Mll-AF4 Induction during Ontogeny Reveals Early Changes in Myeloid and Lymphoid potential and Results in Hematopoietic Malignancies in Adult Mice

Acute leukemia is the most common malignancy of childhood. The pattern of incidence of childhood leukemia reveals how age at diagnosis is associated with particular molecular subtypes, responsiveness to treatment and outcome.

Translocations involving the Mixed Lineage Leukemia gene (MLL) in particular are associated with the diagnosis of acute leukemia in infancy. As with other forms of childhood leukemia, evidence from retrospective cohort analysis of neonatal blood spots suggests that MLL translocations are present at birth in many of these cases, indicating that the initial steps towards leukemia development occurred in utero (Greaves Early Human Development 2005).

Concurrently there has been increasing recognition of the differences between the foetal and adult hematopoietic cells and supportive niches. These differences might have profound effects on a cell’s ability to undergo malignant transformation and its subsequent properties.

Unlike mouse models of myeloid leukemia associated with MLL fusions, previous attempts to mouse model acute lymphoid leukemia of infancy associated with the MLL-AF4 fusion gene have had mixed results, with unsatisfactory disease latencies and phenotypes (Stam Cell Research 2012). However, many of these models have introduced the translocation only within the adult hematopoietic system – discounting the possible effect of this mutation in utero.

The aim of this study was to examine the effect of MLL-AF4 within the context of the varied hematopoietic sites of the developing embryo, and how the induction of MLL-AF4 within these niches might influence subsequent disease development.

For this, we utilised the Cre inducible Mll-AF4 “invertor mouse” that has previously been described (Metzler et al Oncogene 2006). As embryonic stem cell work suggests MLL-AF4 may alter the emergence of Hematopoietic Stem Cells (HSCs) from the hemogenic precursors (Bueno Cell Research 2012), we crossed the invertor mice with Ve-cadherin-Cre and Vav-Cre murine lines to induce Mll-AF4 expression in (pre)HSCs before and after this critical time point. HSC and progenitor populations at several established hematopoietic sites through ontogeny were assessed by flow cytometry and functional assays, including in vitro methylcellulose based colony forming assays and in vivo transplantation assays. Mice were also bred to adulthood and observed for the development of disease.

Despite no statistically significant changes in HSC and progenitor populations within in utero HSC niches, functional assays reveal marked differences in both myeloid and lymphoid colony forming assays with a particularly strong effect seen in lymphoid assays; however, despite evidence of a limited increase in serial repeatability, these cells did not appear to be fully transformed and could not be re-plated successfully beyond the third round. In vivo transplantation assays also showed enhanced multi-lineage engraftment by foetal liver cells carrying the Mll-AF4 fusion gene. Adult mice developed both B and T cell malignancies with long latencies and median survival of 437 day to 551 days for Mll-AF4xVeCadherin-Cre and Mll-AF4xVav-Cre lines, respectively.

We have thus shown for the first time that the induction of Mll-AF4 has an effect on the myeloid and lymphoid output of foetal derived HSCs. Although this effect appears to fall short of full transformation, the development of lymphoid malignancies in adult mice suggests that such cells have pre-malignant potential. We continue to explore the mechanism of these early pre-malignant effects and aim to explore how additional factors may unlock the true malignant potential of MLL-AF4 in murine model systems.