Tài liệu Báo cáo khoa học: Mixed lineage leukemia: roles in human malignancies and potential therapy pdf

MINIREVIEW
Mixed lineage leukemia: roles in human malignancies and
potential therapy
Rolf Marschalek
Biochemistry, Chemistry & Pharmacy, Institute of Pharmaceutical Biology, Goethe-University of Frankfurt ⁄ Main, Germany
Mixed lineage leukemia fusions, acute
leukemia and the HOX signature
Mixed lineage leukemia (MLL) rearrangements define
a small subset of acute leukemia patients, including
those with therapy-induced secondary leukemias. How-
ever, unlike many other types of leukemia, the pres-
ence of distinct MLL rearrangements predicts early
relapse and very poor prognosis [1].
Based on experimental investigations, the ectopic
transcriptional activation of distinct HOXA genes in
conjunction with the MEIS1 gene has been reported
and proposed as a putative cancer mechanism [2–4].
This particular HOXA ⁄ MEIS1 signature was found
to be associated with the ability to show clonal
growth in semi-solid media and confers serial replat-
ing efficiency.
Consistent data, however, have been obtained for
only some tested MLL fusion alleles, most of which
were associated with an acute myeloid leukemia
(AML) disease phenotype. Taking into account that
MLL fusion proteins are associated with acute myeloid
leukemia (AML) and acute lymphoblastic leukemia
(ALL), it argues that other cancer mechanisms may
exist as well. Different committed or permissive cell
types may be malignantly transformed by the huge
number of diverse MLL fusion alleles (see below).

Because different lineages of the hematopoietic system
naturally display specific ‘HOX profiles’, it may well
be that the observed ‘HOX signatures’ reflect only
Keywords
acute leukemia; AF4; AF9; AF10; cancer
stem cells; ELL; ENL; MLL; MLL fusion
proteins; signaling
Correspondence
R. Marschalek, Goethe-University of
Frankfurt ⁄ Main, Department of
Biochemistry, Chemistry & Pharmacy,
Institute of Pharmaceutical Biology,
Biocenter, N230, Max-von-Laue-Str. 9,
D-60438 Frankfurt ⁄ Main, Germany
Fax: +49 69 798 29662
Tel: +49 69 798 29647
E-mail:
(Received 14 November 2009, revised 7
January 2010, accepted 12 January 2010)
doi:10.1111/j.1742-4658.2010.07608.x
The increasing number of chromosomal rearrangements involving the
human MLL gene, in combination with differences in clinical behavior and
outcome for MLL-rearranged leukemia patients, makes it necessary to
reflect on the cancer mechanism and to discuss potential therapeutic strate-
gies. To date, 64 different translocations have been identified at the
molecular level. With very few exceptions, most of the identified fusion
partner genes encode proteins that display no homologies or functional
equivalence. Only the most frequent fusion partners (AF4 family members,
AF9, ENL, AF10 and ELL) are involved in the positive transcription elon-
gation factor b-dependent activation cycle of RNA polymerase II. Biologi-

cal functions remain to be elucidated for the other fusion partners. This
minireview tries to sum up some of the available data and mechanisms
identified in leukemic stem and leukemic tumor cells and link this informa-
tion with the known functions of mixed lineage leukemia and certain mixed
lineage leukemia fusion partners.
Abbreviations
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; DSIF, DRB-sensitivity inducing factor; GSK, glycogen synthase kinase;
H3K4, histone H3 lysine 4; HMT, histone methyltransferase; MLL, mixed lineage leukemia; NELF, negative elongation factor; PI3K,
phosphatidylinositol 3 kinase; P-TEFb, positive transcription elongation factor b; SET, su(var)3-9, enhancer-of-zeste, trithorax; TGF,
transforming growth factor.
1822 FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS
a particular differentiation state in which the
transformed cell has been arrested (e.g. common
myeloid progenitors) [5]. Whether specific HOX
signatures are indeed necessary for leukemogenesis or
are a concomitant phenomenon needs to be answered
on the basis of performed experiments for individual
MLL fusion proteins (see below).
Cellular functions of the MLL protein
The MLL protein has been identified as the mamma-
lian orthologue of the Trithorax protein in inverte-
brates [6]. Disruption of this gene in invertebrates and
vertebrates leads to homeotic transformation and null-
alleles are incompatible with normal embryonic devel-
opment [7,8]. All observed genetic mutations of the
MLL gene (chromosomal translocations, chromosomal
insertions, spliced fusions) seem to occur preferentially
in hematopoietic cells, indicating that this system
imparts unique properties (permissivity, survival and
development of leukemic clones) on a large variety of

different MLL fusion protein variants. Specific signals
are derived from stromal cells during fetal liver and
definitive hematopoiesis. This enables the activation of
anti-apoptotic pathways and stem cell maintenance
[9,10]. Leukemic cells seem to have the ability to inter-
act with these niches in order to receive important
survival signals and to cope with stress caused by the
presence of oncogenic MLL fusion proteins.
The human MLL protein, or its homo ⁄ orthologues in
various biological systems, is a ubiquitously expressed
protein involved in chromatin regulation. MLL expres-
sion is initiated at very early stages of embryogenesis.
The MLL protein is specifically hydrolysed by the endo-
peptidase Taspase1 [11]. This allows it to assembly into
a high-molecular mass complex which confers the meth-
ylation of histone core particles at histone H3 lysine 4
(H3K4) residues [12,13]. This particular signature is
found on nucleosomes localized at the promoter regions
of actively transcribed genes, and enables their tran-
scriptional maintenance. Therefore, MLL is part of an
epigenetic system that guarantees mitotically stable
gene-expression signatures during embryonic develop-
ment, germ layer formation and tissue differentiation in
mammalian organisms. Other proteins that exhibit
H3K4 histone methyltransferase (HMT) activity are
hSET1a, HSET1b, SET7 ⁄ 9, MLL2, MLL3, MLL4,
ASH1, SMYD3 and PRDM9 [14], however, these pro-
teins are not currently known to be subject to genetic
rearrangements in human cancer.
Because the biological activity of MLL is restricted to

