, these results implicate AML1 as one of the transcriptional
regulators of MIR-222/221.
Up-regulation of MIR-222/221 in AC133+Hematopoietic
Stem/Progenitor Cells versus AC133−Cells
To evaluate MIR-222/221 expression in different hematopoietic
cell contexts, we set up a real-time stem-loop RT-PCR assay  that
allowed us to detect with high efficiency and specificity the two
MIRs (Table W1). We used this method to define the expression
Neoplasia Vol. 12, No. 11, 2010Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.
profile of MIR-222/221 and the myelopoiesis-regulator MIR-223 in
different hematopoietic maturation stages in vivo. We analyzed two cell
fractions isolated by immunomagnetic separation from BM-MNCs:
the AC133+fraction enriched for hematopoietic stem/progenitor cells
(HSPCs), and the AC133−fraction, enriched for more differentiated
cells.Ina healthy donor, theAC133−cells displayedsignificantly higher
transcript levels of both MIR-222/221 and MIR-223 relative to the
AC133+HSPCs (Figure 2A), suggesting that all three MIRs are upreg-
ulated during normal myelopoiesis. In contrast, in AML patients, the
up-regulation of MIR-221/222/223 in AC133+(99.6% blast cells,
RSD 0.4%) versus AC133−(88% blast cells, RSD 11.25%) cells was
less pronounced than the one observed in the healthy donor (Fig-
ure 2B). This could be due, in part, to the cellular composition of AC133+
fraction, which is particularly enriched for leukemic blasts expressing
myeloid-associated differentiation antigens.
Up-regulation of MIR-222/221 during In Vitro Granulocyte/
To determine whether MIR-222/221 expression is modulated in
the course of normal myelopoiesis, we evaluated their transcript levels
during in vitro cell differentiation of AC133+hematopoietic progenitor
cells obtained from the BM-MNC cells of a healthy donor. AC133+
Figure 1. AML1 is implicated in the transcriptional control of MIR-222/221. (A) “Logo” representation of the most probable nucleotide com-
binations of the AML1-consensus sequence (top). Four AML1-consensus sequences could be identified on the minus strand (the tran-
scribed strand) of the 2-kb region upstream of the MIR-222/221 transcription start site (bottom). (B) Scheme showing the relative
position of the putative AML1-binding sites identified by in silico analysis of the human and mouse MIR-222/221 cluster. (C) ChIP with anti-
AML1 showingthat endogenous AML1 is bound significantly more to the AML1-consensussequence-containing regionspresent in the MIR-
222/221 and MIR-223 promoter relative to a control region in U937 cells. (D) Reporter assay showing that luciferase expression driven by the
exogenous AML1 (right).
Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.Neoplasia Vol. 12, No. 11, 2010
cells were grown in a semisolid medium containing growth factors that
The differentiating potential of AC133+cells was compared with the
one of nonsorted BM-MNCs grown under the same conditions. In
the absence of EPO, the growth factors present in the medium (includ-
ing GM-CSF) stimulate the formation of granulocyte/monocyte colo-
nies (CFU-GM), which reach full differentiation within 14 days. A
representative CFU-GM is shown in Figure 3A (left). When EPO is
Figure 3. Up-regulation of MIR-222/221 during AC133+HSPCs in vitro granulocyte/macrophage differentiation. (A) Representative CFU-
GM (left) and BFU-E (right) colonies formed by AC133+HSPCs isolated from the BM-MNCs of the healthy donor, after 14 days in a
colony formation assay in the absence or presence of EPO, respectively. (B) Colony quantitative/qualitative analysis shows that
AC133+cells form significantly more CFU-GM colonies than BM-MNC after 14 days of culture in the presence or absence of EPO.
(C) CFU-GM induction of AC133+cells followed by real-time stem-loop RT-PCR shows up-regulation of MIR-222/221 after 14 days (left),
whereas no significant differences in MIR-222/221 expression could be detected when BFU-E and CFU-GM were concomitantly induced
by culturing AC133+cells for up to 14 days in the presence of EPO (right).
