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Aop: 202

AOP Title

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In-utero DNA topoisomerase II poisons leading to infant leukaemia

Short name:

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topoisomerase II poisons, infant leukaemia

Authors

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Olavi Pelkonen Department of Pharmacology and Toxicology and Clinical Research Unit, University of Oulu, Finland

Andrea TerronEuropean Food Safety Authority (EFSA), Parma, Italy. andrea.terron@efsa.europa.eu

Antonion F. Hernandez, University of Granada School of Medicine, Granada, Spain

Pablo Menendez, Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona; b. Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, and c. Centro de Investigación Biomédica en Red en Cancer del ISCIII (CIBERONC), Spain

Susanne Hougaard Bennekou, The Danish EPA, Copenhagen, Denmark

Point of Contact

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Andrea Terron

Contributors

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  • Andrea Terron
  • Clemens Wittwehr

Status

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Author status OECD status OECD project SAAOP status
Open for comment. Do not cite EAGMST Under Review 1.53 Included in OECD Work Plan


This AOP was last modified on August 03, 2017 08:35

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Revision dates for related pages

Page Revision Date/Time
In-utero DNA topoisomerase II “poisons- September 16, 2017 10:17
In utero MLL chromosomal translocation September 16, 2017 10:17
Infant leukaemia April 26, 2017 09:23
DNA double-strand break August 02, 2017 09:52
MLL translocation leads to IFL April 26, 2017 06:19
DNA topoisomerase II “poisons” leads to DSB August 02, 2017 10:45
DSB leads to MLL translocation August 02, 2017 11:11
Etoposide December 16, 2016 11:26
Bioflavonoids April 25, 2017 04:07
Chlorpyrifos April 25, 2017 04:08
etoposide quinone April 25, 2017 04:57

Abstract

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Infant leukaemia is a rare haematological disease (1 in 106 newborns, accounting for 10% of all childhood acute lymphoblastic leukaemias (ALL)) manifesting soon after birth (<1 year) and having a poor prognosis (Sanjuan-Pla et al 2015). Compared to the more frequent childhood leukaemia, infant leukaemia show distinct features:

  • An early neonatal onset linked to its plausible origin as a ‘intrauterine developmental disease’ (Greaves 2015; Sanjuan-Pla et al 2015);
  • Rearrangements of the mixed-lineage leukaemia (MLL; KMT2A) gene on the q23 band of chromosome 11, as the hallmark genetic abnormality (Joannides and Grimwade 2010);
  • However, MLL is not the only translocation gene; for infant ALL, about 60-80% carry an MLL rearrangement (Sam et al.2012; Jansen et al.2007) and the percentage for infant acute myeloid leukaemia (AML) is about 40 %;
  • The MLL rearrangement at an early stage of development; the likely target cells (still unidentified) are the hematopoietic stem and progenitor cells (HSPC) in fetal liver and/or earlier (mesenchymal) stem cells in embryonic mesoderm (Bueno et al 2009; Menendez et al 2009);
  • The infant MLL-rearranged leukaemia carries less somatic mutations (1.3 vs 6.5/case) than the childhood disease (Andersson et al 2015; Dobbins et al 2013), pointing to the lack of a “second hit” and suggesting a “one big hit” origin.

Following these distinct features a molecular Initiating Event (MIE) a Key Event (KE) and an Adverse Outcome (AO) were identified. The MIE was identified as "In-utero exposure DNA topoisomerase II poisons"; relationship to in-utero exposure was considered relevant to make a specific relationship with the AO (Infant Leukaemia) as epidemiological studies suggested that in-utero exposure to topoisomerases 2 may be involved in generation of the KE, in-utero MLL chromosomal rearrangement.

