Upstream eventBinding, topoisomerase II
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
Certain TopoII poisons stabilize the intermediate cleavage complex and prevent the religation with appropriate DNA strands. Covalently DNA end-bound TopoII protein is digested and a hanging end is created. The same process happens in the translocation partner gene. Hanging ends of both genes are processed and subsequently joined by non-homologous end joining (Cowell and Austin 2012). There is evidence that this inappropriate joining of ‘hanging ends’ happens in the same transcriptional factory (hub), and the result is a fusion gene and ultimately protein product (Cowell & Austin 2012; Pendleton et al 2014; Sanjuan-Pla et al 2015). The first part of this description has not been shown in the putative target cell, which is still not unequivocally identified, but for the second part there is ample evidence of formation of MLL-AF4 fusion product that has been a result of a very early chromosomal translocation and rejoining. It is of interest that the simultaneously induced specific DSBs in the MLL gene and two different translocation partners (AF4 and AF9) by engineered nucleases in human HSPCs resulted in specific ‘patient-like’ chromosomal translocations (Breese et al 2016). For the scope of this AOP , this KE relationship should occur in-utero.
Evidence Supporting this KER
Evidence supporting the causal relationship between etoposide-induced TopoII inhibition and the MLL rearrangement leading to the fusion gene is strong regarding treatment-related acute leukaemia (*Cowell and Austin 2012; *Pendleton et al 2014). The bioflavonoid-rich diet in pregnant women has been suggested to initiate infant leukaemia by an analogous causality between in utero inhibition of TopoII enzymes and creation of the fusion gene. However, there is no direct evidence in humans and it is also difficult or impossible to study. Power of epidemiological studies is relatively weak in the case of a very rare disease and case-control or spatiotemporal cluster studies have barely suggested a causal relationship between exposures and disease. Although the empirical support for the chemical stressor etoposide and the metabolite etoposide quinone should be considered strong, this still remains a limitation for the overall strenght of the weight of evidence for the empirical support. However, the biological plausibility linking topoII poisons to MLL rearrangements, when occuring in-utero in the appropiate cell population ie. prehematopoietic stem cell is strong, making the overall weight of evidence as strong. considered strong.
The KER as such is biologically plausible and strong . Type II topoisomerases are ubiquitous enzymes which are essential for a number of fundamental DNA processes. As they generate DNA strand breaks, they can potentially fragment the genome. Indeed, while these enzymes are essential for the survival of proliferating cells they can also have significant genotoxic effects by means of accumulation of DNA strand breaks that, if not resulting in cell death may lead to chromosomal translocation in the surviving cell population (McClendon et al. 2007). DNA breaks and MLL rearrangements by etoposide and bioflavonoids have been demonstrated in human fetal liver haematopoietic stem cells, in human embryonic stem cells and in human prehaematopoietic mesenchymal stem cells as well as in cord blood mononuclear cells (Ishii et al 2002; Blanco et al 2004; Moneypenny et al 2006; Bueno et al 2009; Menendez et al 2009), which clearly shows that TopoII-associated MLL rearrangements are produced in appropriate human cells in utero.
There are animal models for infant leukaemia which recapitulate at least some salient aspects of the disease (Sanjuan-Pla et al 2015). However, for example the MLL-AF4 knock-in mice develop leukaemia only after a prolonged latency (Chen et al 2006), thus not recapitulating the ‘pathognomonic’ feature of infant leukaemia.
Etoposide treatment in vivo in mice at day 13.5 of pregnancy induces MLL breakage in fetal liver haematopoietic stem cells in utero, but MLL-rearranged fusion mRNAs were detected only in mice which were defective in the DNA damage response, i.e. atm knockout mice. A fusion gene analogous to MLL-AF4 was not detectable in the wild type mice. In this study, an intraperitoneal injection of 10 mg/kg of etoposide into pregnant mice at day 13.5 of pregnancy resulted in a maximum fetal liver concentration of about 5 µM. A dose of 0.5 mg/kg did not result in a measurable concentration. A statistically significant increase (about 6-fold) in DSBs in the MLL gene of isolated fetal liver haematopoietic stem cells was observed after a single dose of 1 mg/kg to pregnant mice. A clear activation of DNA damage response was observed at the dose of 10 mg/kg (Nanya et al. 2016).
There is a lot of information about the interaction of etoposide with TopoII enzymes and MLL chromosomal translocation at the cell culture level and in connection with treatment-related leukaemia.
