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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3134-3143
Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits
the Establishment of Normal Definitive Hematopoiesis and Directly
Generates Dysplastic Hematopoietic Progenitors
By
Tsukasa Okuda,
Zhongling Cai,
Shouli Yang,
Noel Lenny,
Chuhl-joo Lyu,
Jan M.A. van Deursen,
Hironori Harada, and
James R. Downing
From the Departments of Pathology and Laboratory Medicine, Tumor Cell
Biology, and Genetics, St Jude Children's Research Hospital, Memphis,
TN.
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ABSTRACT |
The t(8;21)-encoded AML1-ETO chimeric product is believed to be
causally involved in up to 15% of acute myelogenous leukemias through
an as yet unknown mechanism. To directly investigate the role of
AML1-ETO in leukemogenesis, we used gene targeting to create an
AML1-ETO "knock-in" allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO
(AML1-ETO/+) died around E13.5 from a complete absence of
normal fetal liver-derived definitive hematopoiesis and lethal
hemorrhages. This phenotype was similar to that seen following
homozygous disruption of either AML1 or
CBF . However, in contrast to AML1- or
CBF-deficient embryos, fetal livers from AML1-ETO/+
embryos contained dysplastic multilineage hematopoietic progenitors
that had an abnormally high self-renewal capacity in vitro. To further
document the role of AML1-ETO in these growth abnormalities, we used
retroviral transduction to express AML1-ETO in murine adult bone
marrow-derived hematopoietic progenitors. AML1-ETO-expressing cells
were again found to have an increased self-renewal capacity and could
be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the
normal biologic activity of AML1 but also directly induces aberrant
hematopoietic cell proliferation.
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INTRODUCTION |
THE (8;21)(q22;q22) TRANSLOCATION is one
of the most frequent karyotypic abnormalities detected in acute
myelogenous leukemia (AML), accounting for approximately 40% of de
novo AML cases that have M2 morphology in the French-American-British
(FAB) classification.1 As a result of this translocation,
the gene encoding the AML1 (CBFA2) transcription factor
from chromosome 21 is fused to the eight twenty-one gene (ETO,
also known as MTG8 and CBFA2T1) on chromosome 8.2-5
This fusion gene encodes a chimeric AML1-ETO protein consisting of the
N-terminus of AML1 fused in frame to the C-terminus of ETO. The fact
that the t(8;21) is the sole cytogenetic abnormality in the majority of
these cases suggests that AML1-ETO plays a critical role in the
establishment of the leukemia clone.
One of the targets of this translocation, AML1, is the DNA-binding
subunit of the AML1/CBF transcription factor complex and binds the
enhancer core motif, TGT/cGGT.6,7 AML1's DNA-binding affinity is increased through heterodimerization with CBF, and both
its DNA-binding and interaction with CBF are mediated through a
central domain with high homology to the Drosophila
segmentation gene, runt.6,8,9 The AML1-ETO chimeric
product retains this domain and therefore also binds the core enhancer
sequence and interacts with CBF.6,10 Compared with AML1,
relatively little is known about ETO, the other target of this
translocation. Although ETO contains a zinc finger motif and appears to
be the mammalian homologue of the Drosophila gene
nervy,11 no direct DNA-binding activity has been
detected, nor has its function been identified.12
Transcriptional regulation by AML1 through the enhancer core motif has
been shown to be important for the tissue specific expression of a
number of hematopoietic specific genes including interleukin-3
(IL-3),13 granulocyte-macrophage colony-stimulating factor
(GM-CSF),14 the receptor for CSF-1 (CSF-1R),15
myeloperoxidase,16 and subunits of the T-cell antigen
receptor (TCR).8,17 By creating mice deficient in
AML1/CBF, we18 as well as others19-21 have
shown that the AML1/CBF transcription factor complex is essential
for the establishment of definitive hematopoiesis.
Consistent with a critical role in hematopoiesis, the genes encoding
the AML1/CBF transcription factor complex are the most frequent
targets of chromosomal abnormalities in human leukemia. In addition to
t(8;21), AML1 fusion products are generated as a result of the t(3;21)
(AML1-EVI1) in myelodysplasia and blast crisis of chronic myelogenous
leukemia,22-24 and the t(12;21) (TEL-AML1), the most
frequent translocation in pediatric acute lymphoblastic leukemia.25-28 Similarly, the CBF
gene is fused to the smooth muscle myosin heavy chain gene,
MYH11, as a result of the inv(16) or t(16;16) in the majority
of AML cases with M4Eo FAB morphology.29-30 Biochemical
studies of these translocation-encoded fusion products suggest that
both AML1 and CBF chimeric products function, at least in part, to
dominantly interfere with normal AML1/CBF-mediated transcription.10,31-34 For example, AML1-ETO represses
transcription of reporter genes driven by the
TCR enhancer10 or the
GM-CSF promoter,31 and this activity is dependent
on a putative repression domain within the C-terminus of
ETO.32
To directly investigate the in vivo mechanism through which AML1-ETO
contributes to leukemogenesis, we designed a "knock-in" strategy
to generate mice containing a single allele of AML1-ETO (AML1-ETO/+) whose expression is regulated by the endogenous
transcriptional regulatory elements of murine AML1. Our
analysis shows that AML1-ETO both neutralizes normal AML1/CBF
activity and directly generates signals that lead to the generation of
abnormal hematopoietic progenitors.