open chromatin structures, in particular, to active pro-
moter regions, the MLL complex obviously binds to
different promoters in various tissues. In a recent study,
occupancy of MLL protein was investigated using chro-
matin immunoprecipitation experiments and subsequent
analysis on genome-wide tiling arrays [15]. This study
revealed that MLL was bound to > 2000 different pro-
moter regions within the cell line investigated (U937), of
which 99% were also bound by RNA polymerase II.
However, active transcription can be blocked by associ-
ated Polycomb proteins. Several genes belonging to the
HOXA clusters have been identified (HOXA1, A3, A7,
A9, A10, A11) among these promoters. HOXA genes
are downstream targets of wild-type MLL and of
several tested MLL fusion proteins.
Model systems for the analysis of MLL
fusion proteins and patient analysis
Different MLL fusions have been investigated as a
single transgene using a number of different
approaches. Mouse model systems were based on
transgenic techniques (transgenic mice, knock-in mice,
inverter mice, translocator mice, etc.) [16] or used
retroviral gene transfer [17].
Several laboratories have used retroviral transduc-
tion of murine hematopoietic stem cells to functionally
investigate the oncogenic properties of distinct MLL
fusion alleles. Manipulated hematopoietic stem ⁄ precur-
sor cells were tested in methylcellulose assays for their
clonal growth and replating efficiency, and the result-
ing colonies were transplanted into recipient mice of

various genetic backgrounds. Alternatively, manipu-
lated stem ⁄ precursor cells were used directly for trans-
plantation into recipient mice. In sub-lethally
irradiated recipients, such manipulated cells have the
ability to home into the bone marrow or spleen and
engraft there. In different experiments, transplanted
mice developed AML or myeloproliferative diseases
after several months [18]. All successfully tested MLL
fusion alleles displayed deregulated HoxA genes, for
example, HoxA7 and HoxA9. The transforming capac-
ity of the tested MLL fusion constructs was also
dependent on the presence of Meis1 and Pbx proteins,
as well as on the presence of Men1 and Ledgf [19–21].
More recently, it has been demonstrated that overex-
pressed Meis1 results in the establishment of a unique
gene-expression signature that is further enhanced by
the presence of the HoxA9 protein [20]. Men1 binds
directly to the N-terminus of MLL fusions [22] and
was essential for MLL fusion proteins binding to dif-
ferent HoxA target promoters [13].
However, opposing experimental results have been
published when using fusion genes derived from the
chromosomal translocation t(4;11). Enforced expression
R. Marschalek Role of MLL in human malignancies
FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS 1823
of MLL–AF4 in cell lines (stably or conditionally
expressed) resulted in cell-cycle arrest and a senescent
cellular phenotype [23,24]. Most likely, the observed
cell-cycle arrest was based on the strong increase in
CDKN2A ⁄ p16 transcripts caused by the presence of

overexpressed MLL–AF4. Short-term protein expres-
sion of MLL–AF4 in a doxycycline-dependent manner
resulted in the ectopic activation of HoxA7 and HoxA10
(and 560 other genes), whereas the reciprocal
AF4–MLL fusion protein did not activate any Hox gene
(but did activate 660 other genes). Surprisingly, when
both t(4;11) fusion proteins were expressed in the same
cell, not a single HoxA gene was found to be transcrip-
tionally activated (but 800 other genes were). This
indicated that the reciprocal AF4–MLL fusion protein
was dominant over the investigated MLL–AF4 fusion
protein, suppressing the typically observed HOXA
signature [24]. Is this also the case for other genetic rear-
rangements of the MLL gene? With the exception of
t(11;19) translocations, where 50% of all patients carry
only a single MLL–ENL fusion allele [25], most MLL-
rearranged leukemia patients exhibit both MLL fusion
alleles at the genomic DNA level. It is interesting to
note that these reciprocal MLL fusion alleles seem to be
transcribed at lower levels compared with the transcrip-
tional activity of the direct MLL fusion allele
(R. Marschalek, unpublished observation). Therefore,
most investigators tend to analyze transcripts deriving
from the direct MLL fusion allele as diagnostic readout.
This is presumably one reason why reciprocal MLL
fusion alleles have never received much attention. How-
ever, without testing both reciprocal fusion alleles in the
same test system, it is impossible to answer the impor-
tant question about the role of activated HOXA genes
in the leukemogenic transformation process.

Another important argument comes from a recently
performed gene-expression study using paediatric
t(4;11) leukemia patients. About 60% of patients inves-
tigated displayed the typical HOXA signature (HOXA5,
HOXA9 and HOXA10), whereas 40% exhibited a com-
pletely different signature, with  100-fold downregu-
lated HOXA genes. By contrast, both patient subgroups
displayed similar transcriptional activation of the
MEIS1 gene [26]. The immunophenotype, clinical
parameters and response to therapy of both t(4;11)
leukemia subgroups were identical, suggesting that over-
expressed HOXA proteins are not relevant for the
resulting clinical disease phenotype. Another study per-
formed by a different group validated these findings
[27], but demonstrated that the absence of specific HOX
gene signatures was correlated with a fourfold higher
risk of relapse, and thus, predicts a much worse out-
come for these patients. The combined data indicate
that the transformation mechanism in t(4;11) leukemia
is presumably different from those provided by other
MLL fusions that require activated HOX genes, in
particular HOXA9, for malignant transformation [28].
Some tested MLL fusion genes (MLL–FBP17 and
MLL–LASP1) scored negatively in replating assays
and no animal models could be established from these
MLL fusions [29,30]. These data may indicate that not
every tested derivative(11)–derived MLL fusion allele
is capable of conferring clonal growth. Because these
negatively scoring MLL fusion alleles have been identi-
fied and cloned from acute leukemia patients, this may

