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Keywords:

  • acute lymphoblastic leukaemia;
  • acute myeloid leukaemia;
  • MLL gene;
  • fusion partners;
  • MLL-associated infant leukaemia

Summary

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

Infant acute leukaemia is characterised by specific genetic rearrangements and a rapid onset of disease shortly after birth. The vast majority of these cases bear rearranged MLL alleles. However, many facets of MLL-rearranged leukaemia are largely unknown. Basically, there exists a fundamental and evolutionary conserved relationship between the family of MLL/Trithorax proteins and the regulation of HOX gene clusters. Therefore, direct MLL fusion proteins are per se able to deregulate HOX genes, except when reciprocal MLL fusion proteins come into play. This reviews discusses (i) the current situation in MLL-rearranged leukaemia, (ii) the molecular and genetic tools to functionally investigate the many different MLL fusions, (iii) the latency of disease development, (iv) a novel cancer mechanism that has been recently uncovered when different MLL fusion protein complexes were characterized, (v) mutated signalling pathways in MLL-rearranged leukaemia and (vi) presents new ideas on how a given MLL fusion protein may modulate existing signalling pathways in leukaemic cells. The hypothesis is posed that the many different fusion partners of MLL are critically distinct entities for which specific inhibitors should be identified in the future.

Infant acute leukaemias are classified as aggressive tumours that need high amounts of poly-chemotherapy for treatment, although the outcome is still poor. Genetically, the group of paediatric leukaemia patients is characterized by distinct genetic rearrangements of the MLL gene, located at 11q23. Several improvements in treatment have been achieved over the last decades [recent studies from the USA and the Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP) and Berlin-Frankfüt-Münster (BFM) goups], but the general situation for this group of leukaemia patients is still unsatisfying when treated solely by chemotherapy. In line with this argument, a recent study demonstrated the benefits of haematopoietic stem cell transplantation on the overall and disease-free survival of paediatric leukaemia patients that bear MLL rearrangements (Mann et al, 2010). However, alternative strategies are still necessary to further improve outcome. The use of novel therapeutics, however, is only possible if the pathological disease mechanisms are clearly understood and rational drug design comes into play. Therefore, it is of importance to evaluate the existing data and to draw correct conclusions.

Historically, MLL fusion proteins were recognized as transcriptional deregulators of distinct HOX genes (Kawagoe et al, 1999; Imamura et al, 2002; Ayton & Cleary, 2003). Therefore, a ‘common denominator concept’ has been proposed, claiming that all MLL fusion proteins work by a similar concept: they increase and maintain high level transcription of MEIS1 and HOXA gene family members. A physical association of MEN1, LEDGF and MYB protein at the N-terminal portion of the MLL fusion protein is of functional importance (Milne et al, 2005; Caslini et al, 2007; Yokoyama & Cleary, 2008; Jin et al, 2010). Deregulated HOXA gene expression inflicts proper haematopoietic development, and subsequently, initiates the development of pre- or leukaemic cell clones.

Recent data are questioning this general concept because leukaemia cases have been observed without the activation of HOXA genes, but still with high expression of MEIS1 (Trentin et al, 2009). Paediatric leukaemia patients with a chromosomal translocation t(4;11) and low HOXA gene expression display even a worse prognosis (Stam et al, 2010).

This review focuses on recent developments and data derived from molecular analyses and functional studies. Highly complex data on a novel molecular mechanism – exerted by the most frequently occurring MLL fusion proteins – will be presented. Interesting differences in signalling pathways of MLL-rearranged leukaemia cells will also be discussed. The presented data and ideas should neither contradict nor negate exiting data, but rather demonstrate the existence of alternative cancer pathways that are of potential interest when aiming for new therapeutic strategies or drug development.

Genetic situation in MLL-rearranged leukaemia patients

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

MLL-rearranged leukaemias represent about 10% of all leukaemia cases [acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML)]. Today, 71 different MLL fusion partner genes have been characterized world-wide at the molecular level (Meyer et al, 2009 and unpublished data). MLL rearrangements can be subdivided into reciprocal chromosomal translocations (n = 43; 60·5%), 11q23ter deletions (n = 4; 5·7%) and 11q inversions (n = 8; 11·3%). All of these genetic aberrations require two DNA double-strand breaks, either on different chromosomes or on chromosome 11, respectively. Genetic aberrations based on more than two DNA strand breaks are represented by the insertion of 11q23 material into another chromosome. Such events have been described for 13 MLL fusion partners (18·3%), while the recently discovered spliced fusion mechanism (Meyer et al, 2007) seems to represent a rare mechanism that has been identified at the transcript level for three different MLL fusion partners (4·2%; see Fig 1A).