Overall, based on the available evidence, infant leukaemia pathogenesis originates from a single, severe hit to a target cell during early intrauterine development. Whereas the limited epidemiological studies do not allow any firm conclusion on a possible role for chemicals in infant leukaemia (Pombo-de-Oliveira et al 2006; Ferreira et al 2013), exposures to chemicals able to induce MLL rearrangements through topoisomerase II (TopoII) “poison”, particularly etoposide and other TopoII “poisons”, including some bioflavonoids, have been suggested as agents promoting the driver genetic oncogenic event. Experimental models for infant leukaemia have been developed, but a wholly satisfactory model reproducing the phenotype and latency is not yet available.

Nevertheless, the anticancer drug etoposide can be considered as a model chemical for DNA topoisomerase “poison”. Acute leukaemia is an adverse effect recorded in etoposide-treated patients, showing MLL rearrangements that are in many ways analogous to those in infant leukaemia (Bueno et al 2009; Joannides et al 2010, 2011). Therefore the proposed AOP is supported by a number of convincing inferential evidences by means of using etoposide as a tool compound to empirically support the linkage between the proposed molecular initiating event (MIE) and the adverse outcome (AO). In the meanwhile, this AOP identifies several knowledge gaps, the main ones being the identification of the initiating cell and the investigation of TopoII poisons in a robust model; thus, the present AOP may be modified in future on the basis of new evidence. The authors recognize that additional elements are limiting the strenght of this AOP, in particular that the empirical support is mainly based on one chemical stressor and that essentiality data are also limited and difficult to generate; however, the biological plausibility for the proposed sequence of events for this AOP was considered strong. 


Background (optional)

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Infant leukaemia (<1 year old) is a rare disease of developmental origin distinct from adult and childhood leukaemias which fit the classical two-hit cancer model. Both genetic and haematological studies strongly indicate an in utero origin at an early phase of foetal development. investigation of identical twin pairs with infant leukaemia provided evidence of in-utero transfer of leukemic celss from one twin to the other (Ford AM, 1993), and the in-utero origin of this cancer was confirmed by retrospective analyses of neonatal blood spots from affected infants (Gale KB, 1997). The high concordance rate for leukaemia in monozygotic twins and the short latency of the disease suggest that MLL rearrangement in fetal hematopoietic stem cells causes infant leukaemia (Nanya M, 2015). Rearrangements of the mixed lineage leukemia (MLL) gene producing abnormal fusion protein are the most frequent genetic/molecular hallmarks in infant B-cell ALL. In small epidemiological studies, mother/foetus pesticide exposure has been associated with infant leukaemia; however, strength of evidence and power of these studies are weak at best. Despite recent advances in the pathogenesis of pediatric leukemia, surrogate models such as in vitro, ex vivo or animals in vivo do not reproduce the human disease sufficiently and they suffer from difficulties in interpretation and extrapolation of findings and from the intrinsic limitation in cancer bio-assay design to cover relevant window of exposure. This adverse outcome pathway (AOP) is based substantially on an analogous disease – secondary acute leukaemia caused by etoposide, a topoisomerase II (TopoII) poison –, and on cellular and animal models. Etoposide induces DNA double-strand breaks between the S and the G2/M phases of the cell cycle The hallmark of the AOP is the formation of MLL gene rearrangements (MLLr) via TopoII poisoning, leading to fusion genes and eventually acute leukaemia by global (epi)genetic dysregulation. The findings described above suggest the possibility that MLL-rearrangment infant leukaemia is caused by transplacental exposure to topo2 poisons. Although it is considered unusual for a pregnant woman to be directly exposed to drugs such as etoposide, other compounds presents in the environment, including pesticides, may exert  similar effects, and this is considered toxicologically relevant for risk assessment (Nanya M, 2015). The AOP condenses molecular, pathological, regulatory, clinical and epidemiological knowledge in a pragmatic framework with the aspiration of focussing on human specific hazard in the risk assessment process. The AOP enables to identify important gaps of knowledge relevant to risk assessment, including the specific embryonic target cell during the short and spatially restricted period of susceptibility and the role of (epi)genetic features modifying initiation and progression of the disease. Furthermore, the suggested AOP informs on a potential integrated approach to testing and assessment (IATA) to address the risk caused by environmental chemicals in the future and represents a transparent and weight of evidence based tool to define the plausible causative mechanism necessary for the interpretation and integration of epidemiological studies in the process of risk assessment.