argue for the presence of specific mutations in the
cloned constructs, complementing mutations or other
supporting events, for example, the activation of spe-
cific signaling pathways. In order to answer this impor-
tant question, a careful and systematic examination of
available MLL fusion alleles (n > 60) is necessary to
identify and analyze their specific oncogenic potential.
The multitude of MLL fusion partners
A recent study summarized actual knowledge about the
MLL recombinome [31]. This comprehensive study
provided information about  759 analyzed MLL-
mediated leukemia patients and collected a total of 64
different MLL fusion partners. The analyzed MLL
fusion alleles were classified according to their occur-
rence in ALL and AML patients and their putative cel-
lular function. According to this study, 80% of all MLL
rearrangements are caused by AF4 (42%), AF9 (16%),
ENL (11%), AF10 (7%) and ELL (4%). The remaining
20% of MLL-rearranged leukemia patients displayed 59
different fusion partners, most of which were identified
in only single patients. All known MLL fusion partner
genes are categorized in Fig. 1 according to their cellu-
lar localization and their putative function. Twenty-five
of them represent nuclear proteins and 33 represent
cytosolic proteins; one fusion partner could not be clas-
sified. With few exceptions (e.g. the AF4 and SEPTIN
gene family; AF9 and ENL), all these fusion partners
share little or no homology at the protein level, indicat-
ing that different properties are provided by different
fusion proteins. The common denominator in all

different MLL rearrangements is disruption of the MLL
protein in a region that prevents any subsequent pro-
tein–protein interaction between the resulting MLL
fusion proteins. Thus, the MEN1 ⁄ LEDGF-interacting
domain linked to DNA-binding domains (AT-hook and
MT domain) becomes disconnected from the PHD
domains, the FYRN domain, the transactivating
domain, the FYRC domain and the SET domain.
Moreover, most MLL fusion partners have the ability
Role of MLL in human malignancies R. Marschalek
1824 FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS
to bind to several other proteins. Thus, the pattern of
proteins bound to both reciprocal MLL proteins is quite
complex and will influence the biological properties of a
given MLL fusion protein. Known protein interactions
of all yet characterized MLL fusion partners are
summarized in Table S1.
The positive transcription elongation
factor b system – a common
mechanism for the most frequent MLL
rearrangements
The most frequent MLL rearrangements affect a small
group of genes known as AF4, AF9, ENL, AF10 and
ELL. All these gene products participate in a common
biological reaction known as the positive transcription
elongation factor b (P-TEFb)-dependent transcrip-
tional activation cycle of RNA polymerase II, convert-
ing a ‘promoter-arrested RNA polymerase II’ into
‘elongating RNA polymerase II’ [32].
Briefly, RNA polymerase II assembles at the proximal

promoter regions of active genes. These promoter com-
plexes are arrested and characterized by their association
with the inactive DRB-sensitivity inducing factor (DSIF)
protein and the inhibitory negative elongation factor
(NELF) complex. Initial activation of this complex
results in short transcripts of  50 nucleotides. All
further steps require the presence of P-TEFb kinase
(CDK9 ⁄ CCNT1) and TFIIH (CDK7 ⁄ CCNH): phos-
phorylation of the C-terminal domain-tail of the largest
subunit of RNA polymerase II at serine 2 and 5; phos-
phorylation of DSIF (converts DSIF into an activator);
and phosphorylation of components of the NELF com-
plex, which leads to their dissociation and subsequent
destruction.
However, nuclear P-TEFb complexes are mostly
kept in an inactive state because of an interaction with
a nuclear complex (HEXIM1 ⁄ 7SK ⁄ LARP7 ⁄ MEPCE).
Thus, active P-TEFb kinase is not easily available for
RNA polymerase II. Only a small portion of P-TEFb
kinase is already associated with BRD4, an activator
of P-TEFb kinase which is able to directly bind to his-
tone proteins.
Recently, functional analysis of the above-mentioned
fusion partner proteins – AF4 (family members), AF9,
ENL and AF10 – has shed light on the activation cycle
of P-TEFb kinase. All assemble in a high-molecular
mass complex that binds to DOT1L and P-TEFb kinase
[33]. AF4-bound P-TEFb kinase becomes activated and
interacts with promoter-arrested RNA polymerase.
Activated P-TEFb kinase then phosphorylates DSIF

and NELF. Phosphorylation of AF4, AF9 and ENL
turn them into substrates for proteasomal degradation
[34]. DOT1L, P-TEFb kinase and ELL remain with the
elongating RNA polymerase II until the transcriptional
process comes to an end. P-TEFb can then again associ-
ate with available HEXIM1 ⁄ 7SK ⁄ LARP7 ⁄ MEPCE
complexes. Of interest, the MLL fusion proteins MLL–
ENL, MLL–AF9 and MLL–AF10 are able to bind to
the endogenous AF4 complex, thus influencing the
molecular machinery that activates P-TEFb kinase and
RNA polymerase II.
Fig. 1. Cellular localization of all known
mixed lineage leukemia (MLL) fusion
partners and their functions. All known MLL
fusion partners are shown by their normal
cellular localization and function. Gene
names shown in red have been identified
recurrently in MLL-rearranged leukemias,
whereas all others (in blue) have been
identified only once. Thirty proteins reside in
the nucleus, while 33 proteins are localized
in the cytosol, were associated with
the membrane or display extracellular
localization. One protein is currently not
classified.
R. Marschalek Role of MLL in human malignancies
FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS 1825
A common mechanism for the most
frequent MLL fusion partners
The question remains: what are the malignant func-