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MATERIALS AND METHODS |
Construction of AML1-ETO knock-in targeting vector.
A silent A  G mutation at codon 122 was introduced by
site-directed mutagenesis into the human AML1-ETO cDNA (a gift
from Dr S. Hiebert, Vanderbilt University, Nashville, TN) to create a
SacII cleavage site that is in an identical position to a
SacII site within exon 4 of murine AML1. A 1.9-kb
SacII fragment from this cDNA, which included 62 bp of
AML1 exon 4, all of exon 5, and the entire fused portion of
ETO, was then inserted into the murine AML1 exon 4 SacII site within a 12-kb murine genomic AML1 clone.18 This resulted in an in frame fusion of the first
95 bp of murine AML1 exon 4 to the remaining portion of the
human AML1-ETO cDNA. A 470-bp fragment containing the
polyadenylation signal from the rabbit globin gene excised from
pEµSR,35 followed by a 1.7-kb HSV-tk promoter-neomycin
resistance cassette,18 inserted in the opposite
transcriptional orientation was inserted immediately 3 of the 1.9-kb
cDNA fragment. Finally, a 1.2-kb HSV-tk promoter-diphtheria toxin-A
negative selection cassette was inserted at the 3 end of the
construct. The vector was linearized at a unique Not I site
located at the 3 end of the construct and introduced into embryonic
stem (ES) cells.
Production of chimeric mice.
The linearized targeting vector (50 µg) was electroporated into E14
ES cells as previously described.18 G418-resistant clones were analyzed for homologous recombination by Southern analysis of
Xba I-digested DNA hybridized with 5 (0.4 kb) or 3 (0.5 kb) AML1 fragments derived from murine genomic sequences outside
the vector. Clones with homologous recombination of the targeting vector were then assessed by reverse transcriptase-polymerase chain
reaction (RT-PCR) for expression of the fusion gene. Oligonucleotide primers from murine AML1 exon 3 (5-CCAGCAAGCTGAGGAGCGGCG-3) and human ETO (5-AGGCTGTAGGAGAATGG-3) were used for PCR
amplification, and the products were analyzed by Southern analysis
using a murine AML1 exon 4 specific oligonucleotide probe
(5-GTGGTGGCACTGGGGGACGT-3) for detection. AML1-ETO/+ clones
with undifferentiated morphology and normal karyotypes were injected
into C57BL/6 blastocysts. Male chimeras were bred with C57BL/6 females
and tail biopsy specimens of agouti offspring were screened for the
presence of the knock-in allele by Southern analysis.
Histology.
Embryos were removed from the uterus, dissected free from the fetal
membranes, and inspected under a dissecting microscope for evidence of
gross abnormalities. Fetal tissues were then obtained for genotyping
and cells from yolk sacs or fetal livers were isolated under sterile
conditions and used for in vitro hematopoietic colony assays. Embryos
were fixed in Bouin's solution, embedded in paraffin, and sections
stained with hematoxylin and eosin. Peripheral blood was collected in
10 mmol/L EDTA and smears stained with Wright-Giemsa.
In vitro culture of hematopoietic cells.
Cells from yolk sacs, fetal livers, or bone marrows were obtained and
cultured in methylcellulose semisolid media containing a combination of
colony stimulating factors as previously described.18 Cell
aggregates containing more than 50 cells were counted as colonies.
Cytocentrifuge preparations of hematopoietic colonies were stained with
Wright-Giemsa for morphologic examination or for the presence of
nonspecific esterase (alpha naphthyl butyrate) cytochemical activity.
In replating experiments, either individual hematopoietic colonies or
all of the cells from the primary culture were collected, washed, and
then replated at 1 to 3 × 104 cells/plate into new
methylcellulose cultures under conditions identical to those used for
the primary cultures. Colonies were scored as above and subsequent
replatings were performed in an identical fashion. Cell lines were
generated by harvesting colonies from a single plate and growing the
cells in liquid cultures containing RPMI 1640 media containing 10%
fetal calf serum, 2 mmol/L L-glutamine, 50 U/mL penicillin G, 50 µg/mL streptomycin, and 10 ng/mL of IL-6 and SCF and 2 ng/mL of IL-3.
