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Review in translational hematology
Recent insights into the mechanisms of myeloid leukemogenesis
in Down syndrome
Sandeep Gurbuxani, Paresh Vyas, and John D. Crispino
GATA-1 is the founding member of a
transcription factor family that regulates
growth and maturation of a diverse set of
tissues. GATA-1 is expressed primarily in
hematopoietic cells and is essential for
proper development of erythroid cells,
megakaryocytes, eosinophils, and mast
cells. Although loss of GATA-1 leads to
differentiation arrest and apoptosis of
erythroid progenitors, absence of GATA-1
promotes accumulation of immature
megakaryocytes. Recently, we and others
have reported that mutagenesis of GATA1
is an early event in Down syndrome (DS)
leukemogenesis. Acquired mutations in
GATA1 were detected in the vast majority
of patients with acute megakaryoblastic
leukemia (DS-AMKL) and in nearly every
patient with transient myeloproliferative
disorder (TMD), a “preleukemia” that may
be present in as many as 10% of infants
with DS. Although the precise pathway by
which mutagenesis of GATA1 contributes
to leukemia is unknown, these findings
confirm that GATA1 plays an important
role in both normal and malignant hema-
topoiesis. Future studies to define the
mechanism that results in the high frequency of GATA1 mutations in DS and the
role of altered GATA1 in TMD and DSAMKL will shed light on the multistep
pathway in human leukemia and may lead
to an increased understanding of why
children with DS are markedly predisposed to leukemia. (Blood. 2004;103:
399-406)
© 2004 by The American Society of Hematology
Introduction
The incidence of leukemia in children with Down syndrome (DS)
is 1 in 100 to 200, which is 10- to 20-fold higher than in children
without DS. Although in non-DS children lymphoid neoplasms
comprise most of the leukemias arising in this age group, half of the
leukemias in DS are myeloid. Children with DS exhibit a 46-fold
excess incidence of acute myeloid leukemia (AML), with acute
megakaryoblastic leukemia (AMKL) accounting for at least 50%
of these cases.1 In addition to acute leukemia, children with DS are
also predisposed to developing the related transient myeloproliferative disorder (TMD). At least 10% of infants with DS develop
TMD, a disease in which immature megakaryoblasts accumulate in
liver, bone marrow, and peripheral blood; this disorder undergoes
spontaneous remission in most cases.2-4 Of note, approximately
30% of DS infants with TMD develop AMKL within 3 years.1
TMD blasts are morphologically indistinguishable from AMKL
blasts, contributing to the hypothesis that the second disease is
derived from the first.5,6 Often the karyotype of the AMKL blasts is
more complex than that of the TMD, but contains abnormalities
observed in the original TMD clone, consistent with clonal
evolution.7-11 Classified as FAB M7 subtype, these blasts exhibit
classical ␣-naphthyl acetate esterase activity with a multifocal
punctate cytoplasmic staining pattern that is only partially inhibited
by sodium fluoride.12 Immunophenotyping shows that the blasts
express the myeloid markers CD33 and CD13, in addition to at
least one platelet-associated antigen (CD36, CD41a, CD41b, or
CD61). A proportion of these blasts also express erythroid-specific
mRNAs, such as ␥-globin and erythroid ␦-aminolevulinate synthase.13 Taken together, these observations are consistent with the
From the Ben May Institute for Cancer Research, University of Chicago,
Chicago, IL; Department of Haematology, University of Oxford, Weatherall
Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, United
Kingdom.
Submitted May 15, 2003; accepted September 12, 2003. Prepublished online
as Blood First Edition Paper, September 25, 2003; DOI 10.1182/blood-200305-1556.