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Figure 1.  Classification of MLL rearranged leukaemia. (A) Distribution of different recombination events leading to 71 different MLL rearrangements. rTL, reciprocal chromosomal translocatiions; Ins, insertion of 11q23 material into other chromosomes; Inv, inversions on 11q; Del, deletions on 11q23; Spl, spliced MLL fusions after recombination. Numbers for each mechanism are indicated. (B) Distribution of MLL fusion partners in acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) patients. ALL patients (paediatric and adult; n = 487) predominantly display rearrangements with AFF1, MLLT1, MLLT3 and less frequently with MLLT10, MLLT4 and EPS15 (∼94%). Seventeen other MLL rearrangements are unique (u) for the group of ALL patients, while four fusion partners are shared (s) between ALL and AML patients. AML patients (paediatric and adult; n = 280) display rearrangements with MLLT3, MLLT10, ELL, MLLT4, MLLT1 and less frequently with MLLT6, MLLT10, SEPT6 and EPS15 (∼80%). Twenty other MLL rearrangements are unique (u) for the group of AML patients, while four fusion partners are shared (s) with ALL patients.

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Based on the analysis of more than 760 acute leukaemia patients, carrying 51 different MLL rearrangements, the abundance of identified fusion partner genes could be defined. ALL patients (paediatric and adult) were predominantly characterized by t(4;11) (AFF1 [AF4]; n = 319), t(11;19) (MLLT1 [ENL1]; n = 72), t(9;11) (MLLT3 [AF9]; n = 41), t(10;11) (MLLT10 [AF10]; n = 13), t(6;11) (MLLT4 [AF6]; n = 7) and t(1;11) chromosomal translocations (EPS15; n = 7). These six MLL rearrangements account for about 94% of all diagnosed ALL samples (n = 483). The spectrum in AML patients (paediatric and adult) is broader and comprises t(9,11) (MLLT3; n = 84), t(10;11) (MLLT10; n = 40), t(11;19) (ELL; n = 30), t(6;11) (MLLT4; n = 28), t(11;19) (MLLT1; n = 15), t(11;17) (MLLT6 [AF17]; n = 8), t(1;11) (MLLT11 [AF1Q]; n = 7), t(X;11) (SEPT6; n = 7) and t(1;11) translocations (EPS15; n = 6). These nine MLL rearrangements account for about 80% of all diagnosed AML samples (n = 280). All other MLL rearrangements represent rare events (n < 5), and most fusion partners were identified only once (n = 30). All these data are summarized in Fig 1B.

This analysis provides another important insight: about 10 MLL fusion partners are predominantly diagnosed in MLL-rearranged leukaemias. Only two out of these 10 MLL fusions are specifically associated with an ALL disease phenotype (AFF1 and EPS15), while eight out of 10 are predominantly associated with AML disease phenotypes (MLLT11, MLLT4, MLLT3, MLLT10, MLLT6, ELL, MLLT1 and SEPT6). The dominance of AML-associated leukaemia and the fact that nearly all published studies were performed with MLL-MLLT4, MLL-MLLT3, MLL-MLLT10 and MLL-MLLT1, explains why the available information is biased towards the pathological mechanism of acute myeloid leukaemia. Therefore, much less is known about ALL-associated MLL rearrangements and associated pathological disease mechanisms.

Model systems are the basis to understand oncogenic mechanisms

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

In order to understand malignant pathways, different types of experiments can be performed to investigate the properties exhibited by a given oncoprotein (see Fig 2). Classical experiments include the introduction of a potential oncogene in a cell culture model. More complicated is the generation of a mouse model to analyse the effects of oncoproteins. Patient leukaemia cells can be expanded in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The creation of mouse models requires either transgenic approaches or retroviral transduction of a candidate oncogene into a selected target cell population. Certain transgenic approaches (knock-in or inverter models) have the advantage that the endogenous promoter can be used to express a potential oncoprotein. However, the creation of such strains is time consuming, costly and well established only in a few laboratories. Therefore, many researchers use viral gene transfer into enriched target cell populations. However, this experimental system may suffer from unphysiological expression, transcriptional shut-down or integration mutagenesis. The latter is a real problem and rarely controlled in published experiments, although integration into or next to distinct gene loci may cause malignant transformation (reviewed in Bushman et al, 2005). Thus, results obtained with these systems have to be carefully controlled, experimentally evaluated and repeated.

image

Figure 2.  Model systems to investigate potential oncoproteins. Potential fusion genes can be either investigated by in vitro or in vivo experiments. In vitro experiments can be readily performed by using inducible expression constructs to monitor a large variety of biological parameters. In vivo models include the expansion of leukaemic patient cells, retroviral transduction of selected or unselected murine bone marrow cells, and subsequently, transplantation into isogenic mice. Alternatively, transgenic mouse lines are established. In all these systems, disease development is the experimental endpoint. Moribond mice will be sacrificed and isolated organs or cells can be investigated (e.g. by molecular, pathological or cytometric experiments).

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The available data in cell culture systems and mouse model systems are quite diverse and can be separated into two different subgroups. The first subgroup encompasses MLL-MLLT1, MLL-MLLT3, MLL-MLLT10, MLL-CBP, MLL-ELL and other MLL fusions that all cause myeloproliferative disease (MPD) or AML in mice (e.g. Corral et al, 1996; Lavau et al, 1997, 2000a,b; Dobson et al, 1999; DiMartino et al, 2002; Okada et al, 2005). All these MLL fusion proteins activate combinations of distinct Hox genes (e.g. Hoxc8, Hoxa7, Hoxa9, Hoxa10) and display high-level transcription of Meis1 (Kawagoe et al, 1999; Imamura et al, 2002; Ayton & Cleary, 2003). A similar observation has been made for MLL partial tandem duplications (Dorrance et al, 2006) or cytosolic fusion partners that exhibit protein dimerization domains (Martin et al, 2003). The basic mechanism of all these MLL fusion proteins is based on their ability to interact with Menin1 (Milne et al, 2005; Caslini et al, 2007). Menin1 is a transcription factor that binds to several hundred target genes (Scacheri et al, 2006). The association of Ledgf and Myb with Menin1 is also necessary for the transformation capacity of these MLL fusion proteins, because knock-down of either of those interferes with malignant transformation (Yokoyama & Cleary, 2008; Jin et al, 2010). Another key player is Meis1 (Wong et al, 2007; Kumar et al, 2009) but also the remaining wildtype MLL complex (Thiel et al, 2010).

