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Keywords:

  • acute leukemia;
  • AF4;
  • AF9;
  • AF10;
  • cancer stem cells;
  • ELL;
  • ENL;
  • MLL;
  • MLL fusion proteins;
  • signaling

Abstract

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 strategies. 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 elongation factor b-dependent activation cycle of RNA polymerase II. Biological 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 information 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

Mixed lineage leukemia fusions, acute leukemia and the HOX signature

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

Mixed lineage leukemia (MLL) rearrangements define a small subset of acute leukemia patients, including those with therapy-induced secondary leukemias. However, unlike many other types of leukemia, the presence 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 replating 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 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

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

The MLL protein has been identified as the mammalian orthologue of the Trithorax protein in invertebrates [6]. Disruption of this gene in invertebrates and vertebrates leads to homeotic transformation and null-alleles are incompatible with normal embryonic development [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 interact 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 expression is initiated at very early stages of embryogenesis. The MLL protein is specifically hydrolysed by the endopeptidase Taspase1 [11]. This allows it to assembly into a high-molecular mass complex which confers the methylation 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 transcriptional maintenance. Therefore, MLL is part of an epigenetic system that guarantees mitotically stable gene-expression signatures during embryonic development, 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 proteins 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 promoter regions, the MLL complex obviously binds to different promoters in various tissues. In a recent study, occupancy of MLL protein was investigated using chromatin immunoprecipitation experiments and subsequent analysis on genome-wide tiling arrays [15]. This study revealed that MLL was bound to > 2000 different promoter regions within the cell line investigated (U937), of which 99% were also bound by RNA polymerase II. However, active transcription can be blocked by associated 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

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 transduction of murine hematopoietic stem cells to functionally investigate the oncogenic properties of distinct MLL fusion alleles. Manipulated hematopoietic stem/precursor cells were tested in methylcellulose assays for their clonal growth and replating efficiency, and the resulting colonies were transplanted into recipient mice of various genetic backgrounds. Alternatively, manipulated stem/precursor cells were used directly for transplantation 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 capacity 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 overexpressed 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 different 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 expression 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 transcriptionally 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 rearrangements 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 transcriptional 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. However, without testing both reciprocal fusion alleles in the same test system, it is impossible to answer the important 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 investigated displayed the typical HOXA signature (HOXA5, HOXA9 and HOXA10), whereas 40% exhibited a completely different signature, with ∼ 100-fold downregulated 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 overexpressed HOXA proteins are not relevant for the resulting clinical disease phenotype. Another study performed 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 outcome 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 identified 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 specific signaling pathways. In order to answer this important 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

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 occurrence in ALL and AML patients and their putative cellular 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 cellular localization and their putative function. Twenty-five of them represent nuclear proteins and 33 represent cytosolic proteins; one fusion partner could not be classified. 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, indicating 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 protein–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.

image

Figure 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.

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The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 transcriptional activation cycle of RNA polymerase II, converting 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 complexes 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): phosphorylation of the C-terminal domain-tail of the largest subunit of RNA polymerase II at serine 2 and 5; phosphorylation of DSIF (converts DSIF into an activator); and phosphorylation of components of the NELF complex, 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 histone 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 associate 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.

A common mechanism for the most frequent MLL fusion partners

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

The question remains: what are the malignant functions 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 fashion. 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, unpublished 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 cellular process. P-TEFb binds to the N-terminal portion of AF4, whereas the C-terminal portion of AF4 confers 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 methylation activity of DOT1L activity is now conferred in a MEN1-dependent fashion. Thus, all promoters normally 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 rearrangements will not have any effect on transcriptional processes, despite representing 5′-truncated MLL proteins which might be still able to confer H3K4 HMT activity 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 elongation state of RNA polymerase II (A. Benedikt, unpublished data). This is also reflected by the fact that murine hematopoietic stem/precursor cells, transduced with only the AF4–MLL transgene, developed an acute lymphoblastic leukemia within ∼ 6 months [36].

