SNAPSHOT

1252: In utero exposure to DNA topoisomerase II “poisons”

Short Name: In utero exposure to DNA topoisomerase II “poisons”

AOPs Including This Key Event

Evidence for Perturbation of this Molecular Initiating Event by Stressor

A number of drugs, environmental chemicals and natural substances are identified as TopoII “poisons” (Pendleton et al 2014) (Table 21). A well investigated example   is the anticancer drug etoposide; also bioflavonoids, e.g. genistein, (Barjesteh van Waalwijk van Doorn-Khosrovani et al 2007; Azarova et al 2010) bind to TopoII enzymes, induce cleavage in the MLL gene and produce a fusion gene (and its product) in human cells. The organophosphate pesticide chlorpyrifos has been shown to inhibit (‘poison’) the enzyme in vitro (Lu et al 2015).

Chemical class	Examples	References
Anticancer agents
Epipodophyllotoxins	etoposide, teniposide	Montecucco et al 2015
Anthracyclines	doxorubicin, epirubicin, daunorubicin, idarubicin, aclarubicin	Cowell and Austin 2012
Anthacenedione	Mitoxantrone	Cowell and Austin 2012
Acridines	Amsacrine	Cowell and Austin 2012
Bioflavonoids
Flavones	luteolin, apigenin, diosmetin	Ketron and Osheroff 2014
Flavonols	myricetin, quercetin, kaempferol, fisetin	Ketron and Osheroff 2014
Isoflavones	Genistein	Ketron and Osheroff 2014
Catechins	EGCG, ECG, EGC, EC	Ketron and Osheroff 2014
Isothiocyanates	benzyl-isothiocyanate, phenethyl-isothiocyanate, sulforaphane	Ketron and Osheroff 2014
Other phytochemicals	Curcumin	Ketron and Osheroff 2014
Environmental chemicals
Aromatic compounds	benzene, PAHs
Nitrosamines	Diethylnitrosamine	Thys et al 2015
Organophosphates	Chlorpyrifos	Lu et al 2015

Etoposide

Much of the relevant, albeit  indirect, evidence to support this AOP come from the studies on etoposide, an anticancer drug  TopoII “poison”, which is known to induce therapy-associated acute leukaemia (t-AL) in adults (Cowell and Austin 2012; Pendleton et al 2014). It is of interest that the latency of t-AL is <2 years between the treatment of the primary malignancy and the clinical diagnosis of the secondary disease and that the prognosis of t-AL is poor (Pendleton et al 2014). t-AL is characterized by the MLL rearrangements and it is practically certain that these fusion genes are caused by etoposide or anthracyclines treatment, because MLL rearrangements have not been detected in bone marrow samples banked before the start of the treatment of the first malignancy. Also the breakpoints in MLL or partner genes fall within a few base pairs of a drug-induced enzyme-mediated DNA cleavage site (Pendleton et al 2014).

Etoposide can induce MLL rearrangements 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 rearrangements; in addition, undifferentiated human embryonic stem cells (hESCs) were concurrently liable to acute cell death (Bueno et al., 2009). These findings suggest that the MIE should be put into evidence in target cell models with appropriate sensitivity.

 

Bioflavonoids

Bioflavonoids are natural polyphenolic compounds in a large variety of plant-derived food items. TopoII-mediated DNA cleavage has been linked to genistein, kaempferol, luteolin, myricetin and apigenin (Strick et al 2000; Bandele and Osheroff 2007; Azarova et al 2010; Lopez-Lazaro et al 2010), although the concentrations in in vitro studies have been quite high. It has also been demonstrated that several bioflavonoids are capable of inducing the cleavage of the MLL gene in human cell lines (Strick et al 2000; van Doorn-Khosrovani et al 2007). The in vitro effects of bioflavonoids suggested a possible link between dietary intake and infant leukemia (e.g., Azarova et al., 2010; Lanoue et al., 2010); however until now, epidemiological evidence existing to support or refute such a hypothesis is based on small studies (Ross et al 1996; Spector et al 2005).

