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

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 acc

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: Rolf.Marschalek@em.uni-frankfurt.de

(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 funcinforma-tions 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.

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 inverteinverte-brates and

vertebrates leads to homeotic transformation and

null-alleles are incompatible with normal embryonic

devel-opment [7,8] All observed genetic mutations of the

MLLgene (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 functransduc-tionally 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

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,

HOXA9and 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

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.

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

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 p27Kip1 protein levels Because p27Kip1 is a target for wild-type MLL, active GSK3 seems to prevent the growth inhibitory activity of p27Kip1[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.

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.

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

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 MLLrecombinome of acute leukemias Leukemia 23, 1490–1499

32 Peterlin BM & Price DH (2006) Controlling the elonga-tion phase of transcripelonga-tion 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

(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 identified 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