The second subgroup encompasses MLL fusions that do not confer clonogenic growth in replating assays (e.g. MLL-ABI1, MLL-GRAF, MLL-FBP17, MLL-LASP1; Strehl et al, 2003). Thus, they were never tested for their oncogenic potential in mouse models. However, the most prominent example for contradictory results is the chromosomal translocation t(4;11), which represents the most common MLL fusion in ALL patients. When MLL-AFF1 is overexpressed in transfected cells, they become growth arrested and senescent (Caslini et al, 2004; Gaussmann et al, 2007). This is presumably due to fact that specific target genes, e.g. CDKN2A, CDKN2C and CDKN1B, are upregulated in response to the presence of the MLL-AFF1 fusion protein (Milne et al, 2005; Xia et al, 2005; Gaussmann et al, 2007). In addition, the specific knock-down of either MLL-AFF1 or HOXA9, a direct target gene of the MLL-AFF1 fusion protein, led to increased apoptosis in SEM cells which strongly compromises any self-renewing capacity (Thomas et al, 2005; Faber et al, 2009).

By contrast, Doxycycline-driven overexpression of the reciprocal AFF1-MLL fusion protein strongly increases MTT activity, enhances cell cycling but increases also the rate of spontaneous apoptosis (up to 40%). This observation may be related to a phenomenon called ‘oncoprotein-induced apoptosis’. However, when both t(4;11) fusion proteins were expressed together, enhanced growth and cell cycling was observed, while apoptosis was blocked (Gaussmann et al, 2007). Thus, AFF1-MLL and MLL-AFF1 seem to exhibit complementary properties that result in a selective advantage.

When testing t(4;11) fusion alleles in mouse models, the situation becomes even more complicated. Retroviral transduction of MLL-AFF1 into 5-FU treated bone marrow cells never resulted in any kind of disease phenotype over an observation period of 24 months, even when a group of mice was treated with ENU (N-nitroso-N-ethylurea) in the drinking water to enhance the creation of complementary mutations (Catherine Lavau, Duke University Medical School. Durham, NC, USA, personal communication). Knock-in and inverter mice expressing an Mll-AFF1 knock-in allele caused differentiation into the B-lineage and the development of a lymphoma after very long latency (Chen et al, 2006; Metzler et al, 2006). Only when both approaches were combined, retroviral transduction and transgenesis, development of AML and preB ALL was observed (Krivtsov et al, 2008). However, the Mll-AFF1stop allele needed the elimination of a transcriptional stop signal by crossings either with Mx1-Cre mice or by retroviral transduction of Cre recombinase. Overexpressed Cre recombinase may result in genetic aberrations (Loonstra et al, 2001). In principle, this could be analysed by comparing single nucleotide polymorphism (SNP) experiments of leukaemic cells versus wildtype cells; however, this has not been performed. On the other hand, transduction of haematopoietic cells bearing the Mll-AFF1stop allele with a Cre-expressing retrovirus may have caused integration mutagenesis, which would explain the unexpected finding of AML development in most recipient mice. Cloning retroviral integration sites to reassure that the tested fusion gene, not retroviral integrations, caused the observed disease phenotypes would unambiguously demonstrate and help to interpret the obtained data.

Vice versa, our group has demonstrated that the reciprocal AFF1-MLL fusion allele caused proB ALL (ckit+/B220+/CD19), common lymphocytic progenitor leukaemia (B/T BAL; ckit+/B220+/CD3+) and mixed lineage leukaemia (MLL; ckit+/B220+/Mac1+; Bursen et al, 2010). In this study all leukaemic mice were thoroughly investigated, as were the mice that never developed any kind of disease. Thus, it became clear that AFF1-MLL alone is capable of inducing ALL, while expression of MLL-AFF1 displayed no oncogenic potential in this particular mouse model. The multiplicity of infection in our transduction experiments was tested and found to be very low (10−3–10−4), indicating that integration mutagenesis may not have caused a problem for the experimental read-out. Moreover, the leukaemic cells shared a ‘core signature’ that was nearly identical to the ‘core signature’ recently established for paediatric t(4;11) patients (Trentin et al, 2009).