Western blot and immunophenotypic analysis.
Cells were lysed in boiling RIPA lysis buffer, electrophoretically
separated on a 10% denaturing polyacrylamide gel, and transferred to a
nitrocellulose membrane. Proteins were detected using affinity purified
AML-1 N-terminal peptide antiserum6 or affinity purified ETO N-terminal peptide antiserum10 and visualized with
supersignal ULTRA chemiluminescence substrate (Pierce, Rockford, IL).
Cell surface antigens were detected by a standard direct
immunofluorescence assay using phycoerythrin-conjugated monoclonal
antibodies (MoAb) from PharMigen (San Diego, CA) to Ly-6G (Gr-1), CD116
(MAC-1), c-kit, Sca-1, and Thy-1. Fluorescence activity was analyzed on an FACScan (Becton Dickson, San Jose, CA). Isotypically matched MoAbs
at the same protein concentration were used as negative controls in all
experiments.
Production of retroviral stocks and infection of murine bone marrow
cells.
A human AML1-ETO cDNA10 was inserted into the retroviral
vector MSCVneo36 to generate
MSCV/AML1-ETOneo. Helper-free retrovirus was generated by
transfecting the Bosc23 packaging cell line37 with
MSCVneo or MSCV/AML1-ETOneo DNA, and retroviral
containing supernatants were collected and stored at  80°C.
Supernatants were titered on NIH3T3 cells in G418 containing media as
described previously.38
Murine bone marrow cells were obtained from the femur and tibia of 8- to 10-week-old female BALB/cByJ mice 2 days following an
intraperitoneal injection of 150 mg/kg of 5-FU (SoloPak Laboratories, Elk Grove Village, IL). Harvested cells were prestimulated for 48 hours
with IL-6 and SCF as previously described38 and then infected on fibronectin fragment CH-296-(Takara Shuzo, Otsu, Japan) coated bacterial dishes with 10 mL of viral supernatant supplemented with 100 ng/mL SCF and 100 ng/mL IL-6. Fresh viral supernatant was
added after 2 hours and again after 22 hours, and the infection continued for a total of 48 hours. Infected cells were then plated directly in methycellulose medium containing G418 at a concentration of
1.0 mg/mL, and the hematopoietic growth factors IL-3, IL-6, and SCF.
Serial replating of hematopoietic colonies was performed as described
above. Cell lines were established by growing cells in liquid cultures
containing Iscove's media containing 15% fetal calf serum (FCS), 2 mmol/L glutamine, 10 ng/mL of IL-3 and IL-6, 50 ng/mL SCF, 0.1 mmol/L
2-mercaptoenthanol, 10 µg/mL insulin, 200 µg/mL transferrin, and 3 U/mL erythropoietin.
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RESULTS |
Embryonic lethality, absence of definitive fetal liver hematopoiesis,
and lethal hemorrhages in mice heterozygous for the AML1-ETO
knock-in allele.
To create an AML1-ETO chimeric gene that mimics that formed by
the t(8;21), we fused human AML1-ETO sequences in frame to murine AML1 exon 4 (Fig 1). This
targeting strategy resulted in the generation of a murine/human hybrid
AML1-ETO gene whose expression is controlled by endogenous
murine AML1 regulatory sequences. Three independent
AML1-ETO/+ ES cell clones with normal ploidy were analyzed and
shown by RT-PCR to express AML1-ETO (Fig 1). In
undifferentiated ES cells, endogenous murine AML1 is expressed and can
be detected by RT-PCR (data not shown).
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| Fig 1.
Generation of an AML1-ETO chimeric gene by
homologous recombination. (A) Schematic of AML1 cDNA, partial
murine AML1 genomic locus, and replacement vector containing a
partial human AML1-ETO cDNA, polyadenylation signal (pA), and
the positive selection neomycin resistance cassette (Neor)
and negative selection diphtheria toxin-A cassette (DT-A). Arrows indicate the position of the AML1-ETO fusion and the
transcriptional orientation of selection cassettes. The structure of
the targeted allele and the chimeric AML1-ETO cDNA is shown, as
are the primers used for RT-PCR amplification and detection of the
AML1-ETO fusion transcript. Use of AML1 genomic probes
A or B on Xba I-digested DNA allows resolution of wild-type
and targeted alleles. (B) Southern analysis of control (CTR) and
AML1-ETO knock-in (KI) ES cell clones. (C) RT-PCR analysis of CTR and
KI ES cell clones. AML1-ETO mRNA was amplified using primers 1 and 2, and products were hybridized with the murine AML1-specific
oligonucleotide 3. Amplification was also performed for HPRT
mRNA as a control for the presence of amplifiable RNA.
