Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|topoisomerase II poisons, infant leukaemia||KeyEvent|
Key Event Description
Chromosomal rearrangements of the mixed-lineage leukaemia (MLL) gene, located on the q23 band of chromosome 11 (11q23), are the genetic hallmark of most infant leukaemias (Meyer et al 2013; Sanjuan-Pla et al 2015). MLL is located within the fragile site FRA11G; chromosomal fragile sites are regions of the genome susceptible to breakage under conditions of replication stress; interference with TopoII may promote fragile site instability. MLL encodes a protein homologous to the Drosophila trithorax gene, which has relevant functions in embryogenesis and hematopoiesis (Ernest et al 2004, Hess et al 1997).
MLL, a human homologue of the epigenetic transcriptional regulator Trithorax of Drosophila, is an upstream transcriptional effector of HOX genes. The importance of normal MLL protein for normal axial-skeletal developmental process and HOX gene regulation has been demonstrated in the embryos of heterozygous and homozygous MLL knockout and MLL truncation mutant mice. Furthermore, expression of MLL protein is not necessary for turning on transcription of certain HOX genes, but for the maintenance of their transcription. Experiments in vitro using hematopoietic progenitors from embryos of homozygous MLL knockout mice or mice with MLL mutant showed that MLL was also critical for hematopoietic development. Recent findings suggested that MLL is required during embryogenesis for the specification or expansion of hematopoietic stem cells. As HOX genes also play a key role in the regulation of hematopoietic development, the hematopoietic dysfunction of MLL null cells is likely to be attributed to deregulated patterns of HOX gene expression in hematopoietic stem cells or progenitors. This link between MLL, HOX gene regulation, and hematopoiesis is of particular importance (Li et al. 2005).
There are many translocation and fusion partners for MLL; DNA breakage within MLL can lead to rearrangement with over 120 partner genes (Meyer et al 2013). In principle all MLL fusion genes are potential initiating drivers, although clinical studies have shown a preponderance with infant leukaemia for only a few of these rearrangements. For infants diagnosed with ALL, approximately 60-80% carry an MLL rearrangement (Sam et al 2012; Jansen et al 2007), with predominant fusion partners being AF4 (41%), ENL (18%), AF9 (11%) or another partner gene (10%). In particular, the fusion gene MLL-AF4 shows a specific and consistent relationship with the disease (Menendez et al., 2009): however, it has been difficult to reproduce a manifest disease resulting from this rearrangement in in vivo animal models. For AML, about 30 % of the patients carry an MLL rearrangement.
The occurrence of MLL rearrangements at a very early fetal development is highly probable on the basis of neonatal blood spot analysis and by the high concordance rate of infant leukaemia in monozygotic twins (Ford et al 1993; Gale et al 1997; Sanjuan-Pla 2015). Menendez et al (2009) showed that MLL-AF4 fusion gene is present in bone marrow mesenchymal stem cells in infant leukaemia patients, but not in patients of childhood leukaemia, suggesting that the origin of the fusion gene is probably prehaematopoietic. Consequently, the affected cell, the so called leukaemia-initiating cell, may be an early prehaematopoietic mesodermal precursor, a hematopoietic stem cell or hematopoietic progenitor cell residing mainly in the liver (Greaves 2015; Sanjuan-Pla et al 2015).
MLL protein (complexed with a large number of other protein factors) serves as a transcriptional activator or repressor via the binding to promoter regions of active genes, marking these regions by covalent histone modifications (Sanjuan-Pla et al 2015). Translocation and creation of fusion genes and products destroys the intrinsic control mechanisms of the MLL protein. The resulting ‘ectopic’ functions involve promoter hyper-activation and re-acquiring stem cell features (Sanjuan-Pla et al 2015). A schematic presentation of the drastic changes of the MLL product is depicted in the figure below.
Proposed model for the oncogenic conversion of MLL fusion: A. Physiological situation and B: . A chromosomal translocation, which leads to the intrinsic regulatory mechanism of MLL being destroyed. (Sanjuan-Pla et al. 2015).
