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Review article
Beyond Hox: the role of ParaHox genes in normal and malignant hematopoiesis
Vijay P. S. Rawat,1 R. Keith Humphries,2 and Christian Buske1
1The
Institute of Experimental Cancer Research, Comprehensive Cancer Center and University Hospital Ulm, Ulm, Germany; and 2The Terry Fox Laboratory,
British Columbia Cancer Agency, Vancouver, BC
During the past decade it was recognized
that homeobox gene families such as the
clustered Hox genes play pivotal roles
both in normal and malignant hematopoiesis. More recently, similar roles have
also become apparent for members of the
ParaHox gene cluster, evolutionarily
closely related to the Hox gene cluster.
This is in particular found for the caudaltype homeobox genes (Cdx) genes,
known to act as upstream regulators of
Hox genes. The CDX gene family member
CDX2 belongs to the most frequent aberrantly expressed proto-oncogenes in human acute leukemias and is highly leukemogenic in experimental models.
Correlative studies indicate that CDX2
functions as master regulator of perturbed HOX gene expression in human
acute myeloid leukemia, locating this
ParaHox gene at a central position for
initiating and maintaining HOX gene dys-
regulation as a driving leukemogenic
force. There are still few data about potential upstream regulators initiating aberrant CDX2 expression in human leukemias or about critical downstream targets
of CDX2 in leukemic cells. Characterizing
this network will hopefully open the way
to therapeutic approaches that target deregulated ParaHox genes in human leukemia. (Blood. 2012;120(3):519-527)
Introduction
The last decade has seen large advances in our understanding of the
regulatory network in prenatal and adult hematopoiesis. As one
pivotal class of regulatory factors, the highly conserved family of
homeobox genes have been identified as playing major roles not
only in normal hematopoiesis but also in leukemogenesis.1 The
homeobox was first identified in the 1980s as a sequence motif
shared among Drosophila homeotic genes (the HOM-C complex)
that play key roles in embryonic differentiation along the anteriorposterior axis. The existence of homeotic genes was already
proposed in the early 1900s, based on observations of Drosophila
mutants with changes in the body structure.2 The homeobox is
now known to be present in many genes in virtually all
eukaryotic species.3 It is estimated that the human genome
contains ⱖ 200 homeobox genes.4 Unlike the HOM-C/HOX genes,
which are organized in gene clusters, most homeobox genes are
dispersed throughout the genome.5,6 Homeobox-containing genes
constitute a gene family characterized by a highly conserved
183-nucleotide sequence encoding a 61-aa domain, the homeodomain (HD). These HDs are structurally related to the helix-turnhelix motif of prokaryotic DNA-binding proteins and have sequencespecific DNA binding activity.7 Homeobox genes are divided into
2 classes. Class I includes clustered genes (HOX) that comprises
39 members, and the class II divergent homeobox genes are
dispersed through the genome and include smaller families such as
the MSX, ParaHox (CDX), PAX, DLX, and Engrailed (EN) groups.
Although individual members of homeobox families often share
little sequence similarity other than the homeobox, some families
have additional conserved sequence motifs that contribute to their
distinct functional properties.5,8
A key role of homeobox genes for normal and malignant stem
and progenitor cell behavior was first shown for Hox genes. Initial
hints that these genes are essential for regulating HSCs and
progenitor cells came from data showing that Hox genes of the
A and B cluster are highly expressed in normal murine and human
HSCs and progenitor cells, but silenced in their more differentiated progeny.9,10 Studies in experimental models have convincingly proven the functional relevance of Hox genes for normal
hematopoietic behavior in mouse and man; for example,
Hoxa9⫺/⫺ BM cells manifest a severe defect at the level of HSCs
and committed progenitors11,12; however, aberrant expression of
Hox/HOX genes such as Hoxa9 or Hoxa10 can induce acute
myeloid leukemia (AML) in mice, whereas constitutive expression
of Hoxb4 increases the self-renewal capacity of murine HSCs
without initiating leukemic transformation.13-15 Similar data were
obtained for the human system with the use of retroviral gene
transfer techniques and the NOD/SCID xenograft model.16,17 Early
data indicated aberrant expression of HOX genes in patients with
acute leukemia,18 and this was confirmed later by microarray
analyses in large cohorts of patients with AML, thereby associating
aberrant expression of HOX genes such as Hoxa9 with clinical
outcome.19-22 The rich body of data on the role of Hox genes in
normal and malignant hematopoiesis has been reviewed in detail
before.1,7,8,23 In the past years, however, it was shown that other
homeobox genes, not belonging to the clustered Hox genes, have a
crucial role in regulating early hematopoietic cells and facilitating
leukemic transformation. One example is the so-called “threeamino-acid loop extension” superfamily, forming an “atypical
class” of homeobox genes that show low sequence identity with
other classes of homeobox genes.24 Most prominent members of
this family are Meis1 and Pbx1. Both can directly interact with
Hox genes and with each other, thereby forming trimeric complexes, which alter Hox–DNA binding properties and in the case of
Meis1 accelerate Hox-induced leukemogenesis.25 This review
focuses on another homeobox gene family, the ParaHox cluster,
which was shown to be involved in normal as well as malignant
hematopoiesis.6,24
Submitted February 16, 2012; accepted April 23, 2012. Prepublished online as
Blood First Edition paper, April 30, 2012; DOI 10.1182/blood-2012-02-385898.