The study of Trentin et al (2009) revealed another important finding: about half of all investigated paediatric t(4;11) patients displayed no activated HOXA genes, while MEIS1 and HOXC8 were highly expressed. The absence of typical HOXA signatures in about 50% of investigated t(4;11) patients was confirmed by another group which correlated the absence of typical HOXA signatures with a 3–4 times higher risk for relapse (Stam et al, 2010). Despite the presence or absence of HOXA signatures, all t(4;11) patients displayed highly activated MEIS1. In additon, two interesting AML cases have been published recently (Kaltenbach et al, 2010). Both AML leukaemia patients harboured only a reciprocal NUP98-MLL fusion gene (exon13::exon3), but did not exhibit a direct MLL fusion gene. Leukaemic cells of both AML patients displayed a very low level of HOXA and MEIS1 gene transcription, arguing again against the ‘common denominator concept’ in MLL-rearranged leukaemia.

Early and late onset of leukaemia development

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

Another issue is the latency of leukaemia development. Leukaemia development in murine model systems usually takes about 6–12 months. This is quite similar to human beings, where pre-leukaemic cell clones bearing genetic rearrangement of the MLL gene can already be detected in utero (reviewed in Greaves, 2005). However, leukaemia development occurs either immediately after birth or is delayed for years. Therefore, many scientists tend to believe that MLL rearrangements require complementary mutations to allow disease progression. In case of t(4;11) rearrangements, development and progression of disease mostly starts after birth, possibly accompanying the establishment of the immune system in the newborn. This suggests a unique role of t(4;11) translocations in the multitude of MLL-rearranged leukaemia. At least for this particular group of patients, secondary mutations may not be of importance, because recently published SNP experiments, performed with genomic DNA of paediatric t(4;11) leukaemia patients, revealed no gross genomic changes (Bardini et al, 2010).

There is some evidence emerging that other mechanisms may explain latency of leukaemia development. IRF8 has been identified as a downstream target of the Interferon signalling pathway and resembles a transcription factor predominantly expressed in the myeloid lineage and in B cells. In haematopoietic stem cells, IRF8 is highly expressed while the WT1 gene is not expressed. Increased WT1 expression, e.g. by leukemogenic fusion proteins, caused the downregulation of IRF8 (Vidovic et al, 2010). IRF8 transcription was shown to be significantly downregulated in myeloid leukaemia or dysplasia (BCR-ABL1, MPD, therapy-related myelodysplatic syndrome, therapy-related AML [t-AML], etc.) (Diaz-Blanco et al, 2007; Tshuikina et al, 2008; Qian et al, 2010). Moreover, Irf8 knock-out mice developed leukaemia very rapidly when proto-oncogenes or leukaemogenic MLL fusion genes were over-expressed (Konieczna et al, 2008; Schwieger et al, 2009). IRF8 was shown to downregulate PTPN13, which encodes the tyrosine phosphatase FAP1 that dephosphorylates the FAS receptor, thereby blocking FAS-mediated apoptosis (Huang et al, 2008). Thus, high IRF8 expression seems to make cells prone to apoptotic signals, and vice versa, downregulation of IRF8 is associated with increased cell survival (Yang et al, 2007). This implicates that high expression of IRF8 in haematopoietic stem cells may delay leukaemia development because of ‘oncoprotein-mediated’ induction of apoptosis. This would explain a late disease onset without the need of additional mutations. Another study demonstrated that IRF8 expression seems to be significantly downregulated in aged haematopoietic stem cells (Stirewalt et al, 2009). Thus, the likelihood for developing leukaemia naturally increases with age.

Several MLL fusion proteins influence transcriptional processes

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

In recent years, several laboratories have attempted to biochemically purify MLL fusion protein complexes. Beside the purification and characterization of the MLL complex (Nakamura et al, 2002; Yokoyama et al, 2002; Dou et al, 2005) and the identification of Taspase1 (Hsieh et al, 2003), which specifically hydrolyses the MLL protein, little was known about the cellular functions of identified fusion partners. The AFF1 gene was identified in 1992 (Gu et al, 1992), however, the function of AFF1 remained elusive for nearly one decade. Aff1 knock-out mice do not display a severe phenotype (Isnard et al, 2000), presumably due to complementing functions provided by the other members of the same protein family (AFF4 [AF5/MCEF], AFF3 [LAF4] and AFF2 [FMR2]; Nilson et al, 1997). The first hint came from studies on AFF4 (Estable et al, 2002), demonstrating that AFF4 interacts with the heterodimeric protein complex of CyclinT1/2 and CDK9, also known as positive transcription elongation factor b (P-TEFb), which was originally purified in the mid 1990s (Marshall & Price, 1995). All AFF1 family members are controlled in their steady-state abundance by the two E3 ligases SIAH1 and SIAH2 (Bursen et al, 2004). A conserved SIAH binding motif, P-x-A-x-V-x-P (House et al, 2003), is located in an N-terminal domain of AFF1 family proteins (ALF domain, Nilson et al, 1997). Moreover, MLLT3 binds to AFF1 at a conserved motif located in the C-terminal portion (Erfurth et al, 2004), and similarly, MLLT1 also binds next to the MLLT3 binding site (Zeisig et al, 2005).

