Project description:The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well-established that MLL-AF4 arises pre-natally during human development, its effects on hematopoietic development in utero remains unexplored. We have created a human-specific in vitro system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Differentiation and functional studies as well as clonal analyses and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. It enhances the specification of hemogenic precursors from hESCs and impairs further hematopoietic commitment of these precursors in favour of the endothelial cell fate. Similar to that reported in cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs), MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells in vitro or in vivo, indicating that additional events may be required to initiate leukemogenesis or that embryonic hematopoiesis is not the appropriate human cellular target for MLL-AF4-mediated leukemogenesis. This work illustrates how hESCs can provide unique insights into human development and further our understanding of how leukemic fusion genes known to arise pre-natally regulate human embryonic hematopoietic specification. MLL is involved in transcriptional regulation and most MLL translocations appear to result in increased expression of Hox genes and hematopoietic genes. We therefore assessed the impact of MLL-AF4 expression on the transcriptome of hESCs. Gene expression profiling performed in MLL-AF4 hESCs revealed that MLL-AF4 preferentially activates transcription. 1826 out of the 3001 genes (61%) expressed were up-regulated in MLL-AF4 hESCs. Human ESC samples were collected during the exponential cell growth phase and stabilized in RNA later. 500 ng of each total RNA sample was labelled with Cy3 using the Quick-Amp Labelling kit and hybridized with the Gene Expression Hybridization kit to a Whole Human Genome Oligo Microarray (Agilent Technologies) following the Manufacturer’s instructions. Each cell line was analyzed as independent duplicates. NEO-expressing (empty lentivector) hESC line was used as the baseline.

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:MLL-AF4 is a hallmark genomic aberration which arises prenatally in high-risk infant acute lymphoblastic leukemia (ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hemato-endothelial specification but was not sufficient for transformation. Additional cooperating genetic insults seem required for MLL-AF4-mediated leukemogenesis. FLT3 is highly expressed in MLL-AF4+ ALL through activating mutations (FLT3-TKD or FLT3-ITD) or increased transcriptional expression, being therefore considered a potential cooperating event in MLL-AF4+ ALL. Here, we explored the developmental impact of FLT3 activation on its own or in cooperation with MLL-AF4 in the hematopoietic fate of hESCs. FLT3 activation did not impact specification of CD45-CD31+ hemogenic precursors but significantly enhanced the formation of CD45+CD34+ and CD45+ blood cells and blood progenitors with clonogenic potential. Importantly, FLT3 activation through FLT3 mutations or FLT3-WT overexpression completely abrogated hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly impacts hESC specification rather than selective proliferation/survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling supported the limited impact of FLT3 activation on hESC specification towards CD45-hemogenic precursors and the enhanced hematopoiesis upon FLT3 activation, and inhibited hematopoiesis upon MLL-AF4 expression in FLT3-activated hESCs which was associated to large transcriptional changes and regulation of master early hematopoietic genes. Also, although FLT3 activation and MLL-AF4 cooperate to inhibit embryonic hematopoiesis the underlying molecular/genetic mechanisms differ depending on how FLT3 activation is achieved. Finally, FLT3 activation did not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells. 18 samples were analyzed. CD45- hemogenic precursors EV, 2 biological rep CD45- hemogenic precursors FLT3-TKD, 2 biological rep CD45- hemogenic precursors FLT3-WT, 2 biological rep CD45- hemogenic precursors FLT3-TKD/MLLAF4, 2 biological rep CD45- hemogenic precursors FLT3-WT/MLLAF4, 2 biological rep CD45+ blood cells EV, 1 biological rep CD45+ blood cells FLT3-TKD, 2 biological rep CD45+ blood cells FLT3-WT, 2 biological rep CD45+ blood cells FLT3-TKD/MLLAF4, 2 biological rep CD45+ blood cells FLT3-WT/MLLAF4, 1 biological rep

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A novel human fetal liver-derived model reveals that MLL-AF4 drives fetal gene expression programs in infant ALL [ChIP-seq]

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