© 2012 by The American Society of Hematology
BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
RAWAT et al
Table 1. Characteristics of ParaHox genes
Human
gene
GSH1
Protein
size, kDa
Homology between
man and mouse, %*
27
98.01
Chromosomal
translocation
Aberrant expression in
leukemia
—
Unknown
Transforming potential
Not tested
Phenotype of knockout mice
Phenotypically normal at birth, but
75% of mice die at 4 wks of age;
marked growth retardation,
infertile, and low WBC count29
GSH2
32
89.14
CHIC2-ETV630
AML (case reports)30
Transforms NIH 3T3 cells
in vitro30
Born healthy, but death within
24 hours after birth; homozygous
mutants have apnea and severe
brain alterations31
PDX1
30
87.99
—
Unknown
Not tested
Death at 1 dpp; apancreatic32
CDX1
28
85.28
—
Not expressed33
Not tested
Viable and fertile, anterior homeotic
transformations; posterior shifts
of Hox gene expression in the
somitic mesoderm34
CDX2
33
93.89
ETV6-CDX235
AML, ALL, CML in blast
Causes AML in mice37
crises33,35,36
Peri-implantation embryonic
lethality; heterozygous mutants
show growth retardation and
anterior transformation38
CDX4
30
82.98
—
AML39
Overexpression induces AML
in mice39,40
Viable, only a mild anterior
transformation with low
penetrance41
WBC indicates white blood cell; CML, chronic myeloid leukemia; and —, not described.
*At amino acid level.
The ParaHox cluster genes: emerging key
players in normal and malignant hematopoiesis
Genomic organization and molecular characteristics
The ParaHox gene cluster was discovered in 1998 by Brooke et al,
who reported that Gsx (genomic screened homeobox), Xlox
(Xenopus laevis homeobox), and Cdx (Caudal-type homeobox)
genes form their own cluster in the cephalochordate amphioxus.26 This cluster was termed the ParaHox cluster because
it showed homology to the different paralogue groups of the
Hox cluster. In mice and man homologous genes cluster on
chromosome 5 and 13q12, respectively; these comprise the
genes Gsh1/GSH1, Xlox/XLOX (also known as Pdx1/PDX1),
and CDX2.27,28 In contrast to chordates, mammals have additional ParaHox genes, namely Gsh2/GSH2, Cdx1/CDX1, and
Cdx4/CDX4, which are spread over 3 different chromosomes in
the human genome (Table 1; Figure 1A).
The ParaHox and Hox clusters are thought to originate from
an ancient hypothetical ProtoHox cluster by duplication before
the split of protostomes and deuterostomes, characterizing the
ParaHox cluster as one of the evolutionary early homeobox
gene clusters.6,42,43 This also explains the homologies of the
ParaHox genes to paralogue groups of the Hox cluster. The
paralogue groups of the Hox cluster subdivide the Hox genes
into 4 groups: the anterior group (paralogue group [PGs] 1-2),
group 3, central group (PGs 4-8), and posterior group (PGs
9-13).6,43 The Gsx gene is more closely related to Hox anterior
group genes than to other ParaHox genes; Xlox is more similar
to Hox group 3, and Cdx is more similar to the Hox posterior
group genes.
Structure–functional analyses have characterized the molecular
characteristics of ParaHox proteins in detail (Table 1; Figure 1B).
As for homeobox genes, all the ParaHox genes are characterized by
a DNA-binding HD and in part by a N-terminal transactivation
domain. However, homology between the different family
members of the ParaHox genes is relatively low and proteins
display individual features. Thus, the ParaHox gene PDX1
contains an antennapedia-type hexapeptide motif (PFPWMK),
which preferentially binds the DNA motif 5⬘-[CT]TAAT[TG]3⬘. PDX1 is phosphorylated by HIPK2 on Ser-268 on glucose
accumulation, which mediates shifting of the protein from
nuclear periphery to nucleoplasm.44 GSH1 contains an additional Snail/Gfi domain essential for transcriptional repression.