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Chimeric male mice generated from two independent AML1-ETO/+
ES cells were bred with wild-type C57BL/6 females, and agouti pups
were obtained, showing germline transmission of the ES cell-derived genome. Of the 94 live agouti pups genotyped, none contained the AML1-ETO knock-in allele, suggesting that expression of
AML1-ETO was embryonic lethal (Table 1). To
define the stage of embryonic development at which expression of
AML1-ETO is lethal, we analyzed embryos between 10.5 and 14.5 days of
gestation. At E10.5 and E11.5 the majority of AML1-ETO/+
embryos were viable and showed no significant morphologic abnormalities
when compared with their normal litter mates (Table 1 and data not
shown). However, by E12.5 a significant proportion of the
AML1-ETO/+ embryos were dead, and by E14.5 no viable embryos
with the knock-in allele were detected (Table 1). From E12.5 to E13.5
the AML1-ETO/+ embryos showed normal overall organ development
and were equal in size to their control litter mates; however, the
AML1-ETO/+ embryos were identifiable by marked fetal liver
pallor and by massive hemorrhages within the ventricles of the central
nervous system (CNS) and within the pericardial cavity and the soft
tissues of the back (Fig 2).
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| Fig 2.
Phenotype of E12.5 AML1-ETO/+ and wild-type
(WT) embryos. (Top panels) AML1-ETO/+ embryos were identical
in size to wild-type littermates, but were easily identifiable by the
presence of fetal liver pallor and massive hemorrhages within the
ventricles of the CNS and the soft tissues of the back. (Second panels)
Sections showing hemorrhages within the dorsal root ganglia of
AML1-ETO/+ embryos. (Third panels) Sections of the fetal
liver from AML1-ETO/+ embryos showing a complete absence of
hematopoietic precursors with only rare primitive nucleated
erythrocytes seen within hepatic sinusoids. By contrast, sections of
the fetal liver from control littermates show numerous erythroblasts
and scattered myeloblasts and megakaryocytes. (Bottom panels) Smears of
peripheral blood show the absence of definitive erythrocytes and
platelets in the AML1-ETO/+ embryos. By contrast, numerous
enucleated definitive erythrocytes and platelets are seen in the
peripheral blood from wild-type embryos.
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On microscopic examination, the CNS hemorrhages appeared to originate
within midbrain parenchyma, the dorsal root ganglia, and the ganglia of
the VII/VIII cranial nerve complex (Fig 2). Focal hemorrhages were
detected within these structures as early as E11.5. Apoptotic
neural cells were also identified within these ganglia; however, no
differences in the number of apoptotic cells were noted between
normal and AML1-ETO/+ embryos. In addition, we saw no evidence
of necrosis in the developing neural structures before the onset of
bleeds.
The major developmental defect identified in the AML1-ETO/+
embryos was the complete absence of definitive fetal liver-derived hematopoiesis. Primitive yolk sac-derived hematopoiesis was intact, as
assessed by the absence of overt anemia and by normal morphology of
primitive nucleated erythrocytes. By contrast, microscopic sections of
the fetal liver revealed the complete absence of definitive fetal
liver-derived hematopoiesis (Fig 2). Within the liver no erythroid,
myeloid, or megakaryocytic progenitors were identified, and only
scattered primitive nucleated erythrocytes were seen. Peripheral blood
smears from E11.5-E13.5 AML1-ETO/+ embryos contained only
primitive nucleated erythrocytes and lacked visible platelets (Fig 2).
By contrast, platelets and definitive erythrocytes were easily
identified within smears from control litter mates. In addition,
neutrophils and monocytes were seen upon scanning the smears from
normal E11.5-E13.5 embryos but were absent from AML1-ETO/+ smears (data not shown).
Expression of AML1-ETO leads to the generation of dysplastic
hematopoietic progenitors.
To further characterize the hematopoietic defect identified in
AML1-ETO/+ embryos, we analyzed cells from yolk sacs and fetal livers for in vitro hematopoietic colony forming activity. Cells from
the yolk sacs of viable E10.5 embryos were plated in methycellulose media under conditions optimal for the development of myeloid, erythroid, and mixed colonies. At this stage of development no primitive hematopoietic progenitors are detected under the in vitro
culture conditions used,39 and thus all hematopoietic colonies detected in these assays are of definitive origin. Numerous colonies grew in cultures of yolk sacs from E10.5 wild-type embryos, whereas only rare granulocytic or granulocytic-monocytic colonies were
identified in yolk sac cultures from E10.5 AML1-ETO/+ embryos (Table 2). Similarly, numerous definitive
hematopoietic colonies were detected in cultures of fetal liver cells
from E11.5 wild-type embryos. By contrast, 20- to 30-fold fewer cells
were recovered from fetal livers of E11.5 AML1-ETO/+ embryos,
and when these cells were plated in methycellulose at numbers equal to
that used in wild-type cultures, only rare hematopoietic colonies were
identified (Table 2). AML1-ETO expression was confirmed within these
colonies by RT-PCR analysis (data not shown). Taken together, these
data show that expression of AML1-ETO leads to almost a complete
absence of definitive hematopoiesis; however, in contrast to
AML1-deficient embryos,18,19 a few progenitors could be
identified.