tions provided by the above-mentioned MLL fusion
proteins? A first glimpse came from two recent studies.
Krivtsov et al. [35] demonstrated that expression of a
transgenic Mll–AF4 knockin allele confers ectopic
H3K79 signatures on transcribed regions, thereby
changing the epigenetic code in a genome-wide fash-
ion. This is most likely because the tested Mll–AF4
knockin allele encodes a fusion protein that retains the
ability to bind to AF9, ENL, AF10 and DOT1L, and
thus compete with their binding to the AF4 complex.
An as yet unpublished study has demonstrated that
the reciprocal AF4–MLL fusion protein retains its
H3K4 HMT activity and is able to bind to P-TEFb
kinase and RNA polymerase II (A. Benedikt, unpub-
lished data). The presence of the AF4–MLL fusion
protein seems to enhance transcription via activation
of P-TEFb kinase. In line with this, after 5 days of
induction, ectopic expression of AF4–MLL resulted in
the transcriptional deregulation of 660 genes, of which
580 (88%) were transcriptionally activated, whereas
only 80 were downregulated [24].
From the data presented it is clear that AF4 plays a
central role. AF4 serves as a protein-binding platform
for several other proteins to initiate a fundamental cel-
lular process. P-TEFb binds to the N-terminal portion
of AF4, whereas the C-terminal portion of AF4 con-
fers binding to ENL and ⁄ or AF9 (which in turn binds
to AF10 and DOT1L). Therefore, MLL–AF4, MLL–
AF9, MLL–ENL and MLL–AF10 fusion proteins are
all functionally equivalent as they all bind, directly or

indirectly, to the DOT1L protein. Because all the
above-mentioned MLL fusion proteins also retain the
ability to bind to MEN1, the H3K79 histone methyla-
tion activity of DOT1L activity is now conferred in a
MEN1-dependent fashion. Thus, all promoters nor-
mally bound by MEN1 ⁄ MLL complexes may acquire
ectopic H3K79 signatures.
With the exception of AF4–MLL, all reciprocal
MLL fusions of the above-mentioned MLL rearrange-
ments will not have any effect on transcriptional pro-
cesses, despite representing 5¢-truncated MLL proteins
which might be still able to confer H3K4 HMT activ-
ity in a MEN1-independent fashion. AF4–MLL,
however, is the only reciprocal fusion protein that
retains the ability to directly interact with P-TEFb via
the N-terminal portion of AF4, and thus to interfere
with a fundamental mechanism necessary for the elon-
gation state of RNA polymerase II (A. Benedikt,
unpublished data). This is also reflected by the fact
that murine hematopoietic stem ⁄ precursor cells, trans-
duced with only the AF4–MLL transgene, developed
an acute lymphoblastic leukemia within  6 months
[36].
P-TEFb as potential drug target
As outlined above, the most frequent MLL fusion pro-
teins in AML and ALL derive from chromosomal trans-
locations t(4;11), t(11;19), t(9;11) and t(10;11),
respectively. The encoded fusion proteins, MLL–ENL,
MLL–AF9 and MLL–AF10, are all able to directly bind
to the AF4 complex, thus influencing the properties of

an ‘RNA polymerase II activator complex’. By contrast,
MLL–AF4 binds to pre-assembled ENL ⁄
AF10 ⁄ DOT1L, competing for factors that normally
bind to the AF4 complex. The oncogenic AF4–MLL
fusion protein binds directly to P-TEFb and strongly
activates its kinase function (A. Benedikt, unpublished
data). Activated P-TEFb can be inhibited by the potent
CDK9 inhibitor, flavopiridol, an experimental drug
identified in 1992 as an anticancer drug [37]. Flavopir-
idol has been tested in several clinical trials but was
found to be effective in only few malignacies when
administered in a certain way (e.g. chronic lymphoblas-
tic leukemia). Replication of HIV-1 is also strongly
inhibited by flavopiridol in low nanomolar concentra-
tions, because transcription elongation of HIV-1 is regu-
lated by the TAT ⁄ TAR ⁄ P-TEFb system [38]. Therefore,
CDK9 inhibitors may be a promising tool with which to
gain insight into the molecular mechanisms of MLL-
mediated leukemia. Moreover, many CDK inhibitors
are cross-reactive against glycogen synthase kinase
(GSK) proteins [39]. This may allow specific targeting of
two different mechanisms at the same time (see below:
WNT-signaling pathway; P-TEFb mediated elongation
control of RNA polymerase II), both of which seem to
be crucial for MLL-mediated acute leukemia.
Signaling and MLL-mediated leukemias
Very few studies have tried to experimentally investi-
gate signaling pathways that might be important for
MLL-rearranged cells. As a matter of fact, leukemic
cells obtained from MLL-mediated leukemia patients

tend to die very quickly when cultured ex vivo. This
may indicate that MLL-rearranged cells are highly sen-
sitive to environmental changes and depend strongly
on specific extracellular signals. By contrast, leukemia
patients are hard to cure, indicating that MLL-rear-
ranged leukemia cells can survive perfectly in vivo and
display therapy-resistance when in their specific envi-
ronment. Assuming that the bone marrow (or a similar
Role of MLL in human malignancies R. Marschalek
1826 FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS
niche) in leukemia patients provides an environment in
which leukemic cells receive signals to trigger the sur-
vival of cancer stem cells, whereas a loss-of-contact to
this environment may trigger proliferation of the
tumor bulk, one might speculate that leukemia cells
have the general ability to switch between a quiescent
state and massive proliferation.
Tumor stem cells are a challenging issue in leukemia
research and serious efforts have been undertaken to
characterize such cells in MLL-rearranged leukemias.
Leukemic stem cells are steered by several key players
such as BMI-1, p21 and proteins of the FOXO family
that are counter-regulated by the phosphatidylinositol
3 kinase (PI3K) ⁄ AKT signaling pathway [40]. Stem
cells have the ability to control a full repertoire of
mechanisms, for example, pumping different drugs to
the outside of the cell, and thus are hard to address
pharmacologically. The mode of proliferation – resul-
ting in large numbers of tumor cells – is presumably
the target of current chemotherapies, because most