On the basis of bioinformatics analysis it is postulated that GSH
genes bind to DNA as monomers or as homodimers and/or
heterodimers, in a sequence-specific manner. CDX1, in contrast
to other CDX members, does not contain the highly conserved
pentapeptide domain for binding of the three-amino-acid loop
extension cofactor Pbx1.
Lessons from knockout mice: initial clues to function of
ParaHox genes
Knockout mice have been an important tool for our understanding
of ParaHox gene function and their essential role in body development (Table 1). Gsh1 homozygous mutant mice are phenotypically
normal at birth but fail to grow properly after 7-10 days. Homozygous mutants exhibit extreme dwarfism, sexual infantilism, and
significant perinatal mortality with total white blood cell count
5-fold decreased in mutant mice. Both lymphoid and myeloid cells
were similarly reduced, whereas macrophages were not affected.29
Gsh2 knockout mice display severe structural alterations in the
brain, accompanied by apnea and reduced blood oxygen levels,
leading to death within the first day after birth.31 Pdx-1 was shown
to be crucial for the development of the pancreas and anterior
duodenum.32 Cdx1-null mutant mice are viable and fertile, but they
display anterior homeotic transformations of vertebrae accompanied by posterior shifts of Hox gene expression in the somatic
mesoderm.34 Cdx2-null mutant embryos die between 3.5 and
5.5 days after coitum, and heterozygotes have tail abnormalities
and exhibit anterior homeotic transformations that involve the
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
PARAHOX GENES IN NORMAL AND MALIGNANT HEMATOPOIESIS
Figure 1. Characteristics of ParaHox genes. (A) Chromosomal location of human ParaHox genes. Chromosomal locations of murine ParaHox genes are given in
brackets. The direction of transcription is indicated by an
arrow. (B) Protein structure of ParaHox genes; shown is
the protein structure depicting the N-terminal transactivation domain (blue), the Pbx-interacting motif (PIM; brown),
the HD (green), and validated phosphorylation sites
(orange).
A
GSH
PDX
CDX
1
1
2
Chr 13q12 (Chr 5 82cM; 41cM for Gsh1)
1
Chr 5q31-33 (Chr 18 30cM)
2
521
Chr 4q12-13 (Chr 5 82cM)
4
Ch: X13.2 (Chr X 42.5 cM)
B
cervical and upper thoracic vertebrae, ribs, and midgut endoderm,
underlining the crucial role of Cdx genes for anterior-posterior
regional identity. Ninety percent of Cdx2 heterozygote mutant
mice develop multiple intestinal adenomatous polyps, particularly in the proximal colon, suggesting that Cdx2 mutations
might be the primary event in the genesis of some intestinal
tumors.38,45,46 In contrast to Cdx2, Cdx4-null murine embryos
are born healthy and do not show any major structural or
histologic abnormalities.41
Cdx2 and Cdx4 are key regulators of embryonic hematopoiesis
Over the past several years it has become evident that ParaHox
Cdx family members are critical for ordered proliferation and
differentiation of embryonic hematopoietic cells.47 This was
particularly found for the Cdx genes Cdx4 and Cdx2. Evidence
for a key role of Cdx genes in embryonic hematopoiesis initially
came from studies in zebrafish; homozygous mutations in cdx4
caused a severe defect in embryonic hematopoiesis with a
profound reduction of hemoglobin-positive erythroid cells that
resulted in severe anemia.48 These blood defects were accompanied by deregulated or reduced expression of hoxb4, hoxb5a,
hoxb6b, hoxb7a, hoxb8a, hoxb8b, and hoxa9a. Of note, the
blood deficiency in zebrafish embryos could be rescued by
overexpression of hoxb7a or hoxa9a but not by scl, indicating
that the hematopoietic defect is at least partly caused by
perturbation of distinct Hox genes.48 The same group found that
morpholino-mediated knockdown of cdx1a in a cdx4-mutant
background caused a severe perturbation of Hox gene expres-
sion and a complete failure to specify blood in zebrafish.