Chlorpyrifos

Chlorpyrifos is a widely used organophosphate insecticide, which has been suspected as a risk factor for infant and childhood leukaemia after the house-hold exposure of pregnant women. According to Lu et al (2015), chlorpyrifos and its metabolite chlorpyrifos oxon exhibit an inhibitory effect on in vitro TopoII activity. Chlorpyrifos causes DNA double strand breaks as measured by the neutral Comet assay and induces MLL gene rearrangements in human fetal liver-derived CD34⁺ hematopoietic stem cells via TopoII ’poisoning’ as detected by the FISH assay and in vitro isolated TopoII inhibition assay, respectively (Lu et al 2015). Chlorpyrifos also stabilizes the TopoII-DNA cleavage complex. Etoposide was used a positive reference compound in these studies and it performed as expected. The lowest concentration of chlorpyrifos used was 1 µM and it gave a statistically significant effect in many in vitro assays. The point of departure of etoposide, which was calculated to be 0.01 to 0.1 µM (Li et al 2014), is at least 10-fold lower than that of chlorpyrifos.

 

 

 

 

Biological Organization

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.

1253: In utero MLL chromosomal translocation

Short Name: MLL translocation

AOPs Including This Key Event

Stressors

Biological Organization

Life Stage Applicability

Sex 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).

1254: Infant leukaemia

Short Name: IFL

AOPs Including This Key Event

Stressors

Etoposide

Etoposide

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). The effects of etoposide in various model systems will be described in detail in the description of the developed AOP.

 

There should be some information about the concentration-response relationships of the selected tool chemical in the development of the AOP. 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; however, when the number of double strand breaks (DSBs) exceeds a threshold value, ataxia telangiectasia mutated (ATM) is recruited and becomes fully activated through a reversible mechanism, leading to elevated repair capacity as a result of phosphorylation (activation) of several target proteins, including p53 and other tumour suppressor proteins. The model was able to quantitatively capture 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 foetal liver CD34⁺ cells an increase in DSBs was observed at a concentration of 0.14 µM (Bueno et al., 2009) and MLL-r were detectable by FISH or flow cytometry at higher concentrations (Moneypenny et al., 2006). 

Biological Organization

Life Stage Applicability

Sex Applicability

Infant leukaemia is a paediatric leukaemia likely resulting from gene-environmental interactions. The limited data available suggest that dietary and environmental exposure to substances targeting topoisomerases together with reduced ability of the foetus or their mother to detoxify such compounds because of the polymorphic variants of given genes could contribute to the development of this AO (Hernadez  et al. 2016).

In animals the disease is not known and artificial animal models able to reproduce the disease have limitations. Bardini et al (2015) has however developed a xenograft mouse model with patient MLL-AF4-involving leukoblasts transplanted. 

In utero exposure to DNA topoisomerase II “poisons” leads to MLL translocation

Evidence Supporting Applicability of this Relationship

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.

How Does This Key Event Relationship Work

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).

Weight of Evidence

Biological Plausibility

The KER as such is biologically plausible. 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.  

Empirical Support for Linkage

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^[1]. 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).

Uncertainties or 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.  

References

Blanco JG, Edick MJ, Relling MV. Etoposide induces chimeric Mll gene fusions. FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY (FASEB) 2004; 18(1):173–5. doi: 10.1096/fj.03-0638fje

Breese EH, Buechele C, Dawson C, Cleary ML, Porteus MH. Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells. Public Library of Science (PLoS ONE) 2015 Sep 9;10(9):e0136644. doi: 10.1371/journal.pone.0136644.

Buechele C, Breese EH, Schneidawind D, Lin CH, Jeong J, Duque-Afonso J, Wong SH, Smith KS, Negrin RS, Porteus M, Cleary ML. MLL leukemia induction by genome editing of human CD34+ hematopoietic cells. Blood 2015 Oct 1;126(14):1683-1694. doi: 10.1182/blood-2015-05-646398.