Figure 2. Up-regulation of MIR-221/222/223 in AC133+versus AC133−cells is more pronounced in the healthy donor than in AML patients.
Stem-loopRT-PCRshowingMIR-222/221 andMIR-223expressionlevelsin theAC133+andAC133−cellfractionsisolatedfromBM-MNCof
either a healthy donor (A) or non-CBFL patients (shown is the average of five patients) (B). The data represent the mean (±SD) of three
replicates from one representative experiment of three performed.
Neoplasia Vol. 12, No. 11, 2010 Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.
added to the other growth factors, cells are induced to form BFU-E
besides CFU-GMs. A representative BFU-E, characterized by EPO-
of culture, the AC133+cells formed five and three times more CFU-
GMs than BM-MNCs, when grown in the absence and presence of
EPO, respectively (Figure 3B), indicating that the AC133+fraction
has a stronger granulocyte/macrophage differentiation potential relative
to the unsorted BM-MNC cells.
Next, we analyzed the MIR-222/221 expression profiles during
AC133+in vitro differentiation. The expression of MIR-222/221 was
induced about seven times on CFU-GM induction after 14 days of
culture (Figure 3C, left). In contrast, no significant effect on MIR-
222/221 expression was observed in the presence of EPO (Figure 3C,
right). Because down-regulation of MIR-222/221 is known to occur
during EPO , MIR-222/221 up-regulation in CFU-GM may be
masked by MIR-222/221 down-regulation in the BFU-E colonies in-
duced by EPO.
MIR-222/221 Transcriptional Repression by the t(8;21)-CBFL
Fusion Protein AML1-MTG8 (AML1-ETO)
AML1target genes, including MIR-223 , have been reported to
be repressed in CBFL patient samples and CBFL cell lines. To test
whether CBFL rearrangements can induce repression of MIR-222/
221, which is a bona fide AML1 target gene (Figure 1), we chose the
AML1-MTG8 protein, derived from the t(8;21)-CBFL translocation.
AML1-MTG8 is known to exert a repressive action on the transcrip-
tion of several AML1 target genes . ChIP analysis with an anti-
MTG8 antibody of the t(8;21)-positive cell line SKNO-1 showed
significantly more binding of endogenous AML1-MTG8 to the
AML1-consensus sequences of the MIR-222/221 promoter and the
MIR-223 promoter (positive control) relative to a control region with-
out AML1-binding sites (Figure 4A). Further, transient expression of
exogenous AML1-MTG8 in U937 induced a significant (P < .05),
dose-dependent repression of MIR-222/221-luciferase (Figure 4B).
Down-regulation of MIR-221/222 in CBFL Overexpressing
CBFL progression has been reported to be associated with activat-
ing mutations and/or over expression of the tyrosine kinase receptor
KIT [11–14]. Because KIT mRNA is a known target of MIR-221
and MIR-222 , we tested whether KIT overexpression in CBFL
samples is associated with either KIT mutations that may impair
MIR-mRNA binding or defects in MIR-222/221 expression in
We analyzed 26 CBFL samples, which had been tested at diagnosis
both for the presence of mutations in the KIT coding region and for
the expression of the KIT receptor in BM-MNC cells (Table W2
based on Beghini et al. ), and 13 non-CBFL samples. The CBFL
samples displayed higher incidence of KIT mutations (Table W2)
and significantly higher KIT expression (CD117 antigen) relative
to non-CBFL samples (Figure 5A). Further, we could detect by
stem-loop RT-PCR lower expression levels of the CBF MIR target
MIR-223  in CBFL samples relative to non-CBFL samples
When we tested the same samples for MIR-221 and MIR-222 ex-
pression levels, we detected significant down-regulation of MIR-221
and MIR-222 in CBFL versus non CBFL (Figure 5, B and C). The
observed MIR-222/221 down-regulation correlated with KIT/
CD117 overexpression. Comparison of KIT (CD117) and MIR ex-
pression in either inv(16) or t(8;21) CBFL samples versus non-CBFL
samples showed that KIT (CD117) overexpression (Figure 5E ) was
associated with MIR-221, MIR-222, and MIR-223 down-regulation
(Figure 5, F-H) in both inv(16) and t(8;21) samples relative to non-
CBFL samples. Apparently, both AML1-MTG8 (AML1-ETO) and
CBFβ-MYH11 seem to exert a comparable repressive effect on the
transcription of both MIR-223 and MIR-222/221.