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). The model was based on the hypothesis that cells are capable of clearing low-level DNA damage with existing repair capacity, but when the number of DSBs exceeds a certain value; ATM and p53 become fully activated through reversible mechanism, leading to elevated repair capacity. The model was able to capture quantitatively the dose-response relationships of a number of markers observed with etoposide. Especially interesting are the dose-response relationships for activation of p53 and the formation of micronuclei in the target cell model, which indicate point-of-departure concentrations of etoposide in the range of 0.01 to 0.1 µM (Li et al. 2014). This range is in agreement with the finding that in human fetal liver CD34+ cells an increase in DSBs was observed at a concentration of 0.14 µM and MLL translocations were detectable by FISH or flow cytometry at higher concentrations (Moneypenny et al 2006).
 Hypothetically, based on linear extrapolation from the dose of 10 mg/kg, the concentration would be of the order of 0.5 µM.
Uncertainties and Inconsistencies
· A prerequisite for the specific outcome, i.e. creation of chromosomal rearrangement, is that TopoII inhibition has to occur in an especially vulnerable and correct hot spot in the MLL locus; however, details of this process and how it happens are not clear.
· A target cell, i.e. leukaemia-initiating cell, has not been identified with sufficient confidence and consequently there is no target cell model to recapitulate the linkage between TopoII inhibition (‘poisoning’) and the production of DSB in an appropriate target. Recently, by the expression of engineered nucleases (TALENs) to induce simultaneous patient specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), Breese et al (2015) were able to produce specific chromosomal translocations in K562 cells and in primary HSPCs.
· In-utero etoposide-treatment failed to induce leukaemogenesis (Nanya et al 2015). Consequently, the envisaged linkage has not been empirically supported or rejected. However, it should be kept in mind that, whereas etoposide does induce a large number of MLL rearrangements, most of them occur within non-coding regions, therefore not eliciting any direct oncogenic consequence. A MLL-AF4 in frame fusion is a rare event that needs to occur in a target cell within a relatively small and spatially restricted cell population during the appropriate, epigenetically plastic, developmental window; thus it may be difficult to empirically support this process.
· Dose-response relationships between etoposide and treatment-related leukaemia are difficult to unravel, but risk of leukaemia seems to increase with larger total exposure to etoposide. However, comparison of exposures or kinetics of etoposide between leukaemia patients and non-leukemic treated subjects did not reveal any significant differences (Relling et al 1998). Also, it is not known whether the etoposide (or metabolite) concentrations during the treatment are of significance. In child and adult chemotherapy, concentrations are extremely variable between individuals; the lowest through plasma concentrations of etoposide have been of the order of 1 µM and peak concentrations very much higher. For example, in a study of Relling et al (1998), the maximum plasma concentration of etoposide was about 90 µM and that of etoposide catechol about 100-times less, below 1 µM. In another high dose chemotherapy study (Stremetzne et al 1997), the etoposide concentration was 170 µM and that of the catechol metabolite 5.8 µM maximally. However, it is not straightforward to juxtapose plasma concentrations and the tissue or cell concentration which TopoII enzyme ’sees’. Penetration of etoposide or its metabolite through plasma membrane is probably rather slow and it has been shown that the brain cancer tissue (metastasis or glioma) to plasma ratio for etoposide is only 0.1 (Pitz et al 2011). Blood-brain barrier is not necessarily a good model for cross-membrane distribution, but may give some idea about the general distributional behaviour of a drug. Even if the active target concentration of etoposide is only 10 % of the plasma concentration, it is still in the same range as the effective concentrations in cellular studies (see above). A final note on relevant concentrations: etoposide concentrations resulting in DSB and fusion gene are probably within a relatively restricted range. The concentration resulting in a proper fusion gene should be in a range which gives rise to a partially repaired insult and cells bypassing death and accumulating the abnormality.
Quantitative Understanding of the Linkage
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
DNA topoisomerases are ubiquitous enzymes, which control the integrity of double-stranded DNA. They are thus key enzymes at all levels of living organisms. The available evidence suggest that important differences in sensitivity to topoisomerase inhibition might exist among different cell types, depending on the amount of proliferative burden, of the TopoII enzymes and on physiological repair processes. Mesodermal precursor or hematopoietic stem and progenitor cells (HSPCs) are rapidly dividing cells with a high content of TopoII and for these reasons they can be a sensitive target during a critical developmental window (Hernandez and Menendez 2016). In addition, evidence from micronuclei assay studies conducted in untreated and chemical-treated foetuses and newborns show that both the baseline and chemically induced micronuclei frequencies are higher in the foetuses and infants than in adults (Udroiu et al 2016). This is possibly indicating a greater sensitivity to genotoxic insult during development which can be due to the higher proliferation rate and lower ability of DNA repair of the hematopoietic stem cells. However, the role that the different microenvironments (foetal liver, infant bone marrow and adult bone marrow) during ontogenesis can exert on cell sensitivity cannot be ruled out (Udroiu et al. 2016). The existence of relevant interspecies differences is unknown, but it cannot be ruled out presently.
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