P-TEFb as potential drug target

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

As outlined above, the most frequent MLL fusion proteins in AML and ALL derive from chromosomal translocations 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]. Flavopiridol 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 lymphoblastic leukemia). Replication of HIV-1 is also strongly inhibited by flavopiridol in low nanomolar concentrations, because transcription elongation of HIV-1 is regulated 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

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

Very few studies have tried to experimentally investigate 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 sensitive to environmental changes and depend strongly on specific extracellular signals. By contrast, leukemia patients are hard to cure, indicating that MLL-rearranged leukemia cells can survive perfectly in vivo and display therapy-resistance when in their specific environment. Assuming that the bone marrow (or a similar niche) in leukemia patients provides an environment in which leukemic cells receive signals to trigger the survival 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 – resulting 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 expression, 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 necessary 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 oncogenesis. Active GSK3 is indicative of absent WNT-signaling and leads to the proteasomal destruction of GSK3-phosphorylated β-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).

image

Figure 2.  GSK3 signaling. GSK3 is a key molecule 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 physiological reactions such as β-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.

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The mode of action and why two GSK3 inhibitors, lithium and SB216763, had such an impact on the survival 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 myelomonocytic 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 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)β signaling [44]. In line with this, GSK3 has recently been identified in a complex with DDX3 and cellular inhibitor of apoptosis 1 that prevent apoptotic signaling via competitive binding to death receptors [45]. As mentioned above, a second explanation is the inhibitory effect of mostly all GSK3 inhibitors against certain CDKs, including CDK9 [39]. As outlined above, inhibition of CDK9 will presumably impair P-TEFb functions 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 portion 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 different 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

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 myeloid leukemias and their leukemic stem cell compartment [48]. The presence of oncogenic PML–RARalpha or AML1–ETO fusion protein resulted in oncogene-mediated DNA damage in which p21 protein was activated 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 leukemic 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. Activation of p21 in leukemic stem cells resulted in a quiescent phenotype, allowing DNA repair processes and maintenance of the leukemic stem compartment [49]. A complex scenario is depicted in Fig. 3 in which active TGFβ signaling, inactive WNT-signaling (= active GSK3) and several key processes may explain the observed effects. TGFβ 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 directly activate transcription of the CDKN1a/p21 gene, explaining why p53 was not necessary for the transcriptional 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 β-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 TGFβ signaling pathways in MLL-rearranged leukemias, asking whether the absence of active WNT signaling (absence of WNT or FZD/LRP; presence of inhibitory WIF1 or DKK-, SFRP-family members) and active TGFβ 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-mediated reactive oxygen species. Interestingly, GADD45a has recently been shown to be involved in reactivation the OCT4 gene locus [50]. OCT4 transcriptionally activates the NANOG gene locus [51], whereas forced NANOG overexpression led to transcriptional activation 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 transcriptionally activate the NANOG gene [53], whereas KLF4 alone is also able to transcriptionally activate the CDKN1a/p21 gene [54]. Of interest, transcriptional activation of NANOG and OCT4 has recently been identified 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-activation of embryonic stem cell genes and controls CDKN1a/p21 independent of p53. These pathways are highly attractive for future research and have the potential 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].

image

Figure 3.  The FOXO/SMAD switch: regulation of stem cell features. Known pathways involved in WNT and TGFβ signaling, as well as the ‘FOXO/SMAD switch’, are depicted. Regulatory pathways switch between a proliferation 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/transcriptional activation; red arrows, inhibitory function. Tx, act through transcriptional activation.

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References

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Mixed lineage leukemia fusions, acute leukemia and the HOX signature
  4. Cellular functions of the MLL protein
  5. Model systems for the analysis of MLL fusion proteins and patient analysis
  6. The multitude of MLL fusion partners
  7. The positive transcription elongation factor b system – a common mechanism for the most frequent MLL rearrangements
  8. A common mechanism for the most frequent MLL fusion partners
  9. P-TEFb as potential drug target
  10. Signaling and MLL-mediated leukemias
  11. Quiescence of cancer stem cells and the potential role of MLL fusion proteins
  12. Acknowledgements
  13. References
  14. Supporting Information

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 rearrangements; underlined: interacting proteins found to be twice or more as binding partner for different MLL fusion partner proteins.

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