Chen W, Li Q, Hudson WA, Kumar A, Kirchhof N, Kersey JH. A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy. Blood Journal 2006; 108(2):669–77. doi: 10.1182/blood-2005-08-3498

Ishii E, Eguchi M, Eguchi-Ishimae M, Yoshida N, Oda M, Zaitsu M, et al. In vitro cleavage of the MLL gene by topoisomerase II inhibitor (etoposide) in normal cord and peripheral blood mononuclear cells. International journal of hematology. 2002; 76(1):74–9.

Li Z, Sun B, Clewell RA, Adeleye Y, Andersen ME, Zhang Q. Dose-response modeling of etoposide-induced DNA damage response. Toxicological Sciences 2014 Feb;137(2):371-84. doi: 10.1093/toxsci/kft259.

Libura J, Slater DJ, Felix CA, Richardson C. Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion. Blood Journal 2005; 105(5):2124–31. doi: 10.1182/blood-2004-07-2683

Libura J, Ward M, Solecka J, Richardson C. Etoposide-initiated MLL rearrangements detected at high frequency in human primitive hematopoietic stem cells with in vitro and in vivo long-term repopulating potential. European Journal of Haematology 2008; 81(3):185–95. doi: 10.1111/j.1600-0609.2008.01103.x

McClendon AK, Osheroff N. DNA Topoisomerase II, Genotoxicity and Cancer. Mutation Research 2007; 623 (1-2): 83-97.

Moneypenny CG, Shao J, Song Y, Gallagher EP. MLL rearrangements are induced by low doses of etoposide in human fetal hematopoietic stem cells. Carcinogenesis. 2006; 27(4):874–81. Epub 2005/12/27. doi: 10.1093/carcin/bgi322

Montecucco A, Zanetta F, Biamonti G. Molecular mechanisms of etoposide. JOURNAL OF EXPERIMENTAL AND CLINICAL SCIENCES. 2015 Jan 19;14:95-108. doi: 10.17179/Journal - Experimental and Clinical Sciences (EXCLI)2015-561.

Nanya M, Sato M, Tanimoto K, Tozuka M, Mizutani S, Takagi M. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells. Public Library of Science (PLoS ONE). 2015 Dec 11;10(12):e0144540. doi: 10.1371/journal.pone.0144540.

Pitz MW, Desai A, Grossman SA, Blakeley JO. Tissue concentration of systemically administered antineoplastic agents in human brain tumors. Journal of Neuro-Oncology 2011 Sep;104(3):629-38. doi: 10.1007/s11060-011-0564-y.

Relling MV, Yanishevski Y, Nemec J, Evans WE, Boyett JM, Behm FG, Pui CH.Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia. Leukemia. 1998 Mar;12(3):346-52.

Stremetzne S, Jaehde U, Kasper R, Beyer J, Siegert W, Schunack W. Considerable plasma levels of a cytotoxic etoposide metabolite in patients undergoing high-dose chemotherapy. European Journal of Cancer 1997 May;33(6):978-9. 

MLL translocation leads to IFL

Evidence Supporting Applicability of this Relationship

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.

How Does This Key Event Relationship Work

Propagation of a leukaemic cell clone is based on both blockage of differentiation to more mature cells and ability to expand in an uncontrolled way. Formation of the MLL-rearranged fusion genes and their protein products are intimately involved in both the blocked differentiation of HSPCs and the expansion of the fusion gene-carrying clone. It is believed that the fusion gene product block cell differentiation by inhibiting the normal transcriptional programs and recruiting repressor molecules such as histone deacetylase enzymes (Greaves 2002; Teitell and Pandolfi 2009). Furthermore, the fusion gene product activates other key target genes, which ultimately lead to the propagation of transformed cell lines without normal restrictions (Greaves 2015; Sanjuan-Pla et al 2015). Therefore, the potential of both differentiation blockage and clonal expansion are inherent properties of the MLL-rearranged fusion product, based on the preservation of some original functions, even if in a modified form, and on the gain of some other functions due to the sequences from the new fusion partner gene (Marschalek 2010; Sanjuan-Pla et al 2015).

