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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: 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 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. 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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 . MINIREVIEW Mixed lineage leukemia: roles in human malignancies and potential therapy Rolf Marschalek Biochemistry, Chemistry & Pharmacy, Institute. 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