Furthermore, these Cdx-deficient embryos displayed a significant reduction in expression of key hematopoietic genes such as
scl, runx1, and gata1.49 This hematopoietic defect could be
efficiently rescued by hoxa9 overexpression, but not by hoxb7.49
In mouse, ectopic expression of Cdx4 enhances hematopoietic mesoderm formation and further promotes blood progenitor
specification and hematopoietic engraftment of murine embryonic stem cells (ESCs) in adult mice.50 In addition, Cdx4
overexpression rescues defective blood progenitor formation in
mouse ESCs deficient in Mll, a Hox regulator involved in
definitive hematopoiesis.51 Surprisingly, Cdx4 knockout mice do
not show any major hematopoietic defects.40 Indeed, in both
germline and conditional Cdx4 knockout models, there is no
apparent defect in HSCs and progenitor cells as well as mature
blood cells. This indicates that Cdx4 as a single factor is not
essential for normal adult hematopoietic stem cell functions in
mice and that Cdx4 function can be compensated at least partly
by other Cdx genes.40 One candidate for this is Cdx2; Cdx2deficient ESCs show impaired production of multipotential
blood progenitor colonies in vitro consistent with an essential
role of Cdx2 in early embryonic hematopoiesis.52 By injecting
Cdx2⫺/⫺ or Cdx2⫹/⫹ ESCs into normal lacZ⫹ blastocysts,
Wang et al could show an intrinsic requirement for Cdx2 during
primitive embryonic hematopoiesis in vivo.52 According to
genome-wide expression analysis they reported that Cdx2
deficiency dramatically perturbs expression profiles of Hox
genes and genes involved in signal transduction, cell growth and
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
RAWAT et al
A
Normal Hematopoiesis
p
Cdx/CDX
44
1
Malignant Hematopoiesis
Cdx/CDX
B
44
2
1
2
HOX expression
CDX4 expression
CDX2 No expression
CD
X2
CDX1 No expression
Stem cell
Stem cell
Progenitor
Oligo lineage
precursor
Mature
cells
CD
Transformed
stem cells
X2
Leukemia initiating cells
Stem cell
Transformed
Progenitors
Acute Myeloid Leukemia
HOX
CDX4
CDX2
CDX1
0
20
40
60
80
100
% of positive patients
Figure 2. Cdx expression in normal and malignant hematopoiesis. (A) Normal hematopoiesis.Cdx4 and HOX A and B cluster genes are expressed in normal stem and
progenitor cells, whereas Cdx2 and Cdx1 remain silenced. (B) Malignant hematopoiesis. Ectopic expression of CDX2 at the level of stem or progenitor cells induces increased
expression of Hox A and B cluster genes and leukemic transformation of hematopoietic cells. The proportion of patients with AML, which ectopically express CDX2 and
overexpress HOX genes, is indicated. CDX4 is expressed only in a minority of patients, and CDX1 expression is not detectable in patients with leukemia.
proliferation, and hematopoiesis. Expression of several components of the canonical signal transduction pathways such as the
Jak/Stat and sonic hedgehog pathways were altered in Cdx2⫺/⫺
embryonic bodies (EBs). Expression of several hematopoiesisspecific genes, including Scl, Gata1, Runx1, and ␤-H1 globin,
were markedly decreased in Cdx2-defecient EBs,52 supporting
the notion that Cdx2 is a key player during hematopoietic
development.
In contrast to Cdx4 and Cdx2, loss of Cdx1 gene function
does not affect hematopoietic development as shown by homozygous Cdx1 mutant mice,34 but also by studies in zebrafish,
whereby cdx1a had no effect on rostral blood island hematopoiesis and the intermediate cell mass development.49 The distinct
effects of the different members of the Cdx gene family was
elegantly shown in a study,52,53 which ectopically expressed
Cdx1, Cdx2, and Cdx4 in murine ESCs; whereas Cdx1 and Cdx4
both promoted the hematopoietic potential of the EB-derived
CD41⫹c-kit⫹ population, Cdx2 grossly reduced EB-derived
hematopoietic potential. Expression of all the different Cdx
genes increased Hox gene expression with the notable difference that Cdx2 induced changes in both the anterior and
posterior HoxA and HoxB genes, whereas Cdx1 and Cdx4 only
altered posterior Hox gene expression.53
Taken together, these data indicate that Cdx genes play essential
and both distinct and overlapping roles in embryonic hematopoiesis.
ParaHox genes in malignant hematopoiesis
ParaHox genes have emerged as pivotal players in leukemogenesis
with a rich body of evidence to link aberrant expression of the
ParaHox gene Cdx2 and other ParaHox genes with acute leukemia
(Table 1; Figure 2).