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
theory that TMD and AMKL arise from an immature megakaryocytic or a common erythroid megakaryocytic precursor.13
The role of chromosome 21 in DS leukemia
Several models have been proposed to explain the increased
incidence of AMKL and TMD in DS. These include gene dosage,
mutations of genes on chromosome 21, genetic imprinting leading
to homozygosity, and altered folate metabolism. The most popular
of these is increased gene dosage of a leukemia predisposition gene
or hematopoiesis regulatory gene residing on chromosome 21.14-17
This model is supported by the observation that acquisition of one
or more additional chromosome 21 homologs is a common
numerical abnormality in acute leukemia.18-20 Even though there
appears to be no direct correlation between the biologic features of
AMKL and TMD and known function of genes identified on
chromosome 21, it is likely that one or more of these genes are
involved. Alternatively, overexpression of a gene present on
chromosome 21 may alter expression of genes on other chromosomes that subsequently affect hematopoiesis. For example, the
gene encoding cystathionine ␤-synthase (CBS), located on human
21q22.3 (Figure 1), is expressed at a level nearly 12.5-fold greater
in a DS AMKL cell line compared to a similar non-DS AMKL cell
line.21 The increased activity of CBS in DS results in significantly
lower plasma homocysteine, methionine, S-adenosylmethionine
(AdoMet), and S-adenosylhomocysteine (AdoHyc) levels in DS
individuals compared with a non-DS control group.22 Alterations in
Supported in part by a Junior Faculty Award from the American Society of
Hematology (J.D.C.) and by a grant from the National Institutes of Health
(National Cancer Institute).
Reprints: John D. Crispino, University of Chicago, 924 E 57th St, Rm R116,
Chicago, IL 60637; e-mail: [email protected].
© 2004 by The American Society of Hematology
399
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400
GURBUXANI et al
Figure 1. A subset of genes located on chromosome 21. The distal region of
chromosome 21 present in 3 copies in cells from children with DS contains several
putative leukemogenic genes including RUNX1. In addition, the region 21q22.3
contains the gene that encodes the enzyme cystathionine ␤-synthase (CBS), which is
involved in the metabolism of cytosine arabinoside and may be responsible for
increased sensitivity of DS AMKL blasts to this compound.91
AdoMet and AdoHyc levels may result in altered methylation of
CpG islands, thereby affecting gene expression. In addition, low
homocysteine levels perturb folate metabolism by trapping
5-methyl-tetrahydrofolate resulting in a “functional” folate deficiency. This may result in increased rates of DNA mutation,23
predisposing these children to leukemia.24 Interestingly, it may be
that the greater sensitivity to chemotherapy shown by patients with
DS-AMKL results from the overexpression of CBS.21
Perhaps the most interesting candidate leukemia-predisposing
gene on chromosome 21 is RUNX1(AML1). The transcription
factor RUNX1 is required for generation of all definitive hematopoietic lineages.25 RUNX1 gene rearrangements are involved in
some of the recurring translocations associated with leukemia;
these include t(8;21) in acute myeloid leukemia (AML M2), t(3;21)
in chronic myeloid leukemia (CML) in blast crisis, and t(12;21) in
childhood acute lymphoblastic leukemia (ALL).26-28 Mutations in
the DNA-binding Runt domain of RUNX1 result in a familial
platelet disorder with predisposition to AML,29,30 and are present in
about 10% of sporadic cases of de novo AML31,32 and in myeloid
malignancies with acquired trisomy 21.32 Mutations in the RUNX1
Runt domain were not detected in samples from individuals with
DS-AMKL, arguing that RUNX1 mutations are not involved in the
etiology of AMKL.32-34 However, this finding does not exclude a
pathogenic role for increased gene dosage of RUNX1 in the
development of AMKL. Other putative leukemic oncogenes on
chromosome 21 include the interferon ␣/␤ receptor (IFNAR),
cytokine family 2-4 (CFR 2-4), phosphoribosylglycinamide formyltransferase (GART), and the gene with significant homology to
MYC and MOS (SON).4 Several of the interesting genes that reside
on chromosome 21 in the DS critical region are depicted in Figure 1.
Another model for why DS children are predisposed to leukemia is based on genetic mapping studies that suggest an increased
frequency of disomic homozygosity in DS individuals with TMD
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
and AMKL.35 Nondisjunction of chromosome 21 may result in
disomic homozygosity of mutated tumor suppressor genes.36 For
this hypothesis to be true, it is essential that the DS child inherit 2
copies of the same chromosome with a mutated allele. This is
possible in the 25% of DS cases where the disjunction occurs at
meiosis II. To date, some evidence suggests that in DS-AMKL and
TMDs there is a high prevalence of meiosis II errors and increased
disomic homozygosity in the pericentromeric region encompassing
the most proximal 10% of 21q.14,35-37 However, there is very little
evidence to support the silencing of the normal allele on the third
chromosome 21 by genetic imprinting. This model also requires the
unlikely possibility of a mutated tumor suppressor gene to be
present in the general population at high frequency.
The involvement of chromosome 21 in DS leukemogenesis
might be more complex than simple gene dosage or disomic
homozygosity of a mutated allele. Molecular deletions in the long
arm of one chromosome 21 homolog have been reported in
leukemic cells from 5 acute leukemia patients (4 AMKL, 1 ALL).