Different attempts to purify and functionally analyse the murine ‘Aff1 complex’ (Bitoun et al, 2007), ‘MLLT1 complex’ (Mueller et al, 2007, 2009), the ‘AFF1-MLLT1 complex’ (Yokoyama et al, 2010), the ‘AFF4 complex’ (Lin et al, 2010) and the human ‘AFF1 and AFF1-MLL complex’ (Benedikt et al, 2010) finally shed light on an important regulatory mechanism named ‘transcriptional elongation’. When purified protein complexes are compared to the MLL complex (Nakamura et al, 2002; Yokoyama et al, 2002; Dou et al, 2005), it becomes clear that all of them share a common set of proteins (see Fig 3). Thus, it can be concluded that MLL-MLLT3, MLL-MLLT10, MLL-MLLT1 and the reciprocal AFF1-MLL protein complex exhibit similar properties, e.g. competition for factors bound to the parental MLL or AFF1 complexes.

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Figure 3.  Comparison of MLL, MLL fusions and MLL fusion partner complexes. Composition of different chromatin modifying complexes that have recently been characterized. Left: inactive P-TEFb complex, containing P-TEFb. P-TEFb, is able to bind to the MLLT1, AFF1, AFF4, MLL-X (X = MLLT3, MLLT1 and MLLT10) and AFF1-MLL protein complexes. The approximate molecular weight, their chromatin modifying and cellular functions are given for all complexes. Below, the MLL complex is shown. Dotted arrows indicate that the MLLT1 complex (∼400 kDa) may contribute as primordial complex to the formation of AFF1 and AFF4 complexes (both ∼2 MDa). Components binding to MLL and AFF1 were also found to be bound to the AFF1-MLL complex, indicating a highly competitive situation.

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In order to understand these properties in more detail, Fig 4 summarizes the actions of the parental MLL and AFF1 complexes during transcription. First, a given promoter region becomes methylated at histone H3 lysine-4 residues (H3K4) by the MLL complex. The MLL-associated histone acetyltransferases CREBBP and MOF acetylate histone core particles, while BMI1 initiates the mono-ubiquitinylation of H2AK119. In a subsequent step, the AFF1/AFF4 protein complex associates with RNA Pol II that has pre-assembled on the activated promoter region. Next, activated P-TEFb kinase phosphorylates Pol II-associated DSIF (DRB Sensitivity Inducing Factor) and the NELF (Negative elongation factor) complex. The inhibitory protein DSIF converts into an activator of transcriptional elongation, while the phosphorylated NELF complex is targeted for proteasomal degradation. The C-terminal domain (CTD) of RNA Pol II, consisting of 52 repeats of the peptide sequence [YSPTSPS], becomes phosphorylated at serine-2 residues, while the RNA Pol II co-bound TFIIH/CDK7 complex phosphorylates serine-5 residues. This allows binding of other factors involved in RNA splicing and polyadenylation to the RNA Pol II complex. In addition, P-TEFb phosphorylates the inactive UBE2A protein, which then associates with RNF20/40, thereby mediating mono-ubiquitinylation of H2BK120. The AFF1-associated DOT1L HMT starts to methylate H3K79 residues, while RNA Pol II converts from the promoter proximal arrested POL-O- into the elongation POL-E-state. During this process, RNA Pol II binds additional elongation factors (e.g. ELL1-3).

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Figure 4.  Molecular actions of MLL and AFF1/AFF4 complexes. Activation of cellular promoters by MLL and AFF1/AFF4. (A) The MLL complex is able to activate and maintain transcriptional processes. For this purpose, the MLL complex sets several modifications in the promoter region of active genes. (B/C) The AFF1 complex has several functions: first P-TEFb phosphorylates RNA Pol II and associated factors. P-TEFb also activates UBE2A to allow mono-ubiquitinylation of histone H2B. In a second step, the associated DOT1L, NSD1 and CARM1 HMT modify the chromatin at R2, R17, R26, K36 and K79. Finally, additional elongation factors associate with RNA Pol II. These actions convert promotor proximal-arrested RNA Pol II into the elongation form, allowing efficient transcription.

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This highly regulated process is disturbed by the presence of MLL fusion proteins (see Fig 3). MLL-MLLT3, MLL-MLLT10 and MLL-MLLT1 are per se able to bind to DOT1L and AFF1/AFF4-associated P-TEFb (Mueller et al, 2007; Yokoyama et al, 2010), while the N-terminal MLL portion binds to MEN1 (complexed with LEDGF and/or MYB) and BMI1 (Xia et al, 2003; Yokoyama et al, 2004; Yokoyama & Cleary, 2008; Jin et al, 2010). This allows these fusion proteins to perform actions that are normally executed by the parental MLL and AFF1 protein complexes. Moreover, the AFF1/AFF4 protein complex displays a high turn-over, which is not the case for the different MLL fusion protein complexes. This results in ectopic chromatin signatures, e.g. by increasing and extending H3K79 signatures (Krivtsov et al, 2008; Mueller et al, 2009), which is presumably one of the key mechanisms associated with MLL-mediated leukaemia development (Okada et al, 2005).