GSH2
The Gsh2 gene was previously characterized as a brain-specific
ParaHox gene important in forebrain and hindbrain formation of the
mouse. Peter Marynen’s group (Cools et al30) has shown that GSH2 is
not expressed in adult human normal hematopoietic cells. However, the
group has reported ectopic expression of GSH2 in 4 patients with AML
carrying the translocation t(4;12)(q11-q12;p13), which itself generated
no detectable fusion gene. GSH2 is located at 4q11-q12 and thus located
closely to the respective breakpoint, which implies that the translocation
induces ectopic expression of GSH2. Indeed the researchers could
demonstrate that aberrant expression of GSH2 caused focus formation
in the NIH3T3 assay as a surrogate marker for the oncogenic potential of
this ParaHox gene.30
Cdx1
In normal adult hematopoietic tissue, expression of this homeobox
gene was not reported so far, and there are no data to suggest
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
PARAHOX GENES IN NORMAL AND MALIGNANT HEMATOPOIESIS
involvement of Cdx1 in malignant hematopoiesis. However, in the
adult Cdx1 is known to be involved in the intestinal differentiation
and the development of intestinal metaplasias.54-56
bivariate Cox regression.60 These data were confirmed for pediatric
ALL whereby most patients also showed aberrant expression of
CDX2. Also in this study, aberrant expression of CDX2 significantly correlated to an inferior disease outcome.61 Another recent
independent study reported CDX2 expression in 61% of patients
with de novo adult ALL and decrease of CDX2 expression in
patients with complete remission and increase in relapse.62
Similar to AML the mechanism of aberrant CDX2 expression in
ALL is not understood. We have not identified any difference in
the methylation level of the CDX2 promoter in patients with
CDX2-positive and -negative ALL.60 In contrast to AML,
whereby aberrant CDX2 expression correlated with HOX gene
deregulation, there was only a low correlation between the
expression of HOX genes with the expression of CDX2, in line
with reports that HOX gene deregulation is less common in ALL
than in AML.63,64 Taken together, these data link aberrant
expression of CDX2 to myeloid as well as lymphoid leukemia.
So far it is unknown whether the stage of differentiation of the
cell, which is initially hit by aberrant CDX2 expression,
determines the phenotype of the leukemia. Experiments testing
whether aberrant expression of CDX2 in lymphoid progenitor
cells generates ALL in mice are lacking so far.
Cdx2 is highly leukemogenic in experimental models. In a
murine BM transplantation model it was shown that ectopic
expression of the ParaHox gene CDX2, but not expression of the
fusion gene ETV6/CDX2, which was described in a patient with
translocation t(12;13)(p13;q12)–positive AML,35 was highly
leukemogenic.37 This was the first direct evidence that the
ParaHox gene Cdx2 is highly leukemogenic and induces aggressive AML when aberrantly expressed in hematopoietic progenitor cells. Retrovirally enforced expression of Cdx2 in murine
hematopoietic progenitors resulted in increased expression of
leukemogenic Hox genes such as Hoxa5, Hoxa7, Hoxa9,
Hoxa10, as well as Hoxb3, Hoxb6, and Hoxb8.33 These data are
in agreement with previous findings that Cdx2 can deregulate
Hox gene expression in embryonic as well as adult nonhematopoietic tissues.45,65 The ability to up-regulate Hox gene expression and to induce AML clearly depended the N-terminal
transactivation domain of Cdx2, known to harbor serine phosphorylation sites, which affect the transcriptional activity of the
gene.33,66 Interestingly, the ETV6-CDX2 fusion gene also lacks
the N-terminal transactivation domain of CDX2, which would
explain the obvious discrepancy of the oncogenic potential
between Cdx2 and the ETV6-CDX2 fusion.
Aberrant Cdx2 expression also led to an altered expression of a
number of genes involved in lymphopoiesis such as Lef1, Tcf3, or
Id3. These genes are frequently deregulated in lymphoid leukemia60,67,68 and are able in the case of Lef1 to induce ALL in mice
that received a transplant.33
Most data on the leukemogenicity of Cdx2 come from
murine models. However, at least at the level of human leukemia
cell lines it could be shown that leukemic growth depends on
aberrant CDX2 expression; thus, shRNA-mediated depletion of
CDX2 in the human pre-B cell line Nalm-6 and several AML cell
lines significantly impaired leukemic cell proliferation and
clonogenicity.56,60
CDX4 is less leukemogenic than Cdx2. It has been shown that
overexpression of Cdx4 confers enhanced replating and proliferative potential to transduced murine hematopoietic cells. Mice
transplanted with Cdx4-overexpressing BM developed a myeloproliferative disorder, which progressed to AML over time.39 In this
model aberrant expression of Cdx4 had to collaborate with the
CDX2
Aberrant expression of CDX2 is a frequent event in patients with
AML. Several studies have reported that CDX2 is normally not
expressed in adult murine and human normal hematopoiesis