Summary of the AOP

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Stressors

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Name Evidence Term
Etoposide Strong
Bioflavonoids Weak
Chlorpyrifos Weak
etoposide quinone Strong

Molecular Initiating Event

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Title Short name
In-utero DNA topoisomerase II “poisons- DNA topoisomerase II “poisons”

Key Events

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Adverse Outcome

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Title Short name
Infant leukaemia IFL

Relationships Between Two Key Events (Including MIEs and AOs)

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Title Directness Evidence Quantitative Understanding
MLL translocation leads to IFL Directly leads to Strong Not Specified
DNA topoisomerase II “poisons” leads to DSB Directly leads to Strong Not Specified
DSB leads to MLL translocation Directly leads to Strong Not Specified

Network View

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Life Stage Applicability

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Life stage Evidence
Embryo Strong

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens Strong NCBI

Sex Applicability

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Sex Evidence
Unspecific Strong

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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Infant leukaemia is a “hidden” disease quite concretely: initiation occurs in utero at an early phase of foetal development. Studies both in identical twins (Ford et al 1993) and in neonatal blood samples retrospectively (Gale et al 1997) strongly indicate in utero origin of the disease. Consequently, direct studies in pregnant humans are difficult or impossible and one has to resort to surrogate in vitro or ex vivo studies or to animal models which necessarily are associated with difficulties in interpretation and extrapolation. Thus, what is described in this overall assessment is based largely on inferences from analogous diseases using tool chemicals able to reproduce the biological basis of the disease (especially etoposide (a Topoisomerases II poison-caused acute leukaemia in children or adults) or from cellular and animal models.

1. Concordance of dose-response relationship

The only in utero study in mice (Nanya et al 2016) has shown that the dose of 0.5 mg/kg (day 13.5 of pregnancy) does not result in measurable etoposide concentration in foetal liver hematopoietic stem cells (HSCs) whereas the dose of 10 mg/kg leads to a maximal concentration of 5 µM. A statistically significant increase in double strand break (DSBs) in MLL gene was observed at a dose of 1 mg/kg, which would result in a concentration of 0.5 µM by linear extrapolation. In treatment-related acute human leukaemia, various treatment schedules in adults and children give rise to etoposide concentrations between (roughly) <1 µM (through to >150 µM (peak). There are no adequate experimental systems to study dose-response and response-response relationships across MIE, KEs and AO in a single model.

2. Temporal concordance among the MIE, KEs and AO

There are no serious doubts about temporal concordance among MIE, KEs and AO. It is very difficult to see any other sequence of events (among this AOP), which would bring the AO into effect. Another matter is that it has never been shown in human pregnancy (or will be reliably or robustly demonstrated in the foreseeable future). In this respect, it is difficult to envisage whether epidemiological studies that are possible in humans, would ever be able to demonstrate the link without a direct biomarker for the MIE and KE2. Available experimental models (Sanjuan-Pla et al 2015) are in conformation with the AOP, except that in experimental in vivo models a very protracted appearance of leukaemia is not in line with a very short latency of infant leukaemia in human.

It is obvious that there exists a vast gap between wide exposure to potential Topo II poisons and the rarity of infant leukaemia. On the basis of studies in human adult and childhood leukemias, there are a large number of genetic, epigenetic and host factors potentially modifying the link between Topo II poisons and leukaemia. Because of the rarity of the disease, it is difficult to envisage an even partial proofing these factors as of importance for the infant leukaemia.

