MLL translocation leads to IFL

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).

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 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 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.

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 (t(10;11)(p12;q23) translocation, which encode the MLL AF10 fusion oncoproteins, observed in patients with acute myeloid leukemia), 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 disruptor of telomeric silencing 1-like (DOT1L or officially KMT4, a histone methyltransferase that methylates lysine 79 located within the globular domain of histone H3, H3K79) to the promoters of MLL target genes. This recruitment seems to be a common feature of many oncogenic MLL fusion proteins and results in abnormal H3K79 methylation and overexpression of several MLL target genes, such as HOXA genes coding for transcription factors involved in body patterning and hematopoiesis (Chen and Armstrong, 2015). 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.

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.

 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. 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). These unique characteristics indicate that biological domain for this KER is specific to an early in-utero exposure condition.