Notably, the reciprocal AFF1-MLL fusion protein complex displays all these features (binding to P-TEFb and DOT1L). In addition, the AFF1-MLL complex exhibits bona fide H3K4 HMT activity (Benedikt et al, 2010) and strongly activated P-TEFb kinase activity. Thus, the AFF1-MLL fusion protein has properties similar to the above-mentioned MLL fusions, but seems to be ‘autosufficient’, as it perfectly mimics the presence of AFF1 and MLL. To this end, the AFF1-MLL fusion protein seems to assemble the most potent complex concerning its ability to mediate ectopic chromatin signatures and to influence transcriptional processes (Fig 3). Therefore, it may not be surprising that the AFF1-MLL fusion protein was capable of inducing the development of proB ALL without the requirement of MLL-AFF1 (Bursen et al, 2010). It is also noteworthy that a recent study has found quite remarkable differences in promoter methylation when investigating paediatric leukaemia samples, such as t(4;11) and t(11;19), and comparing these data to t(9;11) patients cells (Stumpel et al, 2009). This may indicate different epigenetic mechanisms accompanying leukaemia development.

Potential complementing effects of mutated genes and signalling pathways

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

The analysis of cancer genomes by high-throughput technologies substantially supports the concept of multi-step cancerogenesis (exemplified in Sjöblom et al, 2006). However, different studies demonstrated that there is a significant difference between solid tumours and leukaemia. While most solid tumour genomes harbour between 70 and 80 mutations (of which about 15–20 were so-called ‘driver mutations’), the situation in leukaemic cells is characterized by much fewer mutations (in the range of 1–4; Greenman et al, 2007). Thus, one may hypothesize that leukaemic cells are not dependent on a consecutive series of genetic hits, rather on changing epigenetic imprints leading to ‘reprogrammed cells’.

Despite these facts, several groups have tried to identify complementing mutations in MLL-rearranged leukaemia. These studies revealed the presence of RAS, BRAF and NF1 mutations (KRAS, NRAS: codons 12, 13 and 61; BRAF: V600E; NF1: microdeletion and mutations of 2nd allele; Christiansen et al, 2005; Liang et al, 2006; Balgobind et al, 2008; Chandra et al, 2010). Liang et al (2006) investigated a total of 443 patients and revealed a significant association between paediatric MLL-rearranged BCP-ALL (n = 20) and N- or K-RAS mutations (50%), while in non-MLL-rearranged (n = 293) or AML patients (n = 130) the observed frequency of RAS mutations was either 20% or 30%, respectively. NF1 mutations seem to be a rare event in paediatric leukaemias and were observed in the range of 2–3% (Balgobind et al, 2008). NF1 plays an important role as negative regulator of the RAS/RAF signalling pathway. Chandra et al (2010) also identified a much higher frequency of N- and K-RAS mutation in de novo or t-AML leukaemia patients bearing the t(9;11) translocation (36%).

Recently, a link between certain MLL fusions (AFF1, AFF4, AFF3) and phosphorylation of ELK1, a downstream transcription factor of the RAS/RAF signalling pathway has been created. A dominant-negative RAS mutant (S17N) or the use of the MEK inhibitor U0126 blocked this activity, indicating that all these MLL fusions were able to enhance signalling of this particular pathway (Ng et al, 2010). One explanation for this finding could be the recently identified EPHA7 receptor, which represents again a downstream target of the MLL-AFF1 and MLL-MLLT3 fusion proteins (Nakanishi et al, 2007). Another explanation may derive from the ‘core signature’ of paediatric t(4;11) patients (Trentin et al, 2009): the Connective tissue growth factor gene (CTGF [CCN2]) was found to be highly upregulated (mean +50-fold) in every paediatric t(4;11) patient (see Fig 5A). CTGF belongs to a family of six proteins (CYR61 [CCN1], CTGF, NOV [CCN3], WISP1 [CCN4], WISP2 [CCN5] and WISP3 [CCN6]) that are all involved in matricellular signalling. CTGF binds to transforming growth factor β (TGFβ) via module 2 and enhances two different TGFβ pathways (TGFβ/SMAD3 and TGFβ/RAS/RAF). Both pathways lead to increased transcription of CTGF and secreted CTGF acts as ‘growth factor enhancer’ for EFG, FGF and IGF2, thus affecting surrounding stroma by modulating the growth of fibroblasts, chondrocytes and vascular endothelial cells (Crean et al, 2006). To this end, the RAS/RAF signalling pathway may be quite interesting for future research.

image

Figure 5.  Influence of MLL-AF4 targets on different signalling pathways. Different pathway affected by the expression of MLL-AFF1 are depicted. Green: MLL-AFF1 targets overexpressed in t(4;11) cells. These overexpressed proteins influence the RAS/RAF signalling pathway (A), apoptosis (B), the WNT signalling pathway (C), inflammatory response (D), differentiation of myeloid cells (D), quiescence (D) and cell growth and survival (E).

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FLT3 mutations (FLT3 TKD) were less frequently found in paediatric ALL patients (Armstrong et al, 2004; Taketani et al, 2004). However, a significant portion of MLL-rearranged childhood or relapsed ALL patients seem to carry the FLT3-D835I (TKD) mutation (18%). FLT3-TKD mutations are normally diagnosed in hyperdiploid ALL patients (∼20–25%), while FLT3 ITD mutations are mostly observed in AML patients. Noteworthy, the FLT3 promoter is a direct target gene of the MLL-AFF1 fusion protein (Guenther et al, 2008).