Subsequently, cryptic deletions and complex rearrangements involving the same chromosome 21 segments have been reported in a
non-DS infant with TMD (with acquired trisomy 2138). Interestingly, segments involved in these deletions included the RUNX1
locus 21q22.1-22.2 in the ALL and 3 of 4 AMKL patients.39 A final
hypothesis is that, in addition to a role for genes located on
chromosome 21, factors responsible for nondisjunction of chromosome 21 in DS may predispose to a state of chromosomal
instability. A significant increase in polymorphisms of the enzyme
methylenetetrahydrofolate reductase (MTHFR) has been shown in
mothers of individuals with DS.23 These polymorphisms lead to
reduced enzyme activity and altered folate metabolism and cellular
methylation reactions.40 This is of interest because a recent study
demonstrated folate supplementation in pregnant mothers conferred protection from ALL in childhood.41
Regardless of the mechanism, trisomy 21 certainly plays a role
as a potent first hit in the leukemic process. TMD, characterized by
abnormal proliferation of blasts and a unique association with DS,
is often present at the time of birth or in the early neonatal period.
This observation, coupled with the fact that acute leukemias in DS
occur relatively early, with the highest risk at ages younger than 4
years, suggests that either the secondary mutations needed to fuel
abnormal myelopoiesis and acute leukemia are few in number, or
that trisomy 21 predisposes to a state of genetic instability resulting
in rapid acquisition of other mutations.
GATA-1 in normal and abnormal
hematopoiesis
The development of blood cells of various lineages from hematopoietic stem cells is controlled by cell-restricted transcription
factors (for a review, see Cantor and Orkin42). Among these, several
members of the GATA family play key roles in specification and
maturation of a subset of hematopoietic lineages. For example,
GATA-1 is essential for the maturation of erythroid cells and
megakaryocytes (Figure 2).42 GATA-2 participates in the maintenance and proliferation of hematopoietic progenitor cells as well as
in the specification of mast cells.43 GATA-2 can also substitute for
absence of GATA-1 in early megakaryocyte specification.44
GATA-3, on the other hand, is required for T-cell development and
regulating the decision between Th1 and Th2 cell development.45-47
The most extensively characterized member of the GATA
family of transcription factors, GATA-1, is an X chromosome–
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
GATA1 MUTATIONS IN DS MYELOID LEUKEMIA
Figure 2. GATA factors in hematopoiesis. Schematic representation of the
requirements for GATA-1 and its cofactor FOG-1 during lineage specification and
maturation of erythroid cells and megakaryocytes. Vertical bars represent maturation
arrest observed in the absence of GATA-1 or FOG-1, as determined by murine gene
targeting experiments. GATA-1 is required for differentiation of megakaryocytes and
erythrocytes, as well as for mast cells and eosinophils.
encoded gene that is expressed primarily in erythroid cells,
megakaryocytes, mast cells, and eosinophils, cell types that express
a large number of genes that harbor GATA DNA-binding motifs.
GATA-1 has 3 well-studied functional domains: an N-terminal
transactivation domain and 2 zinc fingers. The C-terminal zinc
finger is required for binding of GATA-1 to DNA, whereas the
N-terminal zinc finger stabilizes binding to a subset of sites, termed
palindromic motifs.48 In addition to binding DNA, the N-finger
plays an important role by recruiting a cofactor named friend of
GATA-1 (FOG-149). Both zinc fingers of GATA-1 are essential for
normal activity, but the role of the N-terminal activation domain is
less clear. This region was initially defined in transient reporter
assays in fibroblasts.50 Results of several studies, however, have
suggested this domain is not required for GATA-1 function. First,
GATA-1 molecules that lack the N-terminal activation domain
rescued the differentiation of a GATA-1–deficient erythroid cell
line.51 Second, the activation domain was dispensable in the
conversion of an early myeloid cell line, 416B, to megakaryocytes.52 Finally, mice engineered to express only a variant of
GATA-1 that lacks the N-terminal transactivation domain were
born and appeared healthy.53 We will return to the question of
whether this activation domain plays an essential role in
hematopoiesis.