MLL translocation sites (breakpoint sequences) in the therapy-related leukaemia fall within a few base pairs of etoposide-induced enzyme-mediated DNA cleavage site (r). Although rearrangements associated with infant leukaemias are often more complex than those observed in treatment-related leukaemias, many are nevertheless associated with stable TopoII-mediated DNA cut sites. Although all these findings are indirect regarding infant leukaemia, they are nevertheless rather persuasive in this respect.
Growing scientific evidence, including the stable genome of the patients, suggests that infant leukaemia originates from one “big-hit” occurring during a critical developmental window of stem cell vulnerability (Andersson et al 2013; Greaves 2015). Therefore, the totality of evidence suggests the essential role of the formation of MLL-AF4 (and other partner) fusion gene and product in causing pleiotropic effects in the affected cell and directing it to the obligatory pathway to the adverse outcome of leukaemia (see KER2).
How It Is Measured or Detected
MLL rearrangements can be identified following different methods. It is worthnoting that different methods will give a different information detail.
- Split-signal FISH: The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity (Van der Burg et al, 2004).
- RT-PCR in combination with long-distance inverse PCR (LDI-PCR) performed on isolated genomic DNA. This method allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. The method uses long-distance inverse PCR (LDI-PCR) to identify MLL translocations independent of the involved partner gene or other MLL aberrations that occurred within the MLL breakpoint cluster region. This method allows high-throughput analyses because genomic MLL fusion sequences can be obtained with a minimum of only four PCR reactions. Moreover, this method requires only small quantities of genomic patient DNA (1 μg) and provides relevant genetic information that can be used directly for quantitative minimal residual disease (MRD) analyses (Meyer et al. 2005).
Assays measuring chromosomal aberrations, micronuclei or DNA and chromosome damage (Comet assay) may indirectly identify the KE through its consequences in experimental systems in vitro and in vivo. FISH staining is however necessary for identification of MLL translocations.
Domain of Applicability
Although the KE deals with the general process of DNA integrity, the available evidence do not allow for evaluating whether any significant difference occurs among cell types or species. It has been shown that the mouse has an analogous fusion gene mll-af4. A recent study has shown that in utero exposure to etoposide induces mll translocations in Atm-knockout mice, which are defective in the DNA damage response, albeit not in wild-type mice; moreover, fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells, pointing out the life stage-related susceptibility in regards to TopoII “poison” also in the mouse (Nanya et al., 2015).
MLL-AF4 fusion gene is present and expressed in bone marrow mesenchymal stem cells in infant patients with t(4;11) B cell-ALL (Menendez et al. 2009). However, other paediatric B cell-ALL-specific translocations/gene fusions were never found in this cell population. This suggests that the origin of the fusion gene in infant B cell-ALL is likely prehaematopoietic. Consequently, the target cell for transformation may be an early prehaematopoietic mesodermal precursor, a haematopoietic stem cell or a haematopoietic progenitor cell residing mainly in the liver (Greaves et al. 2015; sanjuan-Pla et al. 2015).
Evidence for Perturbation by Stressor
There is abundant evidence on the interaction of etoposide with topo II enzymes, resulting in further chromosomal translocations (in particular MLL-r) at the cell culture level and in relation to treatment-related leukaemia (Cowell and Austin, 2012; Ezoe, 2012; Pendleton and Osheroff, 2014; Gole and Wiesmuller, 2015). Etoposide can induce MLL-r in different cell types. Interestingly, embryonic stem cells and their hematopoietic derivatives are much more sensitive than cord blood-derived CD34+ cells to etoposide induced MLL-r. In addition, undifferentiated human embryonic stem cells (hESCs) were concurrently predisposed to acute cell death (Bueno et al., 2009). Molecular dose-response modelling of etoposide-induced DNA damage response, based on comprehensive in vitro high content imaging in the HT1080 cell model, was developed by Li et al. (2014).
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