Response-Response and Temporality Concordance for the tool compound etoposide

Concentration of etoposide

KE1

In utero DNA DSB consequent to topo II inhibition

KE2

In utero MLL chromosomal rearrangement

AO

Infant leukaemia

0.01 – 0.1 µM, in vitro(TopII enzymes and cells in culture)

+++

(DNA damage response in various cells)

-

 

0.1 – 1 µM, in vitro cell cultures

+++

(haematopoietic progenitor and stem cells)

+

 

0.5-5 µM, ex vivo, mouse fetal liver HSC concentration1

+++

(inference from MLL cleavage)

+

(only MLL cleavage)

-

(no leukemia development)

max 5 µM, ex vivo, mouse fetal liver HSC concentration1

+++

(inference from MLL cleavage)

+

MLL fusions detected only in DNA repair deficient mice

-

(no leukemia development)

Max >150 µM, plasma concs in etoposide-treated patients2

+++

(inference from MLL cleavage)

++

MLL-AF4 fusion gene and protein

+

treatment-related acute leukaemia

1a range of concentrations is linearly extrapolated on the basis of the concentration of 5 µM after the dose of 10 mg/kg.

2plasma concentration of etoposide cannot be directly extrapolated to the concentration at the active site. Probably the actual active cellular concentrations of etoposide is much lower, perhaps 10 % or less of the plasma concentration.  

3. Strength, consistency of the experimental evidence, and specificity of association of AO and MIE

Regarding the treatment-related acute leukaemia, strength, consistency and specificity of association of AO and MIE is strong, because only etoposide  have strong evidence for causing acute leukaemia in human via the general process of the AOP described here. Although direct observations on the initial in utero MIE in infant leukaemia are not possible, there is a lot of inferential evidence from animal and in vitro cellular studies suggesting strongly that infant leukaemia recapitulates at least at an apparent process level the treatment-related leukaemia. It is important to recognize that in therapy-related AML this has been clearly demonstrated with abnormalities affecting MLL locus. Chlorpyrifos is reported to be a Topo II poison and to induce MLL translocation in the human liver haematopoietic stem cells (Lu et al. 2015). However, it is probable that the dose dependence of the formation of DSBs and fusion genes is linear only in a very restricted “window” of dose range. Considering the rarity of IFL and the common exposure to Topo II poisons like bioflavonoids, specificity is low. However, this consideration is limited by lack of experimental studies conducted with other than anticancer drugs on the sensitive target cells ie the liver haematopoietic stem cell. 

Domain of Applicability

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DNA topoisomerases are key ubiquitous enzymes at all levels of living organisms. Important differences in sensitivity to topoisomerases inhibition might exist among different cell types and hematopoietic stem and progenitor cells can be a sensitive target during a critical developmental period. foetuses and newborns show that both the baseline and chemically induced micronuclei frequencies are higher in the foetus and infant than in adults.

The available evidence do not allow for evaluating whether any significant difference occurs among cell types or species in regard to the KE event " in utero MLL chromosomal translocation". Fetal liver hematopoietic stem cells are more susceptible to the tool chemical etoposide than maternal bone marrow mononuclear cells and this has been also observed in mouse.

The AO "infant lekaemia" is a pediatric leukaemia and in animals the disease is not known and the artificial reproduction of the disease in animal models have limitations.


Essentiality of the Key Events

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In line with the defining question, essentiality for this AOP is moderate. However, the actual knowledge of the IFL is supporting the evidence that IFL is a “single hit” developmental disease and MLL translocation is an essential KE based on the probability linking MLL translocation and the occurrence of the disease. Based on this the overall essentiality can be considered moderate to strong.

Essentiality of the KEs; WoE analysis

Support for Essentiality of KEs

Defining Question

Are downstream KEs and/or the AO prevented if an upstream KE is blocked?

High (Strong)

Moderate

Low(Weak)

Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.)

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO

No or contradictory experimental evidence of the essentiality of any of the KEs

MIE

In utero exposure to DNA topoisomerase II poison

MODERATE

Although there are no direct experimental studies to demonstrate that blocking action of TopoII poisons would prevent the AOP, there are considerable evidence for the relationship between the concentration of etoposide and the formation of the MLL rearrangements in human (pre)haematopoietic progenitor/stem cells, which strongly suggest the essentiality of TopoII inhibition (e.g. Bueno et al 2009; Nanya et al 2015). In addition, chemical-induced DNA breakpoints are associated with predicted Topo II cleavage sites (ie MLL), supporting an essential role for TOPO II mediate breakage (Hernandez and Menendez 2016; Montecucco et al 2015).