Similar to the results obtained with E11.5 fetal livers, the total
number of cells recovered from fetal livers of viable E12.5 and E13.5
AML1-ETO/+ embryos was also 20- to 30-fold less than that
recovered from wild-type embryos. In contrast to the results obtained
with E11.5 embryos, however, cultures of fetal livers cells from viable
E12.5 and E13.5 AML1-ETO/+ embryos contained numerous abnormal
multilineage colonies (Table 2 and Fig 3).These colonies consisted of large, tightly packed aggregates of
differentiating erythroid precursors, monocytes, megakaryoblasts, and
hypergranular cells of the myeloid lineage. Each lineage showed
evidence of dysplastic morphology with numerous abnormal erythroblast,
megakaryoblast, and multinucleated myeloid cells identified (Fig 3).
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| Fig 3.
Morphology of dysplastic AML1-ETO/+ mixed
hematopoietic colonies. (A) Abnormal mixed colonies derived from
AML1-ETO/+ fetal liver cells. (B) Cytocentrifuge preparations
of cells contained within these colonies. (Left) Numerous hypergranular
myeloid cells are seen with frequent abnormal binucleated and
trinucleated cells detected. (Right) Illustration of the mixed nature
of the colony with maturing erythroid cells, a monocyte, binucleated
myeloid precursors, and a dysplastic megakaryoblast.
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To further characterize these abnormal colonies, we collected cells
from individual AML1-ETO/+ or wild-type fetal liver cultures after 7 to 10 days of growth in methylcellulose, disaggregated the
cells, and then grew them in liquid cultures either in the presence or
absence of IL-3, IL-6, and SCF. Under these conditions, cellular growth
and survival was dependent on the presence of hematopoietic growth
factors, with rapid cell death observed in their absence. To confirm
expression of AML1-ETO within the expanding hematopoietic population,
Western blot analysis was performed using an affinity purified
ETO-specific rabbit antisera.10 As shown in Fig
4, the AML1-ETO chimeric protein was not
observed in cells from control cultures but was easily detected within the hematopoietic cells derived from each of the AML1-ETO/+ fetal liver
cultures (representative results from six cultures presented). The
level of AML1-ETO expression was similar to that of endogenous AML1 as
detected with an AML1-specific antiserum (data not shown).
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| Fig 4.
AML1-ETO expression in the dysplastic hematopoietic
cells. Western blot analysis of total cell lysates prepared from
wild-type (Wt.) or AML1-ETO/+ knock-in (KI) cells. Cell lysates
transferred to nitrocellulose membranes were blotted with affinity
purified ETO N-terminal peptide antisera and visualized with
supersignal ULTRA chemiluminescence substrate.
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To examine the self-renewal capacity of the cells within the fetal
liver-derived colonies, we replated either single colonies or bulk cell
populations in secondary methylcellulose cultures. When these cells
were replated under conditions optimal for the development of
multipotential hematopoietic progenitors, similar numbers of colonies
were observed from both the AML1-ETO/+ and the control cells (Table
3). However, in contrast to the wild-type cells, the AML1-ETO/+ cells formed relatively large dysplastic granulocyte-monocyte and multilineage colonies identical to those seen
in the primary cultures (Fig 5A). Moreover,
upon subsequent replating the AML1-ETO/+ cells continued to form
dysplastic mixed colonies at a high efficiency. Cells from control
cultures failed to form colonies after the seventh replating, whereas
AML1-ETO/+ cells continued to generate colony-forming progenitors in
methylcellulose, without any loss in efficiency of colony formation
beyond 20 passages (results from the initial 10 passages are shown in
Table 3). Although the colony-forming efficiency for the AML1-ETO/+
cells remained relatively constant, the frequency of multilineage
colonies containing erythroid cells and megakaryocytes decreased with
each passage. Remarkably, however, definitive multilineage colonies remained through passage 16 (Fig 5A). The immature dysplastic myeloid
and monocytic cells from the replated colonies readily expanded in
liquid cultures containing IL-3, IL-6, and SCF and coexpressed the
myeloid and monocytic markers GR1 and MAC1 (Fig 5B). Identical results
were obtained from AML1-ETO-expressing fetal liver hematopoietic
progenitors derived from two independently targeted ES cell clones.