401
Various lines of mice with an alteration in Gata1 have been
developed (Table 1 presents a complete list and details of Gata1altered mice). Initial studies with mice that lack GATA-1 expression in all cells (GATA-1 null mice) revealed that GATA-1 is
essential for the proper development of erythroid cells.54 GATA-1
null mice die of anemia at embryonic day 10.5 (E10.5). Subsequently, 3 other gene-targeted mouse strains relevant for this
review have been generated that harbor deletions within the
cis-regulatory elements that regulate GATA-1. In GATA-1.05 mice,
deletion of sequences in the promoter of GATA-1 reduce GATA-1
expression to 5% of normal in all cells.55 This is sufficient to block
erythropoiesis, and as a consequence, both female homozygous
mutant mice and male hemizygous mutant mice die in utero at
E12.5. Female heterozygous mice survive but develop a myelodysplastic syndrome and die prematurely at about 5 months of age.55 In
the other 2 strains of mice, sequences containing an enhancer
located 3.4 kb upstream of the GATA-1 were deleted (GATA-1
knock-down and GATA-1low mice).56,57 In these 2 strains of mice
GATA-1 is virtually absent from megakaryocytes. Studies with
these mice have shown that GATA-1 is required for the proper
maturation of megakaryocytes. In the absence of GATA-1,
megakaryocytes proliferate excessively and fail to generate platelets.56,58 Furthermore, abnormal megakaryocytes accumulate in the
spleen and bone marrow of these GATA-1–deficient mice, resulting
in anemia and thrombocytopenia.56 Analysis of the GATA-1low
mice shows they develop extramedullary hematopoiesis in the liver
and myelofibrosis of the bone marrow and mice have been
proposed as a model for human myelofibrosis.59,60 Finally, more
recent studies with Gata1 gene–targeted mice have also revealed an
essential role for GATA-1 in the development of 2 other hematopoietic lineages: eosinophils61,62 and mast cells.63
The existence of mice that express reduced levels of GATA-1
has facilitated a more detailed structure-function study of GATA-1
domains in vivo.53 Mice harboring wild-type and mutant GATA-1
transgenes were bred to the GATA-1.05 mutant mice. Although the
male GATA-1.05 mutant mice die in midgestation of anemia,
GATA-1.05 mice that harbored a wild-type GATA-1 transgene
were born at the expected frequency and were healthy. As expected,
GATA-1 transgenes that lacked either zinc finger failed to rescue
Table 1. Phenotypes of the various Gata1 gene-targeted mice
Name of line
Effect on GATA-1
Phenotype
GATA-1 null
No GATA-1 expression in any tissue
Midgestation lethality due to anemia (E10.5)
GATA-1 knock-down
Wild-type levels of GATA-1 in
Thrombocytopenia with a significant increase in megakaryocyte numbers in
(⌬neo⌬HS)
erythroid cells; no detectable
Mouse/human pathology
the spleen and bone marrow
expression in megakaryocytes
GATA-1low (neo⌬HS)
Erythroid expression decreased
Most die during embryogenesis. About 5% of the hemizygous males are
5-fold; megakaryocytic expression
born anemic and thrombocytopenic. The few animals that survive
not detectable
recover from anemia but remain thrombocytopenic. Surviving mice
Myelofibrosis
develop anemia with extramedullary hematopoiesis and spleen and bone
marrow fibrosis around 15 mo. Mice also exhibit defects in mast cell
maturation.
GATA-1.05
Erythroid expression decreased
20-fold; megakaryocytic
Hemizygous males die by E12.5 of anemia. Heterozygous females exhibit
Myelodysplastic syndrome
anemia and thrombocytopenia and begin to die by 5 mo.
expression not detectable
GATA-1 Val205Gly
GATA-1 is expressed normally, but
Hemizygous males die of anemia by E12.5.
cannot interact with its essential
anemia and
cofactor FOG-1
⌬dblGATA
Insufficient expression in eosinophil
GATA-1 AMKL
Express GATA-1s in lieu of full
Congenital dyserythropoietic
thrombocytopenia
Selective loss of the eosinophil lineage
precursors*
mutant
To be determined
length GATA-1
*Predicted effect of targeting of the palindromic GATA-1 site within the GATA-1 promoter.62
TMD and/or DS-AMKL (?)
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402
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
GURBUXANI et al
the embryonic lethality of GATA-1.05 mice. Surprisingly, however,
mice that expressed a GATA-1 transgene without the N-terminal
activation domain also survived, but only when the transgene was
expressed at high levels.53 These findings suggest the N-terminal
activation domain does have a critical function in blood cell
development.