In human patients, therapy-related acute leukaemia characterized by MLL rearrangement is predominantly associated with etoposide treatment (Super et al. 1993)

KE1

DNA-DSB

STRONG Topisomerases are nuclear enzymes taht play essential role in DNA replication, transcription, chromosome segregation and recombination. All cells have the type I and type II enzymes. Etoposide, a Topo II inhibitor, kills cells by inhibiting the enzyme to ligate DNA (Smith 2014), which leads to the accumulation of DNA-DSBs. DNA-DSBs are indeed critical lesions resulting in a wide variety of genetic alterations including traslocations (Shirvastav 2008). Persistent or incorrectely repaired DSBs can results in chromosome loss, deletion, translocation, or fusion, which can lead to carcinogenesis (Raynard 2017) 

KE2

In utero MLL chromosomal translocation

MODERATE.

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; Sanjuan-Pla et al 2015; Greaves 2015). Therefore, the totality of evidence suggests the essential role of the formation of MLL-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.

The 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 and essential for development of IFL (Menendez et al 2009).

TopoII ‘poisons’ etoposide and bioflavonoids (and some other chemicals) promote MLL rearrangements in in vitro prenatal cells or in utero. There are in vitro cellular and n vivo xenograph studies demonstrating that upon inhibiting signalling pathways from the fusion product on, cells can resume differentiation or clonal expansion of fusion gene-carrying cells is prevented (Benito et al 2015; Buechele et al 2015; Chen and Armstrong 2015). However, in absence of a relevant in vivo experimental model these findings are suggestive but not yet totally convincing.

Many fusion protein have been shown to recruit disruptor of telomeric silencing 1-like (DOT1L).  Although DOT1L is not genetically altered in the disease per se its mislocated enzymatic activity is a direct consequence of the chromosomal translocation. The enzymatic activity of DOT1L is critical to the occurence of MLL because methyltransferases-deficient DOT1L is capable of suppressinggrowth of MLL rearranged cells. A small-molecule inhibitor of DOT-1L inhibits cellular H3K79 methylation, blocks leukaemogenic gene expression, and selectivity kills cultured cells bearing MLL-translocation (Chen and Amstrong 2015).

Animal models expressing MLL-AF4 fusion gene exist(Chen et al., 2006; Metzler et al., 2006; Krivtsov et al., 2008; Bursen et al., 2008; Tamai et al., 2011) . Leukaemia is ultimately developed  the models though latency is protracted (Sanjuan-Pla et al., 2015).  Expression of the MLL-AF4 (or its reciprocal) fusion gene in these models is capable of triggering leukaemia, but it is unknown whether facilitating or additional changes are required during the long latency in the mouse.

The MLL-AF4 knock-in mouse developed leukaemia only after a prolonged latency (Chen et al., 2006), thus not recapitulating the ‘pathognomonic’ feature of infant leukaemia. Other animal models have been developed with similar results (see Sanjuan-Pla et al., 2015). 

Lin et al. (2016) designed a fusion gene between human MLL and murine af4 and demonstrated that it could transform–via retroviral transduction–human CD34+ cells to generate pro-B-ALL with all the characteristic features of the MLL-AF4 infant leukaemia.

 


Weight of Evidence Summary

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Biological plausibility.

The biological plausibility for this AOP is strong. The relationship between Topo II inhibition, DNA double strand breaks, MLL chromosomal translocation and infant leukaemia is well established. The same pathway is reproducible in chemotherapy-induced acute leukaemia in patients following treatment with etoposide, a known Topo II poison.

1 Support for Biological Plausibility of KERs

Defining Question

High (Strong) 

Moderate low (weak)

Is there a mechanistic (i.e. structural or functional) relationship between KEup and KE down consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance.