(Figure 2). Ectopic expression of CDX2 in AML was reported by
Chase et al in 1999 in a patient with AML M1 carrying the
chromosomal translocation t(12;13)(p13;q12), creating a novel
fusion of ETV6 and CDX2.35 In addition, aberrant expression of
CDX2 was found in a case of blast crisis of chronic myeloid
leukemia (CML-BC) with no detectable chromosomal abnormality.35 The fusion partner ETV6 is an important regulator of HSC
survival and frequently affected by translocations.57,58 Later, we
and others have reported that CDX2 is aberrantly expressed in
⬎ 80% of human AML patients, characterizing CDX2 as one of the
most frequent aberrantly expressed proto-oncogenes in human
leukemia (Figure 2). Aberrant expression of CDX2 was found in all
types of cytogenetic subgroups. Scholl et al reported the highest
expression of CDX2 in patients with t(9;11) followed by normal
karyotype AML (CN-AML) and t(15;17)–positive patients,36 similar to our results with highest expression of CDX2 in CN-AML
followed by t(9;11)– and t(15;17)–positive cases.33 Aberrant expression of CDX2 was not only more common in patients with
CN-AML than in patients with abnormal karyotype (89% vs 64%,
respectively), but the expression level of CDX2 was significantly
and ⬎ 15-fold higher in patients with CN-AML as well. Furthermore, the level of CDX2 expression correlated with aberrant HOX
gene expression in human AML.33 The analyses did not show any
correlation of the NPM1 mutational status with the frequency and
level of aberrant CDX2 expression. Aberrant CDX2 expression is
also detectable in most of the human AML cell lines. The
mechanisms responsible for disruption of the normal CDX2
silencing in human AML are unknown and are not because of
promoter mutation, gene amplification, or abnormal promoter
methylation.36 In contrast to CDX2, all CN-AML cases were
negative for expression of the other members of the CDX gene
family CDX1 and CDX4.33
CDX2 is aberrantly expressed in most patients with acute
lymphoblastic leukemia (ALL). Ectopic expression of CDX2 is
not restricted to patients with AML; we showed that CDX2 is
aberrantly expressed in most adult patients with ALL, with 81%
overall positivity and a higher overall expression level than
detectable in patients with CN-AML. The expression of CDX2
showed high variation between the different ALL subgroups
(classified according to the criteria of the European Group for the
Immunologic Characterization of Leukemias59) with highest median expression in pre-T-ALL, followed by c-ALL and pro-B-ALL.
In contrast, patients with B-ALL/Burkitt lymphoma and thymic
T-ALL exhibited only low CDX2 expression. In addition, the
number of patients aberrantly expressing CDX2 differed considerably between ALL subtypes, with 100% positivity in patients with
pro-B-ALL, c-ALL, and Ph⫹ ALL versus only 40% positivity in
B-ALL/Burkitt lymphoma and ⬃ 70% in thymic and pre-T-ALL.60
The expression level of CDX2 in patients with ALL significantly
correlated with treatment outcome (high expression levels were
associated with inferior overall survival) of the analyzed patients.
Although that study was limited by a low number of patients,
CDX2 remained an independent risk factor even after adjusting for
the risk factors of age and presence of molecular markers by
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
RAWAT et al
homeobox gene Meis1 to induce AML in all experimental
animals,39 thus clearly differing from Cdx2, which caused rapid
disease in all mice that received a transplant.37 In line with this,
the long latency of leukemia development in primary animals
that received a transplant suggested that aberrant expression of
Cdx4 alone is not sufficient to induce leukemia but rather
induces a preleukemic stage. Loss of Cdx4 significantly prolonged the latency of disease onset in a mouse model of
mixed-lineage leukemia-AF9–induced AML and altered the
phenotype of the resultant disease with increased expression of
B- and T-lymphoid markers.40 The mechanism of Cdx4-induced
leukemogenesis is not completely understood. However, recently it has been shown that CDX4 activates transcription of
the Hox gene HOXA10 in myeloid cells and binds to a cis
element of its promoter. Vice versa HOXA10 is able to activate
CDX4 expression, pointing to a positive feedback loop between
CDX4 and a HOX gene, which itself is linked to myeloid
leukemogenesis.17,69,70 It was also found that Cdx4 collaborates
with Menin to activate Hoxa9 expression by binding with Menin
at the same regulatory region at the Hoxa9 locus.71 In summary,
these data categorize CDX4 as an important collaborative
partner in Hox-associated leukemogenesis.
In patients with AML CDX4 is expressed aberrantly in ⬃ 25%
of all cases (Figure 2). CDX4 expression, however, did not
correlate to a certain leukemia subtype, and its expression did not
correlate to HOX gene deregulation.39 This finding suggests that
the ability of CDX4 to deregulate HOX gene expression is of minor
relevance for its role in human leukemia.