Obviously GATA-1 plays important roles in normal hematopoiesis, but do alterations in GATA-1 activity participate in human
blood disorders? Earlier studies have shown this to be the case.
Inherited mutations in the N-terminal zinc finger domain of
GATA-1, which disrupt the interaction between GATA-1 and
FOG-1, cause congenital dyserythropoietic anemia and thrombocytopenia.64-66 Also, mutations in GATA-1 that interfere with normal
DNA binding by the N-finger of GATA-1 have been associated
with dyserythropoiesis and thrombocytopenia in humans.67 Similarly, mice that harbor one of the mutations in GATA-1 seen in
human patients that disrupts binding to FOG-1 (Val205Gly mutants) suffer from a phenotype (anemia and thrombocytopenia) that
mimics the human disease (Table 1).44
Given that numerous missense mutations in GATA1 cause
defective hematopoiesis, it seemed likely that distinct mutations in
GATA1 might result in other hematopoietic diseases, including
myelodysplastic syndromes and AMLs. Recently this has been
borne out because a number of groups have shown mutations in
GATA1 in one form of AML, the megakaryoblastic leukemia
associated with DS.33,68-71 To date, GATA1 mutations have been
detected in a total of 43 of 51 AMKL patients (Table 2).
Importantly, mutations in GATA1 were not detected in leukemic
cells of DS patients with other types of acute leukemia, or in other
patients with AMKL who did not have DS.33 Furthermore, GATA1
mutations were not detected in DNA from other patients with other
subtypes AML or healthy individuals. Finally, these studies demonstrated that these mutations were somatically acquired because
remission samples did not harbor GATA1 mutations. Based on these
observations, it appears that disruption of normal GATA-1 function
is an important, if not essential, step in the pathogenesis of AMKL.
To determine if GATA1 mutations in the presence of trisomy 21
are an early event needed for the proliferative process common to
both TMD and AMKL, or are involved in the transformation of
“benign” TMD to the “malignant” AMKL, several groups have
investigated whether GATA1 mutations were present in TMD
patients. GATA1 was found to be mutated in 66 of 68 TMD patients
examined (Table 2),68-72 suggesting that GATA1 mutagenesis is an
early event in DS myeloid leukemogenesis that contributes to both
TMD and AMKL.
Timing, frequency, and natural history
of GATA1 mutations in DS
These described findings raise important questions about the
timing, frequency, and natural history of GATA1 mutations in DS.
The importance of determining when mutations arise is that if
mutations occur during a restricted temporal window, for example,
exclusively in fetal life, this would suggest that mutations only
occur in a defined hematopoietic developmental context in trisomy
21 cells (see “TMD and AMKL in DS: a model for multiple hits in
childhood leukemia”). Clearly, because TMD presents in the
neonatal period, GATA1 mutations in this case almost certainly
occur in utero.68-70,72 However, what of those cases of AMKL that
are not preceded by TMD? In one informative case identical twins
Table 2. Mutations in TMD and DS-AMKL
Nature of mutations
No. of patients
Consequence
AMKL
Insertions
16
Premature stop
Deletions
14
Premature stop
Insertion ⫹ deletion
2
Premature stop
Missense
3
No translation from Met1
Nonsense
4
Premature stop
Splice
4
Alternate splicing
Polymorphism/normal
2
Indeterminate
6
TMD
Insertions
23
Premature stop
Deletions
24
Premature stop
Insertion ⫹ deletion
1
Premature stop
Missense
1
No translation from Met1
Nonsense
12
Splice
5
Polymorphism/normal
2
Premature stop
Alternate splicing
with an acquired trisomy 21 were shown to have precisely the same
GATA1 mutations in leukemic cells leading the authors to speculate
that a GATA1 mutation must have arisen in a hematopoietic
progenitor with acquired trisomy 21 in one of the twins in fetal life
and was then transferred to the other fetus via a shared embryonic
circulation.70 More generally, the question has been recently
addressed by analysis of neonatal blood spots of 4 patients with
AMKL who did not have clinically overt antecedent TMD phase.
GATA1 mutations were present at birth in 3 of 4 of these patients
who went on to develop AMKL 12 to 26 months later.73 In the
fourth patient failure to detect GATA1 mutation in neonatal blood
spots (where there are on average 30 000 nucleated cells) may
reflect the rarity of a clone containing a GATA1 mutation rather
than its absence. These data would support the contention that most
(if not all cases) GATA1 mutations arise in utero.