Taken together, these data suggest that expression of AML1-ETO during
the establishment of definitive hematopoiesis directly leads to the
generation of multipotential progenitors that have a high self-renewal
capacity and dysplastic maturation.
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| Fig 5.
Morphology and immunophenotype of AML1-ETO/+
hematopoietic cells. (A, left-to-right) Typical dysplastic multilineage
AML1-ETO/+ colony from passage 16 with prominent
hemaglobinization. Wright-Giemsa-stained cytocentrifuge preparation of
this colony showing erythroid, monocytic, and megakaryocytic elements.
Prominent alpha naphthyl butyrate cytochemical activity in a cluster of
myeloid and monocytic cells from within this colony. (B) Flow
cytometeric analysis of surface antigen expression in AML1-ETO/+
hematopoietic cells expanded in liquid cultures. Solid black line
represents staining obtained with antibodies specific for the indicated
hematopoietic antigen. Dashed line corresponds to the signal obtained
with an isotype matched control antibody.
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Murine adult hematopoietic cells transduced with AML1-ETO exhibit a
high self-renewal capacity and readily establish cell lines in vitro.
To further define the role of AML1-ETO in the growth abnormalities
observed in hematopoietic progenitors, we targeted its expression to
adult bone marrow-derived progenitors by retroviral infection (Fig
6). A human AML1-ETO cDNA was inserted into
the murine stem cell retroviral vector (MSCV), which contains a
neomycin resistance gene (neo) under the control of the
phosphoglycerate kinase promoter.36 This vector yields high
titer virus capable of efficiently transducing and expressing genes in
murine hematopoietic stem cells and their progeny and allows direct
selection of infected cells based on resistance to G418. In two
independent experiments bone marrow cells obtained from 5-fluorouracil
(5-FU)-treated mice were infected with helper-free retroviral
supernatants containing MSCV/AML1-ETOneo or MSCVneo.
Infected cells were then plated directly in G418-containing
methylcellulose medium under conditions optimal for the growth of
multilineage hematopoietic colonies (Fig 6A and B). Using viral
supernatants with titers of 1 to 5 × 105 G418-resistant
cfu/mL, between 5% and 10% of colony forming progenitors acquired G418 resistance. Although resistant granulocyte,
granulocyte-macrophage, macrophage, and mixed colonies were identified
in all cultures, fewer colonies were generated following infection with
MSCV/AML1-ETOneo. The cause of this difference is not
immediately apparent.
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| Fig 6.
The effects of AML1-ETO expression on bone
marrow-derived hematopoietic progenitors. (A and B)
Replating efficiencies in two independent experiments. The open
circles with red line depict the results of cell infected with
MSCVneo, whereas the solid squares with blue line represent the
results from cell infected with MSCV/AML1-ETOneo. (C) Flow
cytometric analysis of surface antigen expression on MSCV/AML1-ETOneo-infected hematopoietic progenitors derived
from the 13th replating. These cells were carried in liquid cultures for 3 weeks before immunophenotypic analysis. The solid line
represents the staining obtained with the antibody specific
for the indicated antigen. The dashed line represents the signal
obtained with an isotype matched control antibody. (D)
Wright-Giemsa-stained cytocentrifuge preparation of cells obtained
from the 13th replating. (E) Western blot analysis of cell lysates
prepared from MSCV/AML1-ETOneo-infected hematopoietic cells
using affinity purified antibodies raised against an AML1-N-terminal
peptide.
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Cells were obtained from primary cultures 8 to 10 days after plating
and replated in secondary methycellulose cultures in the presence of
G418. This process was serially repeated and the number and morphology
of colonies analyzed after each passage. As shown in Fig 6A and B, a
slight increase in the efficiency of colony formation was noted through
the first four passages for both MSCVneo- and
MSCV/AML1-ETOneo-infected cells. Following passage 4, however,
the number of colony forming progenitors rapidly declined in the
MSCVneo population, with no colonies detected following the 6th
passage. By contrast, MSCV/AML1-ETOneo cells continued to show
an increase in the efficiency of colony formation through subsequent
passages. In the first experiment (Fig 6A), MSCV/AML1-ETOneo-infected cells formed colonies at a high
efficiency through 22 generations (results from 13 passages shown).
Similarly, in a second more recent experiment that has to date been
carried through a shorter number of generations (Fig 6B),
MSCVneo cells failed to form colonies after the 5th replating,
whereas MSCV/AML1-ETOneo cells continued to efficiently form
colonies through 13 passages (data from 6 passages shown).
