A centromeric location of the MLL breakpoint within the MLL BCR, but not the cellular ontogeny, determines the expression of HOX cluster/MEIS genes in MLL-edited CD34⁺ cells. (A) Heat map showing clustering of MLL-edited CD34⁺ HSPCs based on the location of the MLL breakpoint (Mⁱ¹⁰A4 n = 6, blue vs Mⁱ¹²A4 n = 6, green) irrespective of the cellular ontogeny (FL [n = 6, pink] vs CB [n = 6, yellow]) based on significant differentially expressed HOX cluster genes (false discovery rate < 0.05) among the 26 genes included in the HOX cluster. (B) Representative chromatin immunoprecipitation sequencing tracks at the MA4 target genes HOXA9/A10 and MEIS in FL (left) and CB (right) Mⁱ¹⁰A4- and Mⁱ¹²A4-edited cells. Binding of H3K79me3 and H3K27ac to HOXA9/A10 and MEIS was analyzed as a control. (C) Leukemia incidence in primary and secondary recipient mice transplanted with FL- and CB-derived Mⁱ¹⁰A4- and Mⁱ¹²A4-edited HSPCs. Primary mice, n = 57; secondary mice, n = 42. (D) Representative immunophenotype of a non-leukemic (green) and a leukemic (purple) mouse. Leukemic mice recapitulate many immunophenotypic features of primary MA4⁺ B-ALL, such as pro–B-lymphoid (CD10^–)–biased engraftment (practically depleted of myeloid graft) coupled to the expression of the MLL-specific antigen NG2, and some degree of myeloid-lymphoid lineage infidelity/mixed phenotype (CD33⁺CD19⁺). Cells in gray are mouse cells. (E) Clinical data from the international MLL Recombinome Taskforce reveals a higher frequency of telomeric (i11-i12) MLL breakpoints among iB-ALLs (0-6 months old); n = 526 patients analyzed. (F) Rates of N/K-RAS mutations determined by the Oncomine childhood leukemia mutational panel in leukemic mice (n = 4 of 9; 44%) reproduce those reported in primary iB-ALLs. PCR, polymerase chain reaction; WT, wild-type.