Mechanisms of ParaHox-induced leukemogenesis:
speculations and future directions
Gene expression profiling and functional data clearly support a
critical involvement of CDX2 in human leukemogenesis. However,
molecular mechanisms of Cdx2-induced leukemogenesis are poorly
understood. One obvious possibility is that CDX2 exerts its
leukemogenic potential by deregulating expression of leukemogenic genes of the HOXA and HOXB cluster, known to be
dysregulated in 65% of all AML cases, in particular in normal
karyotype (CN-AML) and mixed-lineage leukemia–rearranged
AML33,36 (Figure 2). This would classify the CDX-HOX axis as
one of the main driving forces in human AML. However, it could
be clearly shown that aberrant Cdx2 expression, besides Hox
deregulation, also led to an altered expression of a number of genes
involved in embryonic development, lymphopoiesis, Wnt pathway,
and genes involved in stem cell function. These data support the
hypothesis that Cdx2 might deregulate multiple pathways in
leukemia, which would also explain the differences between Cdx2
and Cdx4 in their leukemogenic potential.
So far strategies to target Cdx2-induced transformation remain
elusive. One possibility would be to identify signaling pathways
that are able to be affected by drugs on which Cdx2 activity is
dependent. Interestingly, it was shown in a colon cancer cell line
that the transcriptional activity of Cdx2 depends on phosphorylation of the N-terminal S60 site by ERK kinases. We have shown
that the N-terminal activation domain of Cdx2, at which the S60
site is localized, is essential for Cdx2-induced leukemogenesis
and perturbation of Hox gene expression.33 Recent studies
documented that Erk1/2 and their upstream effectors Mek1/2 are
constitutively activated in primary human AML cells.72-74
Elevated p-ERK levels were found in 83.3% of the patients with
AML.75 Because aberrant expression of CDX2 is detected in
80% of all AML cases, regulation of Cdx2 could be indirectly or
directly linked with MEK/ERK signaling72,76 (Figure 3A-B).
Therefore, it will be interesting to test whether posttranscriptional modification of CDX2 is crucial for its leukemogenic properties and whether MAPK inhibitors are able to block
Cdx2-induced leukemogenesis.
Another possibility to block Cdx2-induced leukemogenesis
would be to inhibit mechanisms that induce and maintain aberrant
Cdx2 expression. However, mechanisms that lead to aberrant
expression of CDX2 in leukemogenesis are still a mystery. As
reported by Scholl et al, the methylation levels of the promoter
region of CDX2 in patients with AML did not correspond to its
expression levels.36 In addition, treatment of CDX2-negative cell
lines with demethylating agents did not lead to an induction of
CDX2 expression,77 suggesting that CDX2 expression in leukemia
is independent of promoter hypomethylation as well as of the
methylation level of cis-regulatory elements. Amplification of the
CDX2 locus was detected only in a small percentage of patients
with complex karyotype, however, not in AML patients with
normal karyotype, who are characterized by high CDX2 expression
levels.36,78 Another possibility, leading to aberrant transcription of
CDX2 could be deregulation of trans-acting pathways, which
target cis-regulatory elements in the 5⬘ region of the CDX2 gene as
suggested by Hinoi et al.77 However, it was reported that CDX2
expression is monoallelic in most of the analyzed patients with
leukemia,36 which indicates that the altered CDX2 expression is not
primarily because of differences in the expression of trans-acting
factors but rather because of altered cis-regulatory elements,
which affect only one allele. Mutations were not detected in the
promoter region of CDX2. However, this does not exclude still
unknown mutations in cis-regulatory elements of the CDX2
gene, which cause aberrant expression. It would be interesting,
therefore, to expand the sequence analysis of the CDX2 gene
beyond its coding and promoter region and to conduct a more
comprehensive sequence analysis. One target of genetic alterations, for example, could be negative regulators, which Wang
and Shashikant postulated to be present in an 11.4-kb sequence
flanking Cdx2.79 Disruption of these elements in hematopoietic
cells could lead to an aberrant expression of the normally
silenced CDX2 as well as mutations within 2 enhancer regions in
the first intron of CDX2, described to induce ectopic expression of
Cdx2 in the murine embryo.79 Interestingly, several binding sites
for fibroblast growth factor (FGF) and Wnt signaling were
identified in these regions. Mutations therefore could possibly
affect the binding of factors such as FGF4, which previously
was shown to target Cdx280 (Figure 3). Furthermore, it is
probable that Cdx2 not only regulates but also is targeted by Wnt
signaling as already demonstrated for other ParaHox genes such
as Cdx1 and Cdx4.81,82 Another pathway that was shown to
regulate Cdx2 expression is Ras/MAPK signaling,76 a pathway
that links extracellular stimuli among others to proliferation,
differentiation, and cell survival. Because both the Wnt and
Ras/MAPK pathways are frequently deregulated in leukemia,
this deregulation in combination with genetic alterations in the
cis-regulatory elements of Cdx2 could possibly affect the
transcription of the gene (Figure 3).
