MLL-AF4 perturbs the self-renewal and differentiation potential of HSC during the definitive wave of haematopoiesis: A stage-specific pattern

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The t(4;11) MLL-AF4 infant leukaemia has a fast progression and a poor prognosis, and we need to gain a better understanding of the disease. Patients possess the MLL-AF4 fusion gene at birth, suggesting that leukaemia is initiated during embryonic development. This study aims at characterising the effect of MLL-AF4 on the self-renewal and differentiation of embryonic and foetal haematopoietic stem cells (HSC). Using the conditional invertor technology in mice, we initiated the expression of MLL-AF4 in the haemogenic endothelium (VEC-Cre) and confirmed its expression in haematopoietic cells. Through serial transplantation assays, we found that MLL-AF4+ HSCs derived from E12 placenta and E12-E14 foetal liver both have a higher self-renewal potential compared to their normal counterpart. This effect was not observed in MLL-AF4+ HSCs derived from E11.5 AGM, suggesting that the MLL-AF4 influence is stage-specific. MLL-AF4 also caused a differentiation bias toward the B lymphoid lineage in foetal liver HSCs. This was also confirmed by colony-forming assays, where foetal liver MLL-AF4+ haematopoietic cells (LSK and LMPP) formed more lymphoid colonies compared to their normal counterpart. The cells in these colonies expressed progenitor B cell surface markers, similar to human leukaemia blasts. Furthermore, some of our transplanted mice developed a haematological malignancy within 12 months, which confirms that MLL-AF4 is an oncogenic driver on its own. However, since the human disease has a more rapid progression, we are currently testing additional genetic events that could accelerate leukaemogenesis. We identified two microRNAs in patients that are overexpressed in MLL-AF4+ leukaemia blasts. Both of them act as positive regulators of survival and proliferation in MLL-AF4+ leukaemia cell lines and could act as co-drivers of leukaemogenesis. Further work will focus on elucidating the pathways affected by the overexpression of these miRNAs in patients and how they can accelerate the disease progression of t(4;11) MLL-AF4 leukaemia.