The replated colonies from MSCV/AML1-ETOneo cells were composed
primarily of immature blasts like cells that readily expanded into cell
lines in liquid cultures in the presence of IL-3, IL-6, and SCF. These
cells expressed c-kit but were negative for GR-1, Mac-1, Sca-1, and
Thy-1 (Fig 6C). In addition, the cells had the morphology of immature
hematopoietic cells with only rare mid-mature myeloid and monocytic
cells identified (Fig 6D). Western blot analysis of cell lysates from
MSCV/AML1-ETOneo-infected populations revealed AML1-ETO
expression (Fig 6E). Taken together, these data further show that
expression of AML1-ETO can directly lead to the generation of abnormal
hematopoietic progenitors with a high self-renewal capacity.
| |
DISCUSSION |
To directly investigate the mechanistic role of the t(8;21)-encoded
AML1-ETO chimeric product in leukemogenesis, we used gene targeting to
create mice with an AML1-ETO "knock-in" allele that mimics the t(8;21). We now show that murine embryos heterozygous for
the AML1-ETO knock-in allele have normal yolk sac-derived primitive
hematopoiesis but have a near total absence of fetal liver-derived
definitive hematopoiesis and die at embryonic day 13.5 from massive
bleeding in the CNS, pericardial sac, and soft tissue. This bleeding
diathesis appeared to result, at least in part, from the absence of
circulating platelets compounded by an evolving anemia due to the
inability to make definitive erythrocytes. Thus, the phenotype that
resulted from AML1-ETO expression was nearly identical to the embryonic
lethal phenotype seen following homozygous disruption of either
AML118,19 or
CBF.20,21 This similarity in
phenotypes suggests that AML1-ETO effectively neutralizes the normal
biologic activity of the AML1/CBF transcriptional factor complex and
thus supports published data that suggested that AML1-ETO represses
AML1-mediated transcriptional activity through a dominant-negative
mechanism.10,31-34
Despite the overall similarity between the phenotypes resulting from
the loss of AML1/CBF and expression of AML1-ETO, two significant
differences were observed. First, AML1-ETO-expressing embryos lived 1 day longer than AML1- or CBF-deficient embryos. Second, fetal livers
from AML1-ETO-expressing embryos contained rare hematopoietic
progenitors, whereas no progenitors were detected in the fetal livers
of AML1- or CBF-deficient embryos. The presence of these cells in
AML1-ETO/+ embryos could, in part, be responsible for the longer
survival of these embryos, given that these cells have the capacity to
differentiate into megakaryocytes and presumably functional platelets,
hematopoietic elements capable of providing protection from bleeding.
These progenitors, however, failed to establish a functional
hematopoietic system within the developing embryos. Despite this
apparent inability to expand in vivo, these cells readily grew in
cultures where they displayed significant morphologic dysplasia.
Moreover, these AML1-ETO-expressing cells had an abnormally high
self-renewal capacity, a property not seen in normal fetal
liver-derived progenitors but more typical of leukemic cells. The
identical hematopoietic abnormalities were observed in AML1-ETO/+
heterozygous mice derived from two independent ES cell lines,
suggesting that the phenotype was a direct result of AML1-ETO
expression. This was further confirmed by expressing AML1-ETO from a
retroviral LTR in adult bone marrow-derived hematopoietic progenitors. In these experiments, immature hematopoietic colony forming progenitors were generated that had a high self-renewal capacity, and again could be readily established into cell lines when
grown in liquid cultures in the presence of hematopoietic growth
factors. Taken together, these data suggest that AML1-ETO suppresses
normal AML1/CBF activity and leads to the direct generation of
signals that contribute to the initiation of aberrant hematopoietic cell proliferation.
Although our data do not directly address the mechanism by which
AML1-ETO mediates the expansion of abnormal hematopoietic progenitors,
several possibilities are suggested by our observations: (1) the
AML1-ETO chimeric protein may cause an incomplete repression of normal
AML1 transcriptional activity and thereby result in dysregulated
expression of target genes critical for hematopoiesis; (2) expression
of AML1-ETO may result in novel gain-of-function activities that alter
the transcription of genes normally regulated by AML1/CBF or other
runt homology domain-containing transcription factors; (3) novel
AML1-ETO-mediated activities may affect the expression of genes that
are not normally regulated by this class of transcription factors; or
(4) a combination of these effects. Irrespective of the underlying
mechanism, our data provide direct evidence that expression of AML1-ETO
contributes to the expansion of abnormal hematopoietic progenitors by
increasing the self-renewal capacity of multipotential hematopoietic
progenitors.