therapeutics interfere with DNA synthesis or cause
severe DNA damage.
Two questions related to this topic are: what types
of extracellular signals trigger the switch between the
above-described modes and which signaling pathways
are involved? However, despite the high FLT3 expres-
sion, which might be targeted by the potent inhibitors
PKC412 of CEP-701, very few are currently known.
Therefore, the recently performed study in Michael
Cleary’s laboratory was quite a surprise [41]. Wang
and co-workers demonstrated that active GSK3 is nec-
essary for MLL-mediated leukemia cells to survive.
GSK3 is implicated in different signaling pathways,
for example, protein kinase C, protein kinase A,
RAS ⁄ RAF, WNT-, phosphatidylinositol 3-kinase and
Hedgehog, and thus affects metabolism, the cell cycle,
gene expression, developmental processes and oncogen-
esis. Active GSK3 is indicative of absent WNT-signal-
ing and leads to the proteasomal destruction of
GSK3-phosphorylated b-catenin. Active GSK3 also
phosphorylates members of the MYC family and
inhibits their function, for example, their ability to
transcriptionally activate pro-apoptotic proteins. In the
above-mentioned study, active GSK3 led to a decrease
in p27
Kip1
protein levels. Because p27
Kip1
is a target
for wild-type MLL, active GSK3 seems to prevent the

growth inhibitory activity of p27
Kip1
[41]. Thus, active
GSK3 may counteract the growth-inhibiting properties
of MLL fusion proteins during their proliferation
state, whereas inhibition of GSK3 is presumably linked
to quiescence, as it results in dephosphorylated FOXO
proteins which enable the quiescent phenotype (Fig. 2).
The mode of action and why two GSK3 inhibitors,
lithium and SB216763, had such an impact on the sur-
vival of MLL-rearranged leukemia cells remain
unclear. However, there are two possible explanations
for these findings. First, C-MYC protein is protected
against degradation if PI3K or GSK3 inhibitors block
GSK3 activity. MLL–ENL requires overexpressed
C-MYC protein to cause differentiation arrest in myel-
omonocytic progenitors, whereas a dominant-negative
C-MYC variant neutralized the oncogenic effects
mediated by the MLL–ENL fusion protein [42].
Thus, C-MYC protein initiates proliferation, blocks
differentiation and transcriptionally activates several
pro-apoptotic genes, for example, BAX, BIM and
Fig. 2. GSK3 signaling. GSK3 is a key mole-
cule involved in several pathways (PKA,
PKC, RAS ⁄ RAF, AKT, WNT, HH and mTOR).
GSK3 is normally inactivated by specific
phosphorylation at the serine 9 residue. This
renders GSK3 inactive and allows physiolo-
gical reactions such as b-catenin and insulin
signaling, as well as apoptosis. Active GSK3

blocks HH signaling via SMO, and also
blocks apoptosis and MYC-mediated
actions. Moreover, it allows clonal growth
and stabilizes mitochondria. Inhibition of
active GSK3 by lithium or other GSK3
inhibitors leads to cell growth, but may
block differentiation and cause induction of
apoptosis in MLL-rearranged cell lines.
R. Marschalek Role of MLL in human malignancies
FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS 1827
TNF-ligand [43]. This increases the susceptibility to
pro-apoptotic signals. Moreover, active AXIN ⁄ GSK3
signaling leads to the destruction of SMAD3, and thus
interferes with transforming growth factor (TGF)b sig-
naling [44]. In line with this, GSK3 has recently been
identified in a complex with DDX3 and cellular inhibi-
tor of apoptosis 1 that prevent apoptotic signaling via
competitive binding to death receptors [45]. As men-
tioned above, a second explanation is the inhibitory
effect of mostly all GSK3 inhibitors against certain
CDKs, including CDK9 [39]. As outlined above, inhi-
bition of CDK9 will presumably impair P-TEFb func-
tions associated with several MLL fusion proteins.
This influences cell growth and survival, as recently
demonstrated [46].
Moreover, Fig. 2 summarizes different signaling
pathways that should be strictly controlled or
completely shut-off in MLL-rearranged leukemia cells,
because they would otherwise inactivate GSK3 by
phosphorylation of serine 9. This could be explained

by overexpression of cellular phosphatases that are
able to interfere with these signaling pathways, for
example, PP2A. The phosphatase PP2A has been
described as being associated with the N-terminal por-
tion of MLL [47]. This may indicate that the MLL
complex provides additional functions that are not
restricted to the nucleus, but are also exhibited in the
cytosol of cells. Therefore, functional analysis of differ-
ent signaling pathways in MLL-rearranged leukemia
cells may provide an interesting way to identify
novel targets or potent therapeutics for this type of
leukemia.
Quiescence of cancer stem cells and
the potential role of MLL fusion
proteins
Recent advances in the characterization of leukemic
stem cells in non-MLL leukemias also shed light on a
new mechanism that contributes to the stem cell features
of leukemic cells. Viale and co-workers demonstrated
that the p21 protein plays a central role in specific mye-
loid leukemias and their leukemic stem cell compart-
ment [48]. The presence of oncogenic PML–RARalpha
or AML1–ETO fusion protein resulted in oncogene-
mediated DNA damage in which p21 protein was acti-
vated to very high levels. Suppression of p21 or the use
of hematopoietic stem cells deriving from a p21
) ⁄ )
genetic background resulted in exhaustion of the leuke-
mic stem cell compartment. This was demonstrated by
the inability of transplanted leukemic cells to cause a

leukemic disease phenotype in secondary recipients.
More importantly, transcriptional activation of p21 was
p53-independent, indicating that leukemic stem cells
may use alternative pathways to activate p21. Activa-
tion of p21 in leukemic stem cells resulted in a quiescent
phenotype, allowing DNA repair processes and mainte-
nance of the leukemic stem compartment [49]. A com-
plex scenario is depicted in Fig. 3 in which active TGFb
signaling, inactive WNT-signaling (= active GSK3)
and several key processes may explain the observed
effects. TGFb signaling led to the formation of a protein
complex that consists of unphosphorylated FOXO
proteins 1, 3a and 4 in conjunction with phosphorylated
SMAD3 and SMAD4. This protein complex can
Fig. 3. The FOXO ⁄ SMAD switch: regulation
of stem cell features. Known pathways
involved in WNT and TGFb signaling, as well
as the ‘FOXO ⁄ SMAD switch’, are depicted.
Regulatory pathways switch between a pro-
liferation state (upper) and a quiescent state
(lower). The p21 protein plays a central role
in the maintenance and quiescence of
leukemic stem cells. MLL FA, MLL fusion
allele. Green arrows, functional ⁄ transcrip-
tional activation; red arrows, inhibitory
function. Tx, act through transcriptional
activation.
Role of MLL in human malignancies R. Marschalek
1828 FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS
directly activate transcription of the CDKN1a ⁄ p21 gene,