The KER is plausible based on analogy to accepted biological relationship, but scientific understanding is not completely established. There is empirical support for a statistical association between KES but the structural or functional relationship between them is not understood.

MIE to KE1

In-utero exposure to DNA Topo II inhibitor leads to DNA-DSB

STRONG

Rationale:

Although type II topoisomerases are essential to cell proliferation and survival, they have a significant genotoxic potential consequent to the resulting (double) strand breaks following enzymes inhibition. Mis-repair of accumulated of DNA double strand breaks can result in chromosomal translocations which can persist in survived cells (Mc Clendon et al. 2009, Raynard 2017).

KE1 to KE2

 DNA-DSB leads to in-utero MLL chromosomal translocation

STRONG

Rationale:

Studies on identical twins and neonatal blood samples strongly implicate an in utero occurrence of the KER (Sanjuan-Pla et al 2015). Furthermore, a study in pregnant mice demonstrates that in utero exposure of the foetus to etoposide causes DNA-DSB and MLL chromosomal translocation analogous to the human translocation except the principal fusion partner (Nanya et al 2015). Indirect evidence from human prehaematopoietic/mesenchymal stem cells and foetal liver haematopoietic progenitor and stem cells strengthen the plausibility. Experimental evidence in these cell lines has demonstrated that etoposide as a TopII poison causes DSBs in MLL and partner genes, which leads to the formation of fusion genes and their products (SanjuanPla 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. Although rearrangements associated with infant leukaemias are often more complex than those observed in treatment-related leukaemias, many are nevertheless associated with stable TopII-mediated DNA cut sites (Cowell and Austin 2012; Pendleton et al 2014)

KE2 to AO

In-utero MLL chromosomal translocation leads to Infant leukaemia

STRONG

Rationale:

The basic processes underlying overt leukaemia development are well understood and accepted. There is a general understanding of the molecular and epigenetic mechanisms leading to differentiation blockage and clonal expansion and there is evidence that the principal MLL-fusion genes and proteins harbour the necessary properties to execute the pathways associated with differentiation blockage and clonal expansion (Benito et al 2015; Chen and Armstrong 2015; Chen et al 2015).

 

Empirical support

The overall empirical support, using the chemical tool etoposide, is moderate. In vivo and, mainly in-vitro, experiments exist but they are lacking a clear dose or concentration response relationship.

 

 

3  Empirical support for KERs

Defining Question

Does the empirical evidence support that a change in the KEup leads to an appropriate change in the KE down? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup higher than that for KE down?

Are inconsistencies in empirical support cross taxa, species and stressors that don’t align with expected pattern of hypothesized AOP?

High (Strong)

Moderate

Low(Weak)

Multiple studies showing dependent change in both exposure to a wide range of specific stressors (extensive evidence for temporal, dose-response and incidence concordance) and no or few critical data gaps or conflicting data.

Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.

Limited or no studies reporting dependent change in both events following exposure to a specific stressor (ie endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesized AOP

MIE to KE 1 

In utero exposure to DNA topoisomerase II poison leads to DNA DSB.

STRONG

Rationale:

Experimental evidence in pre-hematopoietic/mesenchymal  cell lines has demonstrated that etoposide as a TopII poison causes DSBs in MLL and partner genes, which leads to the formation of fusion genes and their products (SanjuanPla et al 2015).

KE1 to KE2

DNA-DSB leads to In utero MLL chromosomal translocation

MODERATE

Rationale: Evidence comes from in vitro studies in appropriate human cells and from an in vivo/ex vivo study in pregnant mice; the stressor has been etoposide in most of the experiments (Libura et al 2005; Whitmarsh et al 2003; Lovett et al 201, Nanya et al 2015). Some evidence to back this KER comes from in vitro studies with bioflavonoids, especially quercetin, genistein and kaempferol (Barjesteh et al 2007).