In an extension of this observation, GATA1 mutations were also
detected in neonatal blood spots from 2 of 21 randomly selected
otherwise normal DS neonates.73 Importantly, no GATA-1 mutations were detected in 62 non-DS cord blood samples. The presence
of GATA1 mutations in 2 of 21 otherwise healthy DS neonates
could suggest the presence of clinically silent megakaryoblast
proliferation in these infants. Alternatively, acquisition of GATA1
mutation per se in a fetal trisomy 21 hematopoietic progenitor can
give rise to clinically overt TMD only if the mutation occurs early
enough in gestation to produce a large clone at birth.
The question of the frequency of GATA1 mutations in DS
neonates and children is also unclear. Because GATA1 mutations
can be clinically silent at birth, the overall frequency at which
mutations occur is almost certainly higher frequency than that of
TMD (about 10% of all DS neonates). Moreover, we have recently
observed multiple independent GATA1 mutant clones in 4 of 12 DS
AMKL patients,73 suggesting that GATA1 mutations are present at a
remarkably high frequency in DS.
Nature of GATA1 mutations in DS leukemia
The majority of the reported GATA1 mutations involved small
deletions or insertions in sequences encoding exon 2 of GATA1
(Figure 3; Table 2). Each of these alterations resulted in a
disruption of the normal reading frame of GATA1 and an invariable
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
Figure 3. GATA1 is mutated in TMD and AMKL. Positions of the various alterations
in GATA1. All of the mutations are located within exon 2 or the proximal nucleotides of
the downstream intron. Translation that initiates at Met1 produces the 50-kDa
full-length protein, whereas use of the alternate initiation codon at Met84 generates a
shorter 40-kDa isoform.
introduction of a premature stop codon. As a consequence of these
changes, full-length GATA-1 is not expressed in the leukemic cells.
Surprisingly, however, a short isoform of GATA-1, previously
described as GATA-1s,74 is expressed in both TMD and AMKL
blasts33,71 (S.G. and J.D.C., unpublished observations, December
2002). GATA-1s is likely to arise by alternate translation from
Met84, downstream of each of the patient mutations (Figure 3).
GATA-1s lacks the N-terminal activation domain, but retains both
zinc fingers and the entire C-terminus.50 GATA-1s binds DNA
efficiently in vitro, dissociates from DNA at a rate similar to
full-length GATA-1, and interacts with the FOG-1 cofactor to the
same extent as wild-type GATA-1.33 However, due to the absence
of the N-terminal activation domain, the shorter variant has
reduced transactivation potential.33,53,74
Alternative splicing of the GATA-1 mRNA may provide
another mechanism for production of the short isoform of GATA-1,
because a subset of patients with TMD or AMKL harbored
mutations in GATA1 that disrupt mRNA splicing70 (Figure 3). In
analyzing the mRNA from leukemic cells of 3 patients who
harbored exon 2 splice site mutations, Rainis and colleagues could
only detect the presence of a shorter mRNA that lacked exon 2.70 In
contrast, both the wild-type mRNA and the short mRNA lacking
exon 2 were detected in leukemic samples without splice site
mutations as well as in normal bone marrow. Together, these
observations suggest that wild-type GATA1 produces 2 mRNAs,
which may account for the production of the 2 protein isoforms,
and that alternative splicing may provide the template for subsequent expression of the GATA-1s isoform in the majority of
leukemic patients.
How do the mutations in GATA1
promote leukemia?
Calligaris et al initially proposed that GATA-1s is a normal isoform
of GATA-1 because its expression was observed in a variety of
human cell lines as well as in mouse fetal liver.74 Consistent with
this, we have detected both full-length and GATA-1s in human
CD34⫹ cells cultured to differentiate along the erythroid lineage
(A. Muntean and J.D.C., unpublished observations, March 2003),
as well as in a variety of human hematopoietic cell lines.33 What
possible role could GATA-1s play during blood development?