explaining why p53 was not necessary for the transcrip-
tional activation of CDKN1a ⁄ p21. It also explains the
observations made by Wang and co-workers, because
inhibition of GSK3 by lithium or SB216763 results in
b-catenin stabilization, which in turn will result in the
production of MYC protein. MYC protein, however,
effectively blocks transcription of the CDKN1a ⁄ p21
gene. Thus, it would be of great interest to analyze the
WNT and TGFb signaling pathways in MLL-rear-
ranged leukemias, asking whether the absence of active
WNT signaling (absence of WNT or FZD ⁄ LRP; pres-
ence of inhibitory WIF1 or DKK-, SFRP-family mem-
bers) and active TGFb signaling are necessary for the
survival of MLL-mediated leukemia cells (Fig. 3).
Moreover, the FOXO ⁄ SMAD protein complex is able
to transcriptionally activate BMI-1, which controls p16
and ARF production, as well as GADD45, SOD2 and
some other genes that protect cells against stress-medi-
ated reactive oxygen species. Interestingly, GADD45a
has recently been shown to be involved in reactivation
the OCT4 gene locus [50]. OCT4 transcriptionally acti-
vates the NANOG gene locus [51], whereas forced
NANOG overexpression led to transcriptional activa-
tion of the EGR1 gene in non-embryonic stem cells
(I. Eberle, unpublished data). EGR1 has been shown to
transcriptionally activate the CDKN1a ⁄ p21 gene [52].
Alternatively, KLF4 and PBX1 are also able to trans-
criptionally activate the NANOG gene [53], whereas
KLF4 alone is also able to transcriptionally activate the
CDKN1a ⁄ p21 gene [54]. Of interest, transcriptional acti-

vation of NANOG and OCT4 has recently been identi-
fied in an in vitro model system when both t(4;11) fusion
proteins were present. This finding was then validated in
infant and adult t(4;11) leukemia patients [23]. Thus, the
switch between cell growth and quiescence in MLL-
mediated leukemia cells is possibly controlled by a
‘FOXO ⁄ SMAD switch’ which in turn allows re-activa-
tion of embryonic stem cell genes and controls
CDKN1a ⁄ p21 independent of p53. These pathways are
highly attractive for future research and have the poten-
tial for therapeutic intervention. This model would also
explain recent findings in which ‘leukemic stem cells’ –
able to initiate leukemias in a NOD ⁄ SCID mouse model
– have been identified in sorted cells with quite diverse
immunophenotypes (± CD34, ± CD19), indicating
that stem cell characteristics may not be restricted to a
hierarchic stem cell compartment in ALL [55].
Acknowledgements
I thank Geertruy te Kronnie and Theo Dingermann
for critically reading the manuscript. I want to apolo-
gize for not-citing many references due to a citation
limit for this minireview. This work is supported by
research grant 107819 from the Deutsche Krebshilfe
e.V. to RM.
References
1 Holleman A, Cheok MH, den Boer ML, Yang W,
Veerman AJ, Kazemier KM, Pei D, Cheng C, Pui CH,
Relling MV et al. (2004) Gene-expression patterns in
drug-resistant acute lymphoblastic leukemia cells and
response to treatment. N Engl J Med 351, 533–542.

2 Ayton PM & Cleary ML (2003) Transformation of
myeloid progenitors by MLL oncoproteins is dependent
on Hoxa7 and Hoxa9. Genes Dev 17, 2298–2307.
3 Hess JL (2004) Mechanisms of transformation by MLL.
Crit Rev Eukaryot Gene Expr 14, 235–254.
4 Eklund EA (2007) The role of HOX genes in malignant
myeloid disease. Curr Opin Hematol 14, 85–89.
5 Horton SJ, Grier DG, McGonigle GJ, Thompson A,
Morrow M, De Silva I, Moulding DA, Kioussis D,
Lappin TR, Brady HJ et al. (2005) Continuous MLL–
ENL expression is necessary to establish a ‘Hox Code’
and maintain immortalization of hematopoietic progeni-
tor cells. Cancer Res 65, 9245–9252.
6 Djabali M, Selleri L, Parry P, Bower M, Young BD &
Evans GA (1992) A trithorax-like gene is interrupted by
chromosome 11q23 translocations in acute leukaemias.
Nat Genet 2, 113–118.
7 Mozer BA & Dawid IB (1989) Cloning and molecular
characterization of the trithorax locus of Drosoph-
ila melanogaster. Proc Natl Acad Sci USA 86, 3738–
3742.
8 Breen TR & Harte PJ (1991) Molecular characterization
of the trithorax gene, a positive regulator of homeotic
gene expression in Drosophila. Mech Dev 35, 113–127.
9 Arai F, Hirao A & Suda T (2005) Regulation of hema-
topoietic stem cells by the niche. Trends Cardiovasc
Med 15, 75–79.
10 De Toni F, Racaud-Sultan C, Chicanne G, Mas VM,
Cariven C, Mesange F, Salles JP, Demur C, Allouche
M, Payrastre B et al. (2006) A crosstalk between the