By sequence analysis, we did not detect any mutations in both the
KIT 3′-UTR and the pri-MIR-222/221 genomic sequences of CBFL
samples (data not shown). Thus, MIR-222/221 down-regulation does
not seem to be due to the lack of MIR-222/221 binding to KIT 3′
UTR. On the basis of the evidence gathered so far, KIT (CD117)
Figure 4. MIR-222/221 transcriptional repression by the CBFL fusion protein AML1-MTG8. (A) ChIP with anti-MTG8 showing that
endogenous AML1-MTG8 is bound significantly more to the AML1-consensus sequence-containing regions present in the MIR-222/
221 and MIR-223 promoter relative to a control region in the t(8;21)-positive SKNO-1 cell line. (B) Reporter assay showing that luciferase
expression driven by the MIR-222/221 promoter (from −1600 to +1) is significantly repressed by expression of exogenous AML1-MTG8
in U937 cells.
Down-regulation of MIR222/221 in CBF-leukemia Brioschi et al.Neoplasia Vol. 12, No. 11, 2010
overexpression in CBFL may be traced, at least in part, to MIR-222/
221 down-regulation induced by CBF fusion proteins.
Ectopic Expression of a CBF-Related Fusion Protein Leads to
Down-regulation of MIR-221/222/223
To test whether the down-regulation of MIR-222/221 and KIT
overexpression observed in CBFL samples can indeed be traced to
the action of CBF fusion proteins, we exploited a mouse CBFL cell
model that we previously described . This model consists of
32D/WT1 cells ectopically expressing AML1-MTG16 (RUNX1-
CBFA2T3), the CBF fusion protein of t(16;21)-positive CBFL.
the wild type MTG16 and MTG8 mainly differ in their N-terminal
region, and this region is lost on the fusion of MTG16 to AML1
Figure 5. Down-regulation of MIR-222/221 and MIR-223 in CBFL-overexpressing KIT. (A) Flow cytometry analysis of the CD117 antigen (KIT)
in non-CBFL patients and CBFL patients with either inv(16) and t(8;21). (B-D) Stem loop RT-PCR showing MIR-221, MIR-222, and MIR-223
expression levels in non-CBFL patients and CBFL patients with either inv(16) and t(8;21). (E) Flow cytometry analysis of the CD117 antigen
(KIT) in BM-MNCs of non-CBFL patients, CBFL patients with inv(16), and CBFL patients with t(8;21). (F-H) Stem,loop RT-PCR showing MIR-
221, MIR-222, and MIR-223 expression levels in non-CBFL patients, CBFL patients with inv(16), and CBFL patients with t(8;21). The median
valuesfor eachsample group are indicatedby theblack line(± SD) inthe box plots.Mann-Whitney U test was usedtocalculate the P value;
P < .05 was considered statistically significant.
Neoplasia Vol. 12, No. 11, 2010Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.
(Figure 6A and Rossetti et al. ). Two clones expressing AML1-
MTG16 (A23 and A24) and two clones carrying the control empty
retroviral vector (PL4 and PL5) were used in this study. Although con-
trol clones are induced to differentiate into granulocytes by treatment
with G-CSF, AML1-MTG16–positive clones, cultured under the same
conditions, do not undergo granulocytic differentiation (Figure 6B).