High expression of unmutated FLT3 is presumably of importance for niche interactions and the subsequent induction of quiescence, which translates into chemotherapy-resistance (Furuichi et al, 2007). This particular function of FLT3 is associated with the absence of STAT5 signalling and the stabilization of the p27 protein. By contrast, FLT3 mutations (FLT3 ITD or TKD) are generally characterized by phosphorylated STAT5a (Furuichi et al, 2007). Thus, the rarely occurring FLT3-TKD mutation in MLL-rearranged ALL does not lead to p27 accumulation, cells are less resistant against chemotherapeutics, more vulnerable to apoptotic stimuli, and thus, it seems that mutations in FLT3 are counterselected in paediatric ALL patients.

Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

As outlined above, the AFF1-MLL fusion protein is important for changing epigenetic imprints and strongly activates P-TEFb kinase; both mechanisms are presumably key events for leukaemia development. If so, one has to ask about the functions of the MLL-AFF1 fusion protein – besides its ability to block apoptosis and to transcriptionally activate HOXA genes and TERT (Gessner et al, 2010). In order to answer this question, a detailed analysis of the chromatin immunoprecipitation (ChIP) data presented by Guenther et al (2008) was quite helpful. By simply comparing the ChIP data set with the accompanying gene expression data set, 200 MLL-AFF1 candidate genes were identified to be upregulated at least twofold. This gene list contained HOXA7 (+13·1-fold; signal intensity 262), HOXA9 (+11·1-fold, signal intensity 1181), HOXA10 (10·1-fold, signal intensity 222), MEIS1 (+159·7-fold; signal intensity 479) and FLT3 (+6·5-fold; signal intensity 5687). Beside this expected signature, a few more genes were highly upregulated and displayed interesting biological features: TWIST1 (+284-fold; signal intensity 1138), FAIM (+2·3-fold; signal intensity 2366), TRIB2 (+181-fold; signal intensity 906), PTPRR (+170-fold; signal intensity 1023), PAWR (+90-fold; signal intensity 722), SPRED1 (+76-fold; signal intensity 230), LRRFIP2 (+10·8-fold; signal intensity 876), IRF4 (+8·9-fold; signal intensity 500), CXXC5 (+6·7-fold; signal intensity 914), RPS6KA3 (RSK2) (+4·3-fold; signal intensity 892) and IRS1 (+4·1-fold; signal intensity 178).

As outlined in Fig 5A, overexpressed SPRED1 negatively regulates normal haematopoiesis by blocking the signals downstream from SCF/KIT and IL-3/IL-3R (Nonami et al, 2004). The main action of SPRED1 is the inhibition of receptor signals into the RAS-RAF-MEK-ERK signalling pathway (Wakioka et al, 2001). PTPRR is a brain-specific phosphatase induced by NGF signalling (Ogata et al, 1995). PTPRR binds to ERK family members and inactivates them by dephosphorylation (Pulido et al, 1998). Interestingly, PTPRR has been recently identified as a fusion partner of the ETV6 gene in AML patients bearing an inv(12)(p13q13) and leads to GM-CFS-independent STAT3 activation (Nakamura et al, 2005).

TWIST1, a downstream target of HIF1alpha, NFkB1 and the WNT signalling pathway, encodes a bHLH transcription factor that counteracts oncogene- and p53-induced apoptotic pathways (Fig 5B). TWIST1 overexpression is linked to loss-of-contact inhibition, blocks differentiation and was overexpressed in several solid tumours, where it conferred resistance against Taxol and enhanced metastasis (Maestro et al, 1999; Wang et al, 2004; Yang et al, 2004). TWIST1 regulates miR-199A and miR-214, both of which confer stemness in epithelial ovarian cancer cells (Yin et al, 2010). Interestingly, TWIST1 cooperates with HOXA5 to downregulate the transcription of TP53 (Stasinopoulos et al, 2005) and counteracts MYC-induced apoptosis when strongly overexpressed in solid tumour cells (Valsesia-Wittmann et al, 2004). Thus, TWIST1 and FAIM (regulated by IRF4; Kaku & Rothstein, 2009) are interesting candidates for the observed block of apoptosis in MLL-AFF1 expressing cells.

PAWR is another interesting target gene of the MLL-AFF1 fusion protein (Fig 5C). PAWR binds directly to WT1 and blocks transcriptional activation of WT1 target genes (Johnstone et al, 1996). Moreover, WT1 confers growth inhibition and represses transcription of IRF8 (see above). WT1 is important for the transcriptional activation of genes coding for factors directly involved in the WNT signalling pathway (BTRC, CCND2, DACT1, DKK2, JUN, LEF1, NLK, PP3CB/ß-TrCP and TBL1X; Kim et al, 2009). Thus, PAWR compromises the WNT signalling pathway, which may explain some of the findings by Wang et al (2008). CXXC5 (also upregulated by MLL-AFF1) encodes the ‘WT1-induced Inhibitor of Dishevelled’ (WID; Kim et al, 2010). WID directly binds to the Dishevelled protein and abrogates WNT signalling, thus simulating the absence of FRIZZLED ligands. This is in line with recent findings about the effects when GSK3 was inhibited (Wang et al, 2008, 2010). By contrast, LRRFIP2 should activate the WNT signalling pathway by its association to Dishevelled (Liu et al, 2005), thus augmenting the cellular level of β-Catenin protein and activation of downstream target genes. However, as mentioned above, the presence of PAWR and WID may compromise functions downstream of LRRFIP2.