Because GATA-1s can bind DNA and interact with FOG-1, but
exhibits a reduced transactivation potential, it may act as a
dominant-negative protein. During normal hematopoiesis, when
both GATA-1 and GATA-1s are translated, GATA-1s may downmodulate GATA-1 function. In DS-AMKL blasts, however, only
GATA1 MUTATIONS IN DS MYELOID LEUKEMIA
403
the GATA-1s isoform is produced (because GATA1 is encoded on
the X chromosome in leukemic blasts only the mutant allele is
active and exclusively produces GATA-1s33). Hence, during leukemogenesis GATA-1s may act by repressing the activity of a related
GATA protein, possibly GATA-2. GATA-2 is expressed in hematopoietic progenitors and can substitute for GATA-1 during early
megakaryocyte specification.44 There is at least one other example
in which frame shift mutations in a transcription factor result in
production of a dominant-negative isoform: mutations in the
CEBPA gene have been identified in a subset of AMLs.75 Similar to
GATA1 mutations, mutations in CEBPA result in the introduction of
premature stop codons that block expression of full-length protein,
but allow expression of a protein that is initiated downstream of the
stop codon. These shortened isoforms act as dominant-negative
proteins that interfere with the normal function of full-length
C/EBP-␣ and promote malignancy in the heterozygous state.
Although previous studies have suggested that the N-terminal
activation domain of GATA-1 is dispensable, none of these studies
specifically addressed the requirement for the N-terminus in
megakaryocyte growth. As such, there may be an essential function
for the N-terminus in megakaryocytes. Consistent with this hypothesis, a recent report has shown that GATA-1 physically interacts
with RUNX1 during megakaryocytic differentiation. In this study,
both the N-terminus (amino acids 1-85) and the C-terminus of
GATA-1 (amino acids 319-413) were necessary for the interaction.76 Thus, the short isoform, which is deficient for binding
RUNX1, may have an altered activity in leukemic blasts that could
contribute to the abnormal growth of megakaryoblasts.
Surprisingly, however, recent studies have demonstrated that
expression of high levels of GATA-1s can compensate in vivo for
the absence of full-length GATA-1 in erythroid cells of GATA-1
1.05 mice.53 These rescued mice, which express GATA-1s in the
absence of full-length GATA-1, do not exhibit any hematologic
deficiencies, indicating that the shortened form of GATA-1 can
substitute for full-length GATA-1, when substantially overexpressed. Thus, although we cannot discriminate between a quantitative or qualitative deficiency of GATA1 in DS malignancies, the
finding of Shimizu et al53 favors the model that, although the
leukemic cells produce GATA-1s, the levels are insufficient to
promote proper development. The fact that every single GATA1
mutation identified to date is predicted to abolish expression of
full-length GATA-1, while allowing for expression of GATA-1s,
strongly argues that expression of this short isoform is a key
component of leukemogenesis. Perhaps instead of playing an
oncogenic role, the short isoform may alternatively be required for
survival of mutant megakaryoblasts.70 Interestingly, Xu et al
recently demonstrated that expression of the full-length GATA-1,
but not the GATA-1s, can to some extent, rescue differentiation in
an AMKL-derived cell line.71
GATA1 mutations establish the clonal nature
of TMD and AMKL blasts
It has been suggested previously that TMD and AMKL form 2 ends
of an evolving malignant process, and that AMKL arises from the
initial TMD clone after acquiring additional abnormalities.12,77-80 In
a recent study on the incidence of GATA1 mutations in TMD and
AMKL, TMD resolved spontaneously in a majority of the patients.
Eighteen percent of patients (3 of 17), however, developed AMKL
with a median follow-up of 30 months. Identical GATA1 mutations
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404
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
GURBUXANI et al
were present in both the TMD and AMKL blasts from 2 of these
patients for whom sequential samples were available.70 The
presence of an identical GATA1 mutation has also been reported by
Hitzler and colleagues, in a patient for whom samples were
available from TMD and AMKL.68 In all of these cases, samples
from the same patients during clinical remission did not have
GATA1 mutations.
TMD and AMKL in DS: a model for multiple
hits in childhood leukemia
Although all DS children have trisomy 21, only 10% develop
TMD, and even fewer, less than 1%, develop acute leukemia. These
observations support the model that DS leukemogenesis is a
multistep process in which progenitor cells acquire multiple
genetic lesions during the progression to acute leukemia (Figure 4).