Conclusion
In summary, there is a great body of evidence that ParaHox genes
play a pivotal role in normal hematopoiesis but also in leukemogenesis. These data add another homeobox gene family to the growing
number of homeobox genes known to be critically involved in
orchestrating normal hematopoiesis and promoting leukemogenesis. Thus, the story “homeobox genes in normal and malignant
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BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
PARAHOX GENES IN NORMAL AND MALIGNANT HEMATOPOIESIS
525
Normal hematopoiesis
Wnt Signalling
FGF Signalling
FGF
LRP5/6
Frizzled
FGFR
FGFRL1
Wnt
SEF
A
Cytoplasm
FRS2
Ras/Raf
-Catenin
MAPK
Nucleus
?
-Catenin
?
?
11
Cdx/CDX 4
22
11
22
Hox/HOXA
B
Hox/HOXA
B
?
Cdx/CDX 4
Model based on embryonic studies
B
stimulates
blocks
Malignant hematopoiesis
(hypothetical model)
FGF Signalling
Wnt Signalling
FGF
SEF
LRP5/6
Frizzled
FGFR
FGFRL1
Wnt
FRS2
Mutation
Cytoplasm
Ras/Raf
MAPK
-Catenin
Nucleus
-Catenin
TCF/LEF
Cdx/CDX 44
11
2
Hox/HOX A
B
?
?
?
?
Model based on embryonic studies
Cdx/CDX
44
11
2
Hox/HOX A
B
stimulates
blocks
Figure 3. Hypothetical model of Cdx gene regulation in normal and malignant hematopoiesis. (A) Normal hematopoiesis, based on results from studies in
embryogenesis, the canonical Wnt pathway regulates the expression of CDX4 by the Lef1/Tcf complex in hematopoiesis. The FGF-MAPK pathway is not constitutively active in
stem and progenitors cells. (B) Malignant hematopoiesis, either deregulation of the WNT pathway or FGF signaling induces aberrant CDX2 expression in human leukemia.
hematopoiesis” is not at its end but is still growing rapidly since the
last review on this topic in this journal nearly 10 years ago.1 In
those 10 years dysregulation of homeobox genes has been recognized as one of the molecular hallmarks of human AML. We know
today that homeobox genes are key players for determining
“stemness” and are essential for initiating and maintaining selfrenewal programs on which leukemic stem cell function depends.83
ParaHox genes such as Cdx2 are particularly powerful to transform
hematopoietic progenitors into leukemic stem cells, at least partly
because it stands upstream of Hox genes and is able to perturb
expression of several Hox genes at the same time. It is intriguing to
speculate that targeting dysfunctional ParaHox genes such as
CDX2 in human leukemia might reverse HOX gene dysfunction on
a larger scale, thereby depriving the leukemic stem cell the
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526
BLOOD, 19 JULY 2012 䡠 VOLUME 120, NUMBER 3
RAWAT et al
molecular “environment” it needs to propagate leukemic growth. It
is indeed unfortunate that we know so little about the mechanisms
that induce aberrant CDX2 expression in patients with AML,
because this would open possibilities to develop strategies to
re-silence CDX2 expression in leukemic cells. Another hope is to
identify signaling cascades that are essential for CDX2-induced
transformation. Many questions are still open, and clarification of
the role of other members of the ParaHox gene cluster such as GSH2 for
leukemogenesis was just initiated. We can be confident that the next
years will generate exciting data about the role of ParaHox genes for
normal tissue homeostasis and will improve our understanding of how
to correct ParaHox dysfunction in human leukemias.
Acknowledgments
The authors apologize to authors whose work could not be cited
because of space constraints. They thank all members of their
institutes for vivid discussions on this topic. They also thank Silvia
Thoene, Shiva Bamezai, and Tamoghna Mandal for assisting in the
preparation of figures.
This work is supported by grants from the Else Kröner-FreseniusFoundation, the Deutsche Forschungsgemeinschaft, and the Bundesministerium für Bildung und Forschung (V.P.S.R. and C.B.).
Authorship
Contribution: V.P.S.R., R.K.H, and C.B. contributed to the writing
of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Christian Buske, Institute of Experimental
Cancer Research, University of Ulm, Albert-Einstein-Allee 11,
89081 Ulm, Germany; e-mail: [email protected].
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2012 120: 519-527
doi:10.1182/blood-2012-02-385898 originally published
online April 30, 2012
Beyond Hox: the role of ParaHox genes in normal and malignant
hematopoiesis
Vijay P. S. Rawat, R. Keith Humphries and Christian Buske
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