TRIB2 seems to provide several functions (Fig 5D). It is an adapter protein that recruits E3 ubiquitin ligases to specifically degrade the CEPBA transcription factor, and thus, enables the development of AML (Dedhia et al, 2010). Overexpression of TRIB2 is correlated with the increase of cytosolic FOXO proteins, while siRNA-mediated knock-down of TRIB2 correlates with reduced cell proliferation, colony formation and reduced wound healing of melanoma cells. Moreover, knock-down of TRIB2 impairs tumour growth in a melanoma xenograft model (Zanella et al, 2010), indicating that TRIB2 is a potent oncoprotein. TRIB2 transcript levels were shown to be inversely correlated to IL-8 production, thus interfering with the inflammatory response of monocytes. For this purpose, TRIB2 directly binds to selected ERKs and resembles a negative regulator of the MEK/ERK signalling pathway (Eder et al, 2008). Interestingly, the TRIB2 promoter was identified as a direct target of MEIS1 in NUP98-HOXD13-mediated leukaemia cells (Argiropoulos et al, 2008), and retroviral overexpression of murine Trib2 and murine HoxA9 lead to accelerated onset of AML in a murine model system (Keeshan et al, 2008).

Ribosomal S6 kinase 2 (RPS6KA3) is a downstream target of activated ERK1/2, that forms a negative feed-back loop to control the ERK pathway (Clark et al, 2007). RPS6KA3is involved in the inactivation of several pro-apoptotic proteins and promotes cell growth (Carriere et al, 2008). Moreover, RPS6KA3phosphorylates IRS1 (Fig 5E). IRS1 binds and activates the p85 subunit of PI3K and mediates PI3K/AKT signalling in the absence of external stimuli (Asano et al, 2005). IRS1 becomes repressed in the presence of active S6 kinases that are controlled by the tumour suppressor proteins TSC1/2. Thus, active TSC1/2 proteins block S6 kinase activity, which in turn leads to active IRS1 and to an active insulin signalling pathway associated with PI3K/AKT signalling (Harrington et al, 2004). Constitutive IRS1 phosphorylation was associated with malignant transformation and invasive cell growth (Reiss et al, 2001). Moreover, IRS1 also binds to GRB2/SOS, and thus, may also influence the RAS/RAF signalling cascade, which can be inhibited by PTEN (Weng et al, 2001). However, very low expression of PTEN was found in t(4;11) leukaemia cells (Trentin et al, 2009).

This may indicate that several downstream targets of the MLL-AFF1 fusion protein are able to interfere or modulate a large variety of different signalling pathways. Most likely, the above mentioned proteins represent ‘signalling modifyers’. NFkB1, a downstream target of the PI3K/AKT pathway, confers survival but is also a constituent of the AFF1-MLL protein complex (Benedikt et al, 2010). Thus, proper function of the AFF1-MLL complex seems to be dependent on functions exerted by the MLL-AFF1 fusion protein.

Concluding remarks

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

Changing epigenetic imprints seems to be a novel hallmark for leukaemias that are initiated by the presence of MLL-MLLT3, MLL-MLLT10, MLL-MLLT1 or the reciprocal AFF1-MLL fusion protein. However, it is well documented that the typical activation pattern of HOXA genes and MEIS1 in MLL-mediated leukaemia is important for all MLL-rearranged leukaemias displaying a myeloid phenotype. In line with this argument, overexpression of HoxA9 in conjunction with Meis1 is able to drive myeloid transformation in mice (Faber et al, 2009). As a matter of fact, all MLL fusions tested to date – including the MLL-AFF1 fusion protein – lead to the transcriptional activation of distinct HOXA genes.

However, this simple scheme changes when reciprocal MLL fusion proteins are investigated. As an example, the AFF1-MLL fusion protein causes proB ALL in mice but does not transcriptionally activate Hoxa genes. Similar observations have been made for the NUP98-MLL fusion protein, associated with AML disease development (Kaltenbach et al, 2010). This raises some questions about the ‘common denominator concept’ of MLL-rearranged leukaemia. One should rather recognize that ALL-MLL and AML-MLL, and the many different fusion partners of MLL, are critically distinct entities. Thus, future work should concentrate on the pathological mechanisms that derive from different MLL fusion proteins. This will help to categorize the different entities, identify critical targets and develop new drugs for treatment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References

I thank Olaf Heidenreich and Owen Williams for critically reading the manuscript. This work is supported by grants 107819 and 108400 from the Deutsche Krebshilfe e.V. and 01GS0875 from the BMBF to RM. RM is PI within the CEF on Macromolecular Complexes funded by DFG grant EXC 115.

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  1. Top of page
  2. Summary
  3. Genetic situation in MLL-rearranged leukaemia patients
  4. Model systems are the basis to understand oncogenic mechanisms
  5. Early and late onset of leukaemia development
  6. Several MLL fusion proteins influence transcriptional processes
  7. Potential complementing effects of mutated genes and signalling pathways
  8. Potential functions of the MLL-AF4 and AF4-MLL fusion proteins in t(4;11) leukaemia
  9. Concluding remarks
  10. Acknowledgements
  11. References
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