Final Report Summary - LEUKAEMOGENESIS (Developmental impact of MLL-AF4 fusion gene linked to infant acute lymphoblastic leukaemia on human stem cell fate)
Normal blood cell development begins in the bone marrow with the formation of Hematopoietic stem cells (HSC), which are capable of developing into the full range of blood cells, namely red cells, or erythrocytes, white cells, or leukocytes, and platelets, in a process called hematopoiesis. Leukaemia develops when the Deoxyribonucleic acid (DNA) of a leukocyte precursor gains genetic or epigenetic abnormalities and the cell becomes malignant acquiring uncontrolled growth properties.
Acute lymphoblastic leukaemia (ALL) is a rapidly progressing tumour in which the cancerous change begins in a lymphocyte precursor, producing leukemic blasts that are not fully developed. The Mixed lineage leukaemia (MLL) gene located on chromosome 11q23 is one of the most frequently mutated genes in infant acute leukaemias. MLL is involved in chromosomal translocations leading to fusions involving more than 50 different partners. Detection of MLL translocations at diagnosis is a strong negative prognostic marker of the disease. The translocations t(4;11), t(9;11) and t(11;19) that lead to MLL-AF4, MLL-AF9 and MLL-ENL fusions cover over 60 % of all MLL fusions. Although murine models for MLL leukaemias are informative they fail to replicate many of the features of infant leukaemia, suggesting that some essential ingredients of leukaemogenesis during early human development may be missing.
In this work, we aimed to determine the cell population, in the developmental or hierarchical context, that was most vulnerable to transformation by MLL-AF4. As a first approach, we explored the in vitro and in vivo developmental impact of MLL-AF4 on the fate of human neonatal CD34+ Hematopoietic stem progenitor cells (HSPCs). The expression of MLL-AF4 in human CB-derived HSPCs augmented the in vivo multilineage hematopoietic engraftment and homing as well as the in vitro clonogenic potential and enhanced their proliferation. However, MLL-AF4 was not sufficient for leukaemogenesis on its own, indicating that additional hits were required to develop leukaemia or that CB-HSPCs did not constitute the appropriate target for MLL-AF4 mediated ALL.
Human Embryonic stem cells (hESC) are pluripotent cells derived from early stage embryo, able to differentiate to all tissues, including blood cells. In this study, we explored the developmental impact of MLL-AF4 in hematopoietic differentiation from hESC. In this second approach, MLL-AF4 expression was not sufficient to transform hESC derived hematopoietic cells in vitro or in vivo. This suggested that either additional hits might be required for leukaemogenesis or that hESCs were not the appropriate cellular target for MLL-AF4 mediated transformation. Despite its inability to transform by its own, MLL-AF4 induced developmental defects in the hematopoietic lineage as shown by a reduced production of hematopoietic cells. Unexpectedly, MLL-AF4 expression led to an enhanced mature endothelial cell fate of the hemogenic precursors. This data suggested that MLL-AF4 skewed the hemato-endothelial potential of these hemogenic precursors towards a pronounced endothelial cell fate.
Introducing a set of transcription factors linked to pluripotency could directly reprogram human somatic cells to produce induced pluripotent stem cells (hiPSC), which were remarkably similar to human ESC. As a third approach in this work, we attempted to generate MLL rearranged iPSCs from infant patient blasts. By the time of the project completion we were not yet successful in the iPSC generation from patient blasts, even thought we had already generated iPSC from CD34+ CB 'healthy' cells. However, we were still exploring the way to experimentally bring the leukaemic blast back to an embryonic-like pluripotent stage.
Our work established a new platform for the study of cellular and molecular mechanisms underlying MLL-AF4 mediated early embryonic development. Long-term, large scale culture of MLL-AF4 derived hematopoietic cells was anticipated to provide an unprecedented system for drug screening and toxicity.
Acute lymphoblastic leukaemia (ALL) is a rapidly progressing tumour in which the cancerous change begins in a lymphocyte precursor, producing leukemic blasts that are not fully developed. The Mixed lineage leukaemia (MLL) gene located on chromosome 11q23 is one of the most frequently mutated genes in infant acute leukaemias. MLL is involved in chromosomal translocations leading to fusions involving more than 50 different partners. Detection of MLL translocations at diagnosis is a strong negative prognostic marker of the disease. The translocations t(4;11), t(9;11) and t(11;19) that lead to MLL-AF4, MLL-AF9 and MLL-ENL fusions cover over 60 % of all MLL fusions. Although murine models for MLL leukaemias are informative they fail to replicate many of the features of infant leukaemia, suggesting that some essential ingredients of leukaemogenesis during early human development may be missing.
In this work, we aimed to determine the cell population, in the developmental or hierarchical context, that was most vulnerable to transformation by MLL-AF4. As a first approach, we explored the in vitro and in vivo developmental impact of MLL-AF4 on the fate of human neonatal CD34+ Hematopoietic stem progenitor cells (HSPCs). The expression of MLL-AF4 in human CB-derived HSPCs augmented the in vivo multilineage hematopoietic engraftment and homing as well as the in vitro clonogenic potential and enhanced their proliferation. However, MLL-AF4 was not sufficient for leukaemogenesis on its own, indicating that additional hits were required to develop leukaemia or that CB-HSPCs did not constitute the appropriate target for MLL-AF4 mediated ALL.
Human Embryonic stem cells (hESC) are pluripotent cells derived from early stage embryo, able to differentiate to all tissues, including blood cells. In this study, we explored the developmental impact of MLL-AF4 in hematopoietic differentiation from hESC. In this second approach, MLL-AF4 expression was not sufficient to transform hESC derived hematopoietic cells in vitro or in vivo. This suggested that either additional hits might be required for leukaemogenesis or that hESCs were not the appropriate cellular target for MLL-AF4 mediated transformation. Despite its inability to transform by its own, MLL-AF4 induced developmental defects in the hematopoietic lineage as shown by a reduced production of hematopoietic cells. Unexpectedly, MLL-AF4 expression led to an enhanced mature endothelial cell fate of the hemogenic precursors. This data suggested that MLL-AF4 skewed the hemato-endothelial potential of these hemogenic precursors towards a pronounced endothelial cell fate.
Introducing a set of transcription factors linked to pluripotency could directly reprogram human somatic cells to produce induced pluripotent stem cells (hiPSC), which were remarkably similar to human ESC. As a third approach in this work, we attempted to generate MLL rearranged iPSCs from infant patient blasts. By the time of the project completion we were not yet successful in the iPSC generation from patient blasts, even thought we had already generated iPSC from CD34+ CB 'healthy' cells. However, we were still exploring the way to experimentally bring the leukaemic blast back to an embryonic-like pluripotent stage.
Our work established a new platform for the study of cellular and molecular mechanisms underlying MLL-AF4 mediated early embryonic development. Long-term, large scale culture of MLL-AF4 derived hematopoietic cells was anticipated to provide an unprecedented system for drug screening and toxicity.