Molecular mechanisms

The MLL is the most common translocation gene in infant leukaemia. The N-terminal part of MLL becomes fused in frame to one of a large number of fusion partners, but in most cases, this fusion occurs between the N-terminal MLL and either AF4, AF6, AF9, AF10, or ENL (Krivtsov and Armstrong 2007). Due to the DNA-binding properties of the N-terminal MLL motif, these fusion proteins are always nuclear and bind to target genes controlled by MLL irrespective of the normal location of the C-terminal partner.

Many fusion proteins have been shown to recruit DOT1L (catalyzing methylation of histone H3K79) to the promoters of MLL target genes and this recruitment seems to be a common feature of many oncogenic MLL fusion proteins. Although DOT1L is not genetically altered in the disease per se, its mislocated enzymatic activity is a direct consequence of the chromosomal translocation. Thus, DOT1L has been proposed to be a catalytic driver of leukemogenesis (Chen and Armstrong 2015). The enzymatic activity of DOT1L is critical to the pathogenesis of MLL, because methyltransferase-deficient Dot1L is capable of suppressing growth of MLL-rearranged cells. A small-molecule inhibitor of DOT1L inhibits cellular H3K79 methylation, blocks leukemogenic gene expression, and selectively kills cultured cells bearing MLL translocations (Chen and Armstrong 2015). One of the target gene of DOT1L is BCL-2, belonging to a family of anti-apoptotic genes, which maintains the survival of the MLL-rearranged cells (Benito et al 2015). Expression of BCL-2 is high in human MLL-AF4 leukemia cells from a large number of patients.  A specific BCL-2 inhibitor, ABT-199 is capable of killing MLL-AF4 leukaemia cells and prevents cell proliferation in xenograft mouse leukaemia models (Benito et al 2015). Furthermore, a MLL-AF4 cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. The figure below provides a schematic representation of the molecular pathway.

 

MLL-rearranged acute lymphoblestic leukemisa activate BCL-2 through H3K79 methylation and are sensitive to the BCL-2 specific antagonist ABT-199 (benito et al, Cell Rep 2015).

 

Possible facilitating mutated genes

Recurrent activating mutations in the components of the PI3K-RAS signalling pathway have been detected in almost half of the tested MLL-rearranged ALLs in one study (Andersson et al 2015). Prenatal origin of RAS mutations have been demonstrated also in other studies of infant leukaemia with frequencies of about 15-25 % of cases (Driessen et al 2013; Prelle et al 2013; Emerenciano et al 2015).  Emerenciano et al (2015) are of the opinion that RAS mutations seem not to be driver mutations, but may aid disease onset by accelerating the initial expansion of cells.

Overall the activation of the RAS pathway could support the extremely rapid progression of the infant leukaemia. Under this view the mechanism may represent a factor modulating (i.e., increasing) the progression and severity of the adverse outcome, rather than a necessary key event (second hit) for infant leukaemia. In the transgenic MLL-AF4 mouse model, activated K-RAS accelerated disease onset with a short latency (Tamai et al 2011), possibly by augmenting the upregulation of HoxA9. In a recent study of Prieto et al (2016),the activated K-RAS  enhanced extramedullary haematopoiesis of MLL-AF4 expressing cell lines and cord blood-derived CD34+ hematopoietic stem/progenitor cells that was associated with leucocytosis and central nervous system infiltration, both hallmarks of infant MLL-AF4 leukaemia. However, K-RAS activation was insufficient to initiate leukaemia, supporting that the involvement of RAS pathway is an important modifying factor in infant leukemia. It has also been demonstrated that MLL-AF6 fusion product sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukaemia cells and it is possible to attenuate the activation by tipifarnib, a RAS inhibitor (Manara et al 2014).