The expression of both MIR-221 and MIR-222 was significantly
downregulated in AML1-MTG16–positive clones relative to control
clones, both in the absence and the presence of G-CSF (Figure 6C).
Similarly, MIR-223, which is highly conserved between human and
mouse (Figure W2), was significantly downregulated in AML1-
MTG16–positive clones after induction by G-CSF (Figure 6D).
Next, we tested whether there was a correlation between down-
regulation of MIR-222/221 and level of KIT expression by cyto-
fluorimetric analysis of the mouse CD117 antigen. Interestingly, we
found that the two AML1-MTG16–positive clones showed a signifi-
cantly (P < .05) higher KIT level relative to both wild type 32D cells,
and a control 32D clone (Figure 6E). Apparently, ectopic AML1-
MTG16 expression leads to both increased KIT expression and
down-regulation of MIR-222/221 transcription.
This study extends previous studies, including ours, showing that
there is a significant association between rearrangements involving
the CBF subunits and overexpression of either wild type or mutant
involved in the overexpression of (wild type or mutant) KIT has been
an open question.
We hypothesized that MIR-222/221 could be the molecular link
between rearranged CBF subunits and KITreceptor up-regulation in
CBFL because there was evidence that MIR-222 and MIR-221 can
act as regulators of KIT protein expression by targeting the 3′UTR of
KITmRNA  and because we found that the MIR-222/221 pro-
moter harbors conserved consensus sequences for AML1, the CBFα
subunit. The t(8;21) CBF fusion protein AML1-MTG8 (AML1-
ETO) was shown to be a direct transcriptional regulator of MIR-
223, a regulator of myeloid differentiation, capable of inducing
MIR-223 epigenetic down-regulation . We demonstrate here
that MIR-222/221 is an AML1-regulated MIR cluster and that
AML1-MTG8 can bind AML1-consensus sequences of the MIR-
222/221 promoter and induce transcriptional repression of a MIR-
222/221-luciferase reporter gene. This observation strengthened our
hypothesis that CBF rearrangements, by down-regulating MIR-222/
221, can induce overexpression of the KIT receptor.
We set up a stem-loop RT-PCR assay, which was specific and sen-
sitive to detect a differential expression of MIR-221 and MIR-222 in
AC133+and AC133−fractions from BM-MNC cells of a healthy do-
nor. The AC133−cell fraction, enriched for more differentiated cells,
displayed a higher level of both MIR-221 and MIR-222 expression
Figure 6. Ectopic expression of a CBF-related fusion protein leads to concomitant MIR-222/221 down-regulation and KIT up-regulation. (A)
Scheme or the CFBL-related fusion protein AML1-MTG16. (B) 32D/WT1 cells stably expressing AML1-MTG16 are unable of proper granulo-
cytic differentiation in response to treatment with G-CSF for 6 days. (C) Two representative AML1-MTG16–positive clones (A16) display
down-regulation of MIR-221/222 relative to two representative control clones (PL), both in the presence and absence of G-CSF. (D)
G-CSF–induced MIR-223 expression is downregulated in AML1-MTG16–positive clones relative to control clones. (E) Cytofluorimetric analy-
sis with PE-labeled anti-CD117 antibody (representative plots are shown on top) showing that two representative AML1-MTG16–positive
clones express significantly higher KIT levels than wild type and control cells.
Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al.Neoplasia Vol. 12, No. 11, 2010
relative to the AC133+cell fraction, which is enriched for stem/pro-
genitor cells and positive for stem cell antigens, including CD117.
The stem-loop RT-PCR assay let us also detect an increasing expres-
sion of MIR-222/221 in the course of AC133+granulocyte/mono-
cyte differentiation, which results in a decrease of CD117-positive cells
(data not shown; and Ruzicka et al. ).
