MLL-AF9-Induced Leukaemia Changes during Ontogenic Stages

MLL-AF9 causes acute lymphoid leukaemia (ALL), acute myeloid leukaemia (AML) and mixed phenotype acute leukaemia (MPAL) in infants and children. Infant leukaemia originates in utero, yet this has never been formerly shown for MLL-AF9-driven disease. Multiple mouse models invariably revealed that MLL-AF9 can induce myelo-monocytic AML, however, they failed to recapitulate the ALL and MPAL phenotypes as presented in paediatric patients. To explore the impact of developmental stage for MLL-AF9 leukaemic burden and phenotypes, we established a new inducible (“Tet-off”), reversible, transplantable mouse model in which expression of the fusion is controlled by the 3' stem cell leukaemia (SCL)-enhancer element.

We crossed SCL-tTA mice(1) with inducible MLL-AF9 mice(2) to generate double transgenic SCLtTA/iMLL-AF9 mice. To determine the impact of developmental stage, MLL-AF9 was induced from the conceptional stage for prenatal (E0), in neonates for postnatal (P0) and at 20 weeks for adult (≥20 weeks) cohorts. Following induction for 6-8 weeks in vivo MLL-AF9 expression was measured in haematopoietic stem cells (HSCs, (Lin-cKit+Sca-1+CD150+CD48-), lymphoid-myeloid primed progenitors (HPC-1/LMPPs, LSK CD150-CD48+/Flt3+), and granulocyte-monocyte progenitors (GMPs, Lin-cKit+Sca-1CD34+CD16/32hi). We found that MLL-AF9 expression levels were comparable within each population across the age groups but observed higher MLL-AF9 expression in the GMPs compared to HSCs in the postnatal model, concomitant with a moderate expansion in the GMP clonal size.

Upon postnatal MLL-AF9 induction, all the mice developed leukaemia with a short latency of 13 weeks and complete penetrance. In contrast, E0 prenatal- or adult-induction resulted in longer latencies of 32 and 24 weeks respectively. Despite sustained MLL-AF9 expression, not all the mice developed leukaemia in prenatal- and adult-induced cohorts. Postnatal induced mice developed myelo-monocytic (AML-M4), megakaryoblastic (AML-M7), or MPAL, whereas prenatal and adult induction of MLL-AF9 developed AML-M4 exclusively. Regardless of leukaemia phenotypes, all diseased mice displayed thrombocytopenia and splenomegaly, and leucocytosis. Independent from the age at which MLL-AF9 was induced, all AML-M4 mice had clonal expansion of GMPs. Adult-induced AML-M4 mice had an increase in HSCs while postnatal-induced mice had an increase in HPC-1/LMPPs. In postnatal-induced MPAL mice, there was expansion of GMPs and HPC-1/LMPP populations, and postnatal-induced AML-M7 mice had clonal expansions of HSC, HPC-1/LMPP, and MEP populations with no expansion in GMPs. Thus, the age when the MLL-AF9 fusion is induced does not only determine the disease latency and penetrance, but also affects lineage determination, and the amount of leukaemic HSPCs.

Leukaemic-GMP (L-GMP) and leukaemic-HSC (L-HSC) have been characterized in adult models as leukaemic stem cells (LSC). Transplantation of bulk and L-GMPs from adult-induced AML-M4 cells were able to cause AML with complete penetrance, in line with published data and validating our model. However, prenatal- and postnatal-induced AML-M4 cells were not transplantable. In all age cohorts, bulk AML cells formed serially plateable large and compact colonies and were reversible. Collectively, LSCs exist within a GMP-like population of adult-induced MLL-AF9 AML, they may however reside in different HSPC populations in prenatal- and postnatal-induced disease.

In summary, this novel inducible model recapitulates several features of infant, paediatric and adult MLL-AF9 AML demonstrating that ontogenic stage is a determining factor in differences in disease biology.