A possibility that MLL fusions render cells susceptible to additional chromosomal damage upon exposure to etoposide was studied by introducing MLL-AF4 and AF4-MLL via CRISPR/Cas9-genome editing in HEK293 cells as a model to study MLL fusion-mediated DNA-DSB formation/repair (Castano et al 2016). In short, the expression of fusion genes does neither influence DNA signaling nor DNA-DSB repair.

 

Weight of Evidence

Biological Plausibility

The biological plausibility linking the MLL translocation to infant leukaemia is strong. Rearrangement in the MLL gene is commonly associated with infant acute leukaemia and the disease has unique clinical and biological feature (Ernest et al. 2002). An in utero initiation, an extremely rapid progression, and a silent mutational landscape of infant leukaemia suggest that the MLL-translocation-associated gene fusion product is itself sufficient to spawn leukaemia and no “second hit” is required. Therapy-related leukaemias following exposure to the topo II poisons such as etoposide are characterized by the MLL chromosomal translocation (Libura et al. 2006, Super et al.1993) and translocations involving MLL are associated with a gain of function and leukemogenic effect (Yu et al. 1998). A critical developmentally early window of stem cell vulnerability, involving perhaps lesions based on epigenetically controlled regulatory factors, has been suggested to explain a rare occurrence and an exceptionally short latency of infant leukaemia (Greaves 2015; Sanjuan-Pla et al 2015).  In primary HSPCs genome engineered for patient specific MLL translocations it was possible to show that this specific ‘artificial’ initiation can induce a selective advantage in survival in extended culturing and a higher clonogenic potential in colony forming assay (Breese et al. 2015).

Empirical Support for Linkage

A number of MLL-fusion products, such as MLL-AF9 and MLL-ENL, have shown leukemogenic potential in cord-blood stem cells. Although the MLL rearrangement is essential to develop leukaemia, it alone may not be sufficient and activation of cellular proliferation might be necessary for overt leukaemia (Nanya et al. 2015).

There are several animal models, in which MLL-AF4 fusion gene has been expressed (Chen et al 2006; Metzler et al 006; Krivtsov et al 2008; Bursen et al 2008; Tamai et al 2011). In all these models leukaemia is ultimately developed, but latency has been very protracted. In any case, one could conclude that the expression of the MLL-AF4 fusion gene is capable of developing leukaemia, but it is unknown whether facilitating or necessary changes are required during the long latency in mouse.

Gene engineered human HSPCs carrying MLL rearrangements showed that a subset of cells persisted over time and demonstrated a higher clonogenic potential in colony forming assay (Breese et al. 2015).

Transcription activator-like effector nuclease (TALEN)-mediated genome editing generated 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. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts, expressed elevated levels of crucial MLL-fusion partner target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays.

Uncertainties or Inconsistencies

·         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. Also other animal models have been developed with similar results. Thus, an adequate experimental model for infant leukaemia is still in need.

·         The role of a reciprocal fusion gene AF4-MLL in leukemias is controversial: it has a transformation potential in animal model (Bursen et al 2010), but it is not expressed in all MLL-AF4 patients (Andersson et al 2015). The potential role of other reciprocal fusion genes has not been studied.

·         Beyond MLL rearrangements, activation of cellular proliferation by mutation or other (epi)genetic insults might be necessary for overt leukaemia. Further studies are necessary to fully understand which factors would contribute to convey a proliferative advantage, as observed in cells with MLL translocation, to leukaemia.

Quantitative Understanding of the Linkage

Relationships between different fusion genes and subsequent leukemia types are incompletely understood.  Although roughly 70-80 % of infant B-ALL leukemias carry MLL rearrangements, in 20-30 % of the cases there are no MLL rearrangements. In AML and T-ALL leukemia cases MLL rearrangements are even rarer.

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