Fig. 2: MLL-ENL fusion gene lacks leukaemic transforming ability in Flt3L and β-estradiol culture condition.

a Outline of experimental strategy. Single cells -Parental and ME-Parental cells cultured in the presence of Flt3L and β-estradiol, ME-Transformed and MLL-ENL BM cells cultured long term in the presence of IL-3- were sorted into 96-well plates and processed for scRNA-seq using Smart-Seq2 protocol. b PCA plot based on highly variable genes of 166 cells. Parental (47 cells) and ME-Parental (55 cells) cells are Hoxb8/Flt3L dependent while MLL-ENL BM (32 cells) and ME-Transformed (32 cells) are cultured long term in the presence of IL-3. c Expression of selected genes in cells in (b). Selected genes are important in defining PC1 component. Top genes are MPP4/LMPP-specific genes; bottom genes represent myeloid and leukaemogenic genes. Cells are coloured according to the expression levels of denoted genes. Colour scheme is based on log10 scale of normalised counts from 0 (grey) to 4 (red). d Projection of transcriptomic profiles of Parental, ME-Parental, ME-Transformed and MLL-ENL BM cells onto force-directed graph obtained from single HSPCs. First panel shows the cell type landscape and clustering generated by Dahlin et al.²⁴. Following panels show the most similar cells of the landscape to Parental, ME-Parental, ME-Transformed and MLL-ENL BM cells. e Violin plots showing expression levels (log10 scale of Normalised counts on y-axis) of key leukaemic target genes across Parental, ME-Parental, MLL-ENL BM and ME-Transformed cells. f, g MA plots showing the comparison between counts obtained at Parental and ME-Parental accessible regions defined via ATAC-seq at day 6 (f) or day 9 (g) of culture in the presence of β-estradiol and Flt3L. No statistical differences can be detected except for regions corresponding to the transduced MLL-ENL (depicted in red).