KE2 to AO

In utero MLL chromosomal translocation leads to Infant leukaemia

MODERATE

Rationale:  There are a number of factors and pathways linking the fusion products with differentiation blockage and clonal expansion (Marschalek 2010; Sanjuan-Pla et al 2015). MLL encodes a protein homologous to the Drosophila trithorax gene, which has relevant functions in embryogenesis and haematopoiesis (Ernest et al 2004, Hess et al 1997). Studies with MLL-AF4, MLL-AF9 and MLL-ENL (Barabe et al 2007; Mulloy et al 2008) have clearly demonstrated how MLL chromosomal rearrangements block differentiation and enhance clonal expansion. However, there is a specific need to execute these studies in an appropriate experimental system with a proper target cell within a proper molecular and physiological environment.

 

There are several animal models, in which MLL-rearranged fusion genes have been expressed and leukaemia developed (Chen et al 2006; Metzler et al 006; Krivtsov et al 2008; Bursen et al 2008; Tamai et al 2011). Engineered human hematopoietic stem and progenitor cell carrying an MLL rearrangement showed that a subset of cells persisted over time and demonstrated a higher clonogenic potential in colony forming assay (Breese et al. 2015). Cells engineered to carry MLL-AF9 and MLL-ENL fusions demonstrated leukaemogenicity especially after ex vivo and repeated transplantation (Buechele et al 2015).

 

Uncertainties and Inconsistencies

  • In utero evidence of the disease is difficult to obtain in humans and one has to resort to in vitro cellular systems, which may be inadequate to take into consideration the potential effects of proposed microenvironments, rapidly changing developmental stages and facilitating and modifying factors
  • Animal models are a possibility (e.g. Nanya et al 2015), but are naturally prone to species-specific factors.
  • An important problem is to provide a convincing and experimentally justified explanation for the dilemma between the rarity of disease in the face of pervasive exposure to topoII inhibitors
  • The treatment-related AML apparently is a true surrogate for the infant leukaemia, at least mechanistically. Is it only because of etoposide as a principal chemical intiator has provided many crucial findings for understanding the infant leukaemia.
  • The ‘poisoning’ of the TopoII-DNA cleavage complex has not been shown in the putative target cell, which is still not unequivocally identified. 
  • MLL-AF4 knock-in mice develop leukaemia only after a prolonged latency (e.g. Chen et al 2006), thus not recapitulating the ‘pathognomonic’ feature of infant leukaemia.
  • The inability of available in vivo models to recapitulate the whole AOP process is due to a crucial factor which has not yet been found, or to model-specific peculiarities. 
  • In the face of the rarity of the disease, epidemiological studies especially concerning aetiology and risk factors are not powerful enough to provide robust answers. For instance, investigating the hypothesized relationship of bioflavonoids with infant leukaemia will have to consider the gap between the widespread intake of these phytochemicals and the very rare occurrence of the disease.
  • The biology of the disease (i.e. IFL) and the experimental studies conducted with etoposide, indicate in-utero exposure of hematopoietic stem cells (HSC) as the most critical, if not essential, factor for the development of the A . However, a clear comparative quantification in terms of dose response vs different time of exposure and cell systems is lacking.
  • The very early embryonic structure and the liver haematopoietic stem cells in particular, are representing the target cell for this AOP. A clear understanding of a higher sensitivity of HSC vs, mature hematopoietic cells, particularly in the standard genotoxicity test battery is lacking and more chemicals and comparative assays should be tested to scientifically validate this cell system.
  • The role of fusion partners in the process of leukaemogenesis has not been completely elucidated and is representing an important uncertainties for this AOP. Normally, all of them participate in chromatin modifying complex, for example, acting on the transcriptional regulation of target genes. The MLL fusion proteins are dysregulating this highly regulated process and probably different fusion partners are working in a distinct way with variable modulatory effect on signalling pathways in leukaemic cells. Recruitment of DOT1L or officially KMT4, a histone methyltransferase, seems to be a common feature of many oncogenic MLL fusion proteins, resulting in the over methylation and overexpression of several MLL target genes encoding for transcription factors involved in body patterning and hematopoiesis. It is indeed possible that an additional (epi)genetic KE would occur downstream to MLLtranslocation, but a better understanding of the role of fusion partners in the process of leukaemogenesis would be necessary before  adding it and at the moment this should be considered as a knowledge gap for this AOP.
  • On the basis of studies in human adult and paediatric leukaemia, there is a large number of genetic, epigenetic and host factors potentially modifying the link between various chemical exposures and leukaemia. Because of the rarity of the disease, it is difficult to envisage, even partially, aetiological factors as of importance for the infant leukaemia.​
  • Transcription activator-like effector nuclease (TALEN)-mediated genome editing was used to generate endogenous MLL-AF9 and MLL-ENL oncogenes in primary human HSPCs derived from human umbilical cord plasma (Buechele et al., 2015). Engineered HSPCs displayed altered in vitro growth potential and induced acute leukaemias following transplantation in immunocompromised mice at a mean latency of 16 weeks.​