Wnt and the adhesion-dependent signaling pathways
governs the chemosensitivity of acute myeloid leukemia.
Oncogene 25, 3113–3122.
11 Hsieh JJD, Cheng EH & Korsmeyer SJ (2003)
Taspase1: a threonine aspartase required for cleavage
of MLL and proper HOX gene expression. Cell 115,
293–303.
12 Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia
T, Wassell R, Dubois G, Mazo A, Croce CM & Canaani
E (2002) ALL-1 is a histone methyltransferase that
assembles a supercomplex of proteins involved in
transcriptional regulation. Mol Cell 10, 1119–1128.
R. Marschalek Role of MLL in human malignancies
FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS 1829
13 Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero
DJ, Kitabayashi I, Herr W & Cleary ML (2004) Leuke-
mia proto-oncoprotein MLL forms a SET1-like histone
methyltransferase complex with menin to regulate Hox
gene expression. Mol Cell Biol 24, 5639–5649.
14 Ruthenburg AJ, Allis CD & Wysocka J (2007) Methyl-
ation of lysine 4 on histone H3: intricacy of writing and
reading a single epigenetic mark. Mol Cell 25, 15–30.
15 Guenther MG, Jenner RG, Chevalier B, Nakamura T,
Croce CM, Canaani E & Young RA (2005) Global and
Hox-specific roles for the MLL1 methyltransferase.
Proc Natl Acad Sci USA 102, 8603–8608.
16 Lobato MN, Metzler M, Drynan L, Forster A, Pannell
R & Rabbitts TH (2008) Modeling chromosomal trans-
locations using conditional alleles to recapitulate initiat-
ing events in human leukemias. J Natl Cancer Inst

Monogr 39, 58–63.
17 Van Beusechem VW & Valerio D (1996) Gene transfer
into hematopoietic stem cells of nonhuman primates.
Hum Gene Ther 7, 1649–1668.
18 Wong P, Iwasaki M, Somervaille TC, So CWE &
Cleary ML (2009) Meis1 is an essential and rate-limit-
ing regulator of MLL leukemia stem cell potential.
Genes Dev 21, 2762–2774.
19 Milne TA, Dou Y, Martin ME, Brock HW, Roeder
RG & Hess JL (2005) MLL associates specifically with
a subset of transcriptionally active target genes. Proc
Natl Acad Sci USA 102, 14765–14770.
20 Wang GG, Pasillas MP & Kamps MP (2006) Persis-
tent transactivation by meis1 replaces hox function in
myeloid leukemogenesis models: evidence for co-occu-
pancy of meis1–pbx and hox–pbx complexes on pro-
moters of leukemia-associated genes. Mol Cell Biol 26,
3902–3916.
21 Yokoyama A & Cleary ML (2008) Menin critically
links MLL proteins with LEDGF on cancer-associated
target genes. Cancer Cell 14, 36–46.
22 Caslini C, Yang Z, El-Osta M, Milne TA, Slany RK &
Hess JL (2007) Interaction of MLL amino terminal
sequences with menin is required for transformation.
Cancer Res 67, 7275–7283.
23 Caslini C, Serna A, Rossi V, Introna M & Biondi A
(2004) Modulation of cell cycle by graded expression of
MLL–AF4 fusion oncoprotein. Leukemia 18, 1064–
1071.
24 Gaussmann A, Wenger T, Eberle I, Bursen A, Bracharz

S, Herr I, Dingermann T & Marschalek R (2007) The
combined effects of the two reciprocal t(4;11) fusion
proteins, MLL•AF4 and AF4•MLL, confer resistance
to apoptosis, cell cycling capacity and growth transfor-
mation. Oncogene 26, 3352–3363.
25 Meyer C, Burmeister T, Strehl S, Schneider B, Hubert
D, Zach O, Haas O, Klingebiel T, Dingermann T &
Marschalek R (2007) Spliced MLL fusions: a novel
mechanism to generate functional chimeric MLL–
MLLT1 transcripts in t(11;19)(q23;p13.3) leukemia.
Leukemia 21, 588–590.
26 Trentin L, Girodan M, Dingermann T, Basso G, te
Kronnie G & Marschalek R (2009) Two independent
gene signatures in pediatric t(4;11) acute lymphoblastic
leukemia patients. Eur J Haematol 83, 406–419.
27 Stam RW, Schneider P, Hagelstein JA, van der Linden
MH, Stumpel DJ, de Menezes RX, de Lorenzo P,
Valsecchi MG & Pieters R (2009) Gene expression pro-
filing-based dissection of MLL translocated and MLL
germline acute lymphoblastic leukemia in infants. Blood
doi: 10.1182/blood-2009-07-233049.
28 Faber J, Krivtsov AV, Stubbs MC, Wright R, Davis
TN, van den Heuvel-Eibrink M, Zwaan CM, Kung AL
& Armstrong SA (2009) HOXA9 is required for sur-
vival in human MLL-rearranged acute leukemias. Blood
113, 2375–2385.
29 Fuchs U, Rehkamp G, Haas OA, Slany R, Ko
¨
nig M,
Bojesen S, Bohle RM, Damm-Welk C, Ludwig WD,

Harbott J et al. (2001) The human formin-binding pro-
tein 17 (FBP17) interacts with sorting nexin, SNX2, and
is an MLL-fusion partner in acute myelogeneous leuke-
mia. Proc Natl Acad Sci USA 98, 8756–8761.
30 Strehl S, Borkhardt A, Slany R, Fuchs UE, Ko
¨
nig M
& Haas OA (2003) The human LASP1 gene is fused to
MLL in an acute myeloid leukemia with
t(11;17)(q23;q21). Oncogene 22, 157–160.
31 Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J,
Trka J, Ben Abdelali R, Macintyre E, De Braekeleer E,
De Braekeleer M et al. (2009) New insights into the
MLL recombinome of acute leukemias. Leukemia 23,
1490–1499.
32 Peterlin BM & Price DH (2006) Controlling the elonga-
tion phase of transcription with P-TEFb. Mol Cell 23,
297–305.
33 Zeisig DT, Bittner CB, Zeisig BB, Garcı
´
a-Cue
´
llar MP,
Hess JL & Slany RK (2005) The eleven-nineteen-leuke-
mia protein ENL connects nuclear MLL fusion partners
with chromatin. Oncogene 24, 5525–5532.
34 Bitoun E, Oliver PL & Davies KE (2007) The mixed-
lineage leukemia fusion partner AF4 stimulates RNA
polymerase II transcriptional elongation and mediates
coordinated chromatin remodeling. Hum Mol Genet 16,