Next, we searched for an association between the level of MIR-
222/221 expression and expression of CD117 KIT receptor antigen
in leukemic samples with CBF rearrangements. By comparing samples
of CBFL and non-CBFL with significant differences in the expression
level of the KIT CD117 antigen, we found a significant difference in
the expression of MIR-223, known for being downregulated by the
CBF fusion protein AML1-MTG8 (AML1-ETO) . Further, we
found a significantly lower level of expression of both MIR-221 and
MIR-222 in the CBFL group versus the non-CBFL group, showing
that there is a significant correlation between down-regulation of
MIR-221 and MIR-222 and the expression of different CBF fusion
proteins. Interestingly, both the t(8;21)-positive CBFL group and the
inv(16)-positive CBFL group showed comparable down-regulation in
the expression of MIR-223 as well as MIR-221 and MIR-222. Thus,
MIR-223 and MIR-221/222 down-regulation does not seem depen-
dent on a specific CBF subunit rearrangement. How rearrangements
of different CBF subunits exert similar repressive activity on the pro-
moter regions of both MIR-223 and MIR-222/221 remains to be in-
vestigated. It is interesting to note that the promoter regions of both
MIR-223  and MIR-222/221 (data not shown) also contain a pu-
tative CEBPA-binding sequence and that both AML1-MTG8 and
CBFβ-MYH11 can interfere with CEBPA expression at the transcrip-
tional and translational levels, respectively [34–36]. Thus, it is possible
that the down-regulation observed for all these MIRs are due to direct
targeting of the fusion proteins at AML1 sites in the MIR promoter
regions and/or indirectly by the fusion proteins affecting CEBPA-
mediated regulation of the MIRs.
Because of the high conservation between the mouse and human
a CBF-related fusion protein (AML1-MTG16) can concomitantly in-
duce both MIR-221/222/223 down-regulation and KITup-regulation
in the mouse myeloid cell model 32D/WT1. AML1-MTG16, like
AML1-MTG8, maintains the DNA-binding domain of AML1 (the
Runt domain), and the same four repressive domains of the MTG8
protein . We found that AML1-MTG16 leads to down-regulation
of MIR-223, MIR-221, and MIR-222 in the course of mouse granu-
locytic differentiation. Thus, AML1-MTG16, as AML1-MTG8, in
addition to directly targeting and downregulating the expression of
hematopoietic protein-coding genes containing AML1 consensus se-
quences [23–33,37], can target MIR genes important for myelopoiesis,
In conclusion, this study supports a model in which CBF genetic
abnormalities can lead to the overexpression of (wild type or mu-
tated) KIT receptor by direct down-regulation of CBF-regulated
MIRs. This mechanism would explain, at least in part, the concerted
contribution of class I and class II mutations to the pathogenesis pro-
cess of CBFL.
The authors thank T. Mancuso (Bio-Rad Laboratories, Italy) for
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Down-regulation of MIR222/221 in CBF-leukemiaBrioschi et al. Neoplasia Vol. 12, No. 11, 2010
Table W1. Discrimination Power of 222/221 MIR Assay.
Synthetic MIR Target
0.0 Relative Detection (%)
Relative detection (%) calculated based on Ctdifference between perfectly matched and mis-
matched targets (red). A total of 8.4 × 108copies of synthetic RNA were added to the RTreaction.
Table W2. Features of Leukemia Samples.
Patient No.SexAge (years) FAB Karyotypec-KIT
46,XX,−5,−17, tas(13;?) (pter;?),+mar,50dim
FAB indicates French-American-British classification.
Figure W2. SchemeshowingthepositionoftheputativeAML1-bindingsitesidentifiedbyinsilicoanalysisofthehumanandmouseMIR-223.
Figure W1. MIR-223 expression modulation during CFU-GM and BFU-E induction of AC133+HSPCs in a colony-forming cell (CFC) assay.
(A) CFU-GM induction of AC133+cells at 7 and 14 days followed by real-time quantification showed a downregulation of MIR-223 while
(B) Concomitant BFU-E and CFU-GM induction of AC133+cells at 7 and 14 days followed by real-time quantification showed a weak mod-
ulation of miRNAs expression.