Somewhat surprising was the observation that the dysplastic
AML1-ETO/+ hematopoietic cells failed to expand to appreciable levels within the embryos. Similarly, in preliminary experiments the
AML1-ETO/+ cells fail to induce leukemia in sublethally
irradiated syngeneic or severe combined immunodeficiency disease (SCID)
mice (Downing and Cai, unpublished data, March 1998).
Despite this lack of growth in vivo, these cells readily expanded in
vitro when cultured in the presence of IL-3, IL-6, and SCF. Moreover, the cells are growth factor-dependent both for survival and
proliferation. One possible explanation for these results is that
aberrant expression of AML1-ETO may lead to an altered sensitivity to
growth factor-induced proliferation or differentiation, with high
growth factor concentrations such as those used for in vitro cultures
required for the expansion of the abnormal AML1-ETO progenitors. This
possibility is strengthened by the known role that AML1/CBF plays in
the transcriptional regulation of growth factors such as GM-CSF and
IL-313,14 and growth factor receptors such as
CSF-1R.15 The role of growth factor signaling pathways in
the expansion of this abnormal hematopoietic population remains to be
determined.
Although expression of AML1-ETO resulted in an abnormally high
self-renewal capacity in both fetal liver- and adult bone
marrow-derived hematopoietic progenitors, the phenotypes of the
expanding cells differed between these populations. Immature
myelomonocytic cells were obtained following expression of AML1-ETO in
fetal liver-derived progenitors, whereas more undifferentiated
hematopoietic progenitors were obtained following expression of
AML1-ETO in bone marrow-derived progenitors. These differences may
result from expression of AML1-ETO in distinct stem or progenitor cell
populations. Alternatively, the difference may result from subtle
differences in the pattern of expression of AML1-ETO in the expanding
hematopoietic population that result from the different promoters used
to drive its expression. Nevertheless, despite the observed phenotypic
differences both populations showed some evidence of terminal
differentiation. Significantly, in the fetal liver-derived cells,
terminal hematopoietic differentiation was observed along the myeloid,
monocytic, erythroid, and megakaryocytic lineages. Thus, these data
clearly show that although AML1-ETO expression leads to abnormal
proliferation and maturation, it does not block terminal hematopoietic
differentiation.
A number of investigators have recently used knock-in approaches to
replace one gene with another40 or to create chimeric oncogenes.41-43 Yergeau and colleagues43
generated mice heterozygous for an AML1-ETO knock-in allele by using a
strategy that was slightly different from ours. Their data also showed
that expression of AML1-ETO resulted in the death of embryos at
midgestation from CNS hemorrhages and a severe impairment of fetal
liver-derived hematopoiesis. In contrast to our results, however, they
did not observe dysplastic hematopoietic progenitors within the fetal liver but instead identified rare apparently normal macrophage colonies
in yolk sac cultures. Several differences exist between the targeting
strategies used by the two groups, including the exon targeted and the
transcriptional orientation of the neomycin resistance gene in
relationship to AML1. These differences could result in significant
changes in the level of expression of the chimeric gene and thus may be
responsible for the phenotypic differences observed. Although the level
of AML1-ETO expression was not documented in Yergeau studies, using our
strategy we consistently observed levels of AML1-ETO that were similar
to that of endogenous AML1. Moreover, easily detectable levels of
AML1-ETO were obtained following retroviral mediated expression in bone
marrow-derived progenitors, and again this level of expression
resulted in an increase in the self-renewal capacity and in vitro
immortilization of these cells.
In summary, we have presented data that suggest that expression of
AML1-ETO results in both the neutralization of normal
AML1/CBF-mediated activities required for definitive hematopoiesis
and the direct generation of signals that result in the expansion of
dysplastic hematopoietic progenitors. These AML1-ETO-expressing
progenitors are likely to represent the immediate precursors to full
blown leukemic populations. An investigation of the growth properties and leukemic potential of the AML1-ETO/+ cells derived through these
experiments should provide valuable insights into the pathways through
which AML1-ETO contributes to human acute myeloid leukemia.
| |
FOOTNOTES |
Submitted December 3, 1997;
accepted February 12, 1998.
Supported by National Instituted of Health (NIH) Grant P01 CA71907-01,
NIH Cancer Center CORE Grant CA-21765, and the American Lebanese and
Syrian Associated Charities of St Jude Children's Research Hospital.
Address reprint requests to James R. Downing, MD, Department of
Pathology and Laboratory Medicine, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, TN 38105.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank W. Paul Conn, W. Kent Williams, Cristy Nagy, and John
Swift for excellent technical assistance and Drs Scott Hiebert, Darin
O'Brien, and Gerard Grosveld for helpful discussions and critical
reading of the manuscript.
| |
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Abstract
Introduction
Abstract