Quantitative Considerations

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The WOE analysis indicates that many KEs and KERs lack especially experimental evidence, but overall the analysis supports the qualitative AOP. The strong element in the development of the qualitative AOP is the biological plausibility of the overall pathway that it can partially be based on studies in human treatment-related disease recapitulating many crucial features of the infant leukaemia. The lack of sufficient experimental data and uncertainties in quantitative information from treatment-related acute leukaemia makes it problematic to build convincing dose (concentration)-response and response-response relationships and to identify possible practical thresholds for stressors. The MIE is expected to show a dose response relationship to a certain extent. However, it is probable that the dose dependence of the formation of DSBs and fusion genes is linear only in a very restricted “window”. In too-low concentrations the outcome of the stressor is a successful repair of the break, in too-high concentrations the outcome is cell death. It should be kept in mind additionally that the quantification of dose-responses should also consider the different sensitivity of cell systems that should be also representative of the specific time-window of exposure (i.e. in-utero).

The most pressing future need is an adequate and robust experimental model system for the evaluation of relationships between doses, concentrations and responses within a temporal framework of the AOP. 

 


Considerations for Potential Applications of the AOP (optional)

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Applicability of the AOP

The proposed AOP is strictly life stage-dependent, being linked with in utero exposure and early embryogenesis. However, the surrogate disease (i.e. chemotherapy-related acute leukaemia) is not life stage restricted as well as the genotoxic hazard is not expected to be life stage related.

 Potential regulatory applications of the AOP

This AOP was initiated with the intention to use an epidemiologically proposed human health outcome as AO and build back an AOP leading to this. Infant childhood leukaemia is a human disease and consequently apical regulatory endpoints can only explore the hazard by means of surrogate testing. These include carcinogenesis assays and blood cell analyses in the in vivo toxicology assessment. Considering the unique biology of this AO, these tests  show some technical limitations and also the sensitivity and specificity of the available tests for the AO is limited. Additionally, experimental animal models replicating the AO are limited. Technical limitations of the standard regulatory tests include: Standard carcinogenesis studies do not include an early in-utero exposure time, blood cell analysis is not a standard requirement in the extended multi-generation reproductive toxicity study and no cancer-related endpoints are included in this study. In addition, considering the rarity and the complexity of the disease, the sensitivity and specificity of these tests to capture this hazard is likely to represent a big hurdle and the regulatory tests are unlikely to represent the best way to explore this AO. 

This AOP is however indicating that the MIE and the KE1 can be measured in scientific and/or regulatory validated test assays.

With these premises, the authors support the use of this AOP during the process of assessment of epidemiological studies and the use of the AOP framework to support the biological plausibility of  the effects observed in the epidemiological studies when experimental and toxicological studies are indicative that the AOP is affected and this should guide on which additional studies should be performed, if the case, to integrate the AOP framework into the MOA framework for specific chemical entities.

In addition, this AOP should serve in guiding testing strategy. This include the exploration of Topo II poison characteristics of a chemical and, if the genotoxicity standard regulatory testing battery is negative, considerations should be made on the sensitivity of the cell system used in the assay (i.e.liver HSPC).


References

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