92–106.
35 Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati
S, Sinha AU, Xia X, Jesneck J, Bracken AP, Silverman
LB et al. (2008) H3K79 methylation profiles define mur-
ine and human MLL–AF4 leukemias. Cancer Cell 14,
355–368.
36 Bursen A, Schwabe K, Ru
¨
ster B, Henschler R,
Ruthardt M, Dingermann T & Marschalek R (2009)
The AF4•MLL fusion protein is capable of inducing
ALL in mice without requirement of MLL AF4. Blood.
37 Kaur G, Stetler-Stevenson M, Sebers S, Worland P,
Sedlacek H, Myers C, Czech J, Naik R & Sausville E
Role of MLL in human malignancies R. Marschalek
1830 FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS
(1992) Growth inhibition with reversible cell cycle arrest
of carcinoma cells by flavone L86-8275. J Natl Cancer
Inst 84, 1736–1740.
38 Chao SH, Fujinaga K, Marion JE, Taube R, Sausville
EA, Senderowicz AM, Peterlin BM & Price DH (2000)
Flavopiridol inhibits P-TEFb and blocks HIV-1 replica-
tion. J Biol Chem 275, 28345–28348.
39 Knockaert M, Greengard P & Meijer L (2002) Pharma-
cological inhibitors of cyclin-dependent kinases. Trends
Pharmacol Sci 23, 417–425.
40 Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon
DH, Cullen DE, McDowell EP, Lazo-Kallanian S,
Williams IR, Sears C et al. (2007) FoxOs are critical
mediators of hematopoietic stem cell resistance to physi-

ologic oxidative stress. Cell 128, 325–339.
41 Wang Z, Smith KS, Murphy M, Piloto O, Somervaille
TC & Cleary ML (2008) Glycogen synthase kinase 3 in
MLL leukaemia maintenance and targeted therapy.
Nature 455, 1205–1209.
42 Schreiner S, Birke M, Garcı
´
a-Cue
´
llar MP, Zilles O,
Greil J & Slany RK (2001) MLL–ENL causes a revers-
ible and myc-dependent block of myelomonocytic cell
differentiation. Cancer Res 61, 6480–6486.
43 Kotliarova S, Pastorino S, Kovell LC, Kotliarov Y,
Song H, Zhang W, Bailey R, Maric D, Zenklusen JC,
Lee J et al. (2008) Glycogen synthase kinase-3 inhibi-
tion induces glioma cell death through c-MYC, nuclear
factor-kappaB, and glucose regulation. Cancer Res 68,
6643–6651.
44 Guo X, Ramirez A, Waddell DS, Li Z, Liu X & Wang
XF (2008) Axin and GSK3- control Smad3 protein sta-
bility and modulate TGF-signaling. Genes Dev 22, 106–
120.
45 Sun M, Song L, Li Y, Zhou T & Jope RS (2008)
Identification of an antiapoptotic protein complex at
death receptors. Cell Death Differ 15, 1887–1900.
46 Mueller D, Garcı
`
a-Cue
`

llar MP, Bach C, Buhl S,
Maethner E & Slany RK (2009) Misguided transcrip-
tional elongation causes mixed lineage leukemia. PLoS
Biol 7, e1000249, doi: 10.1371/journal.pbio.1000249.
47 Adler HT, Nallaseth FS, Walter G & Tkachuk DC
(1997) HRX leukemic fusion proteins form a hetero-
complex with the leukemia-associated protein SET and
protein phosphatase 2A. J Biol Chem 272, 28407–28414.
48 Viale A, De Franco F, Orleth A, Cambiaghi V, Giuliani
V, Bossi D, Ronchini C, Ronzoni S, Muradore I,
Monestiroli S et al. (2009) Cell-cycle restriction limits
DNA damage and maintains self-renewal of leukaemia
stem cells. Nature 457, 51–56.
49 Seoane J, Le HV, Shen L, Anderson SA & Massague
´
J
(2004) Integration of Smad and forkhead pathways in
the control of neuroepithelial and glioblastoma cell pro-
liferation. Cell 117, 211–223.
50 Barreto G, Scha
¨
fer A, Marhold J, Stach D, Swamina-
than SK, Handa V, Do
¨
derlein G, Maltry N, Wu W,
Lyko F et al.
(2007) Gadd45a promotes epigenetic gene
activation by repair-mediated DNA demethylation.
Nature 445, 671–675.
51 Kuroda T, Tada M, Kubota H, Kimura H, Hatano

SY, Suemori H, Nakatsuji N & Tada T (2005) Octamer
and Sox elements are required for transcriptional cis
regulation of Nanog gene expression. Mol Cell Biol 25,
2475–2485.
52 Ragione FD, Cucciolla V, Criniti V, Indaco S, Borriello
A & Zappia V (2003) p21Cip1 gene expression is modu-
lated by Egr1: a novel regulatory mechanism involved
in the resveratrol antiproliferative effect. J Biol Chem
278, 23360–23368.
53 Chan KK, Zhang J, Chia NY, Chan YS, Sim HS, Tan
KS, Oh SK, Ng HH & Choo AB (2009) KLF4 and
PBX1 directly regulate NANOG expression in human
embryonic stem cells. Stem Cells 27, 2114–2125.
54 Rowland BD & Peeper DS (2006) KLF4, p21 and
context-dependent opposing forces in cancer. Nat Rev
Cancer 6, 11–23.
55 le Viseur C, Hotfilder M, Bomken S, Wilson K,
Ro
¨
ttgers S, Schrauder A, Rosemann A, Irving J, Stam
RW, Shultz LD et al. (2009) In childhood acute
lymphoblastic leukemia, blasts at different stages of
immunophenotypic maturation have stem cell
properties. Cancer Cell 14, 47–58.
Supporting information
The following supplementary material is available:
Table S1. All protein-protein interaction data were
obtained from the biogrid Database (www.thebiogrid.org)
and listed according their order [45]. Bold: MLL fusion
partner that has been i dentified recurrently in MLL rear-

rangements; underlined: interacting proteins found to be
twice or more as binding partner for different MLL
fusion partner proteins.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
R. Marschalek Role of MLL in human malignancies
FEBS Journal 277 (2010) 1822–1831 ª 2010 The Author Journal compilation ª 2010 FEBS 1831