Project description:Malignant transformation in a multipotential precursor has recently been shown to underlie mixed phenotype acute leukaemias. In contrast, and despite the conclusive demonstration that MLL-AF4 infant ALL arises in utero. In order to address this we purified haematopoietic stem/progenitor cell (HSPC) populations from four non-lineage switching presentation infant ALL cases and one ALL presentation which went on to lineage switch at relapse. The specific MLL-AF4 fusion event was determined in unsorted samples by LDI-PCR and identified in purified populations by nested PCR. In three cases where non-lineage restricted populations were able to be purified, MLL-AF4 was identified in the CD34+CD38-CD45RA+ population (LK228, LK230, LS01), the CD34+CD38-CD45RA-CD90- multipotential progenitor population (MPP, LS01) and even a candidate CD34+CD38-CD45RA-CD90+ haematopoietic stem cell (HSC) population (LK228) Interestingly, in a single non-switched MLL-AF9 case analysed for external comparison, the fusion was only identified in CD19+ blast/B cell populations, not in any HSPC population. Unexpectedly, we were also able to identify the presence of the MLL-AF4 fusion in apparently normally differentiated CD34-CD19/3-HLA-DR+CD14/11c+ monocytes/dendritic cells from the peripheral blood/bone marrow of 4/5 cases examined. This finding was further supported by single cell analysis of LS01PALL which identified the fusion in 2/22 CD34-CD19/3-HLA-DR+CD14/11c+ monocytes/dendritic cells analysed. Furthermore, the matched AML sample (LS01RAML) also contained MLL-AF4 positive mature T lymphoid cells. These data imply that MLL-AF4 does not impose a complete block on normal haematopoietic differentiation, and raise the question of what additional factors contribute to leukaemic lineage determination in the setting of the MLL-AF4 translocation. To examine the functional capacity of MLL-AF4 transformed precursor cells we generated a patient-derived xenograft model using NOD-scid/IL2Rγ-/- (NSG) mice. Serial xenotransplantation identified an MLL-AF4 positive clone persisting within the human CD34+CD38-CD45RA-CD90+ compartment across four generations of mice, confirming self-renewal of this population. Together these data identify an early multipotent progenitor or HSC as the putative cell of origin for MLL-AF4 ALL. Furthermore, they demonstrate that MLL-AF4, uniquely amongst MLL fusion genes, imposes a profound lymphoid bias on the developing leukemia but without completely abolishing broader, non-malignant, haematopoietic differentiation.

Project description:Purpose: We designed this study to evaluate the feasibility of using only one factor to respecify human induced pluripotent stem cells (iPSCs)-derived blood cells into long-term engraftable hematopoietic stem and progenitor cells (HSPCs). We also parallelly compared iPSC-derived HSPCs (iHSPCs) with primary HSPCs under the same induction and transplantation condition in order to examine the functional equivalency between iHSPCs and bona fide HSPCs. Methods: In vitro derived human iPSC-HSPCs are induced with or without (w/o) doxycycline for the expression of MLL-AF4. In vivo derived bone marrow cells are harvested and FACS-sorted for human populations from primary transplants over nine to sixteen weeks after transplantation. Either iPSC-HSPCs or primary HSPCs, both of which are induced by MLL-AF4, is used for the transplantation experiments. iPSCs are either derived from peripheral blood mobilized CD34+ HSPCs (CD34-iPSC) or mononuclear cells (MN-iPSCs). Normal human CD34+ HSPCs and mononuclear cells are set as control groups. Results: MLL-AF4 can impart HSC and lymphoid potential to iPSC-derived blood cells in vitro, and induction of MLL-AF4 leads to the cellular identity transition from common myeloid progenitors to hematopoietic stem and progenitor cells. MLL-AF4 alone is sufficient to realize the potent engraftment of induced HSPCs (iHSPCs) from iPSCs, and multilineage and long-term hematopoiesis could be observed. Primary HSPCs with the induction of MLL-AF4 could gain significantly enhanced engraftability. During the long-term engraftment period, leukemic mutations with a bias to B-cell leukemia could be found in the iHSPCs and their derivatives. By contrast, MLL-AF4 induced primary HSPCs maintain the normal hematopoiesis without leukemic transformation. Conclusions: Our study has demonstrated for the first time, to our knowledge, that the pluripotency-dependent conversion of somatic cells to long-term engraftable HSPCs can be achieved by using a single factor in a non-integrative way. This study also suggests that iPSC-derived HSPCs are more prone to leukemic mutations during the long-term engraftment period, which provides a necessary caveat of using them in the actual therapies.

Dataset Information

Transcription profiling of mouse activated or control 5-FU bone marrow from MLL-AF4stop knockins

Publications

H3K79 methylation profiles define murine and human MLL-AF4 leukemias.

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