Trisomy 21 is the first event but is not sufficient for the expansion
of megakaryoblasts seen in TMD. The recent discovery that
mutations in GATA1 occur in nearly every individual with either
TMD or AMKL33,68,70,72 suggests that disruption of wild-type
GATA-1 activity is an essential second step in the development of
leukemia in DS. We hypothesize that mutagenesis of GATA1, in
conjunction with trisomy 21, is sufficient to promote the transient
expansion of immature megakaryoblasts seen in TMD and that
additional mutations are involved in the transformation to acute
leukemia. This model of multiple hits is consistent with accumulating evidence suggesting that at least 2 cooperating mutations, one
that promotes enhanced proliferation and a second that disrupts
differentiation, are necessary for the development of AML.81
Regardless of the mechanism of production or action of GATA-1s,
it is likely that the GATA1 mutations contribute to leukemia by
providing a block in megakaryocytic differentiation. GATA1 mutations in megakaryoblastic leukemia may act in a manner analogous
to those in CEPBA in AML, whereas the increased dosage of genes
on chromosome 21 likely impart a survival advantage to progenitors, in a manner similar to the activating mutations in FLT3 found
in other AMLs.82
Even though the presence of trisomy 21 and GATA1 mutations
appears to be adequate for the excessive proliferation of megakaryoblasts seen in TMD, it appears to be insufficient for leukemogenesis
because the majority of TMDs resolve spontaneously. The sponta-
neous disappearance of these immature cells shortly after birth
suggests that a specific environment might be essential for the
proliferation of these cells. An attractive hypothesis to explain this
has been the suggestion that TMD arises in the fetal liver (for a
review, see Gamis and Hilden4). The spontaneous regression of
TMD shortly after birth could then be explained by the loss of a
permissive fetal hematopoietic environment. Interestingly, oncostatin M, which cooperates with glucocorticoids to induce hepatic
differentiation and termination of fetal liver hematopoiesis,83,84 is
also an inducer of myeloid differentiation in vitro.85
Based on the data now available from the recent studies on
TMD and AMKL in DS, we propose a model of leukemogenesis in
which trisomy 21 cooperates with a nonfunctional/dysfunctional
GATA-1 transcription factor to specifically target a megakaryocytic
or common erythroid-megakaryocytic precursors resulting in excessive proliferation. These cells are not frankly leukemic and likely
need a specific milieu to proliferate (eg, fetal liver). However, these
excessively proliferating immature cells are prone to acquire
additional lesions, which, in turn, result in overt leukemia. These
additional lesions could be mutations in p53,86 altered telomerase
activity,87 or additional acquired karyotypic abnormalities, trisomy
8 being the most common in DS-AMKL.70,88,89 Additional studies
investigating mutations in genes commonly involved in AML, such
as FLT3 and KRAS1/NRAS,90 in sequential samples from TMD
and AMKL patients are warranted and will provide important
insights into the mechanism of leukemogenesis in DS as well as
possible tools for identifying TMD patients at a high risk of
developing AMKL.
Summary
Although GATA1 normally promotes the development of
megakaryocytes, erythroid cells, mast cells, and eosinophils,
mutagenesis of GATA1 is an early event in megakaryoblastic
leukemia. In retrospect, perhaps, the observation that mutations in
GATA1 interfere with the normal development of megakaryocytes
is not surprising; however, what is very surprising is that these
mutations are only found in those individuals with DS. Even more
intriguing is the finding that these mutations have been identified in
nearly every patient, and both in TMD and AMKL. Despite this
recent advance, many questions regarding leukemia in children
Figure 4. Model for the progression of TMD and AMKL in DS.
Trisomy 21 likely represents the initiating event in leukemogenesis of
DS because it occurs very early in embryogenesis. The subsequent
mutagenesis of GATA1 and selection of progenitor cells that harbor
GATA1 mutations may be the second initiating event that leads to the
neonatal appearance of TMD in at least 10% of infants with DS. In the
majority of infants with TMD, the GATA1 mutant clones disappear
during remission, but 30% of the time, the mutant clones acquire
additional mutations that promote the development of acute leukemia
by the age of 4 years.
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
GATA1 MUTATIONS IN DS MYELOID LEUKEMIA
with DS remain unresolved: What are the factors contributed by
chromosome 21? What is the mechanism of mutagenesis of
GATA1? Why is GATA1 targeted to such a high degree exclusively
in children with DS? Future studies will address these important
questions and shed light on the mysterious predisposition of
individuals with DS to leukemia.
405
Acknowledgments
The authors thank Drs Michelle Le Beau and Mitchell Weiss for
their helpful comments.
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2004 103: 399-406
doi:10.1182/blood-2003-05-1556 originally published online
September 25, 2003
Recent insights into the mechanisms of myeloid leukemogenesis in
Down syndrome
Sandeep Gurbuxani, Paresh Vyas and John D. Crispino
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