Lef-1 expression in murine hematopoietic tissue and human leukemia. (A) Lef-1 mRNA expression in FACS-sorted murine hematopoietic BM subpopulations. Data represent means plus SD of triplicates (n = 3). K⁺S⁻L⁻, cKit⁺Sca⁻Lin⁻; GM⁺⁺, Gr1⁺Mac1⁺; B⁺43⁺24⁺, B220⁺CD43⁺CD24⁺; B⁺19⁺, B220⁺CD19⁺; B⁺43⁻IgM⁻, B220⁺CD43⁻IgM⁻. (B and C) Expression levels of LEF-1 were determined in two microarray datasets: 120 samples from 13 different leukemia subtypes (B) and a series of 194 expression profiles from AML samples with normal karyotype (C). (B) Comparison of relative LEF-1 mRNA expression in AML and ALL patients. Bars indicate the median. Red dots indicate CALM/AF10⁺ AML/ALL cases. (C) Relative expression of LEF-1 in 194 AML patient samples with normal karyotype (NK).

Lef-1 severely perturbs normal hematopoietic differentiation. (A) Retroviral constructs for expression of WT and mutant constitutive active (CA) Lef-1 proteins. As control, the empty EGFP vector was used. BCBD, β-catenin binding domain; CAD, context-dependent activation domain; HMG, high mobility group DNA binding domain; NLS, nuclear localization signal; LBDBC, Lef-1 binding domain of β-catenin; L, linker; LTR, long-terminal repeat; IRES, internal ribosomal entry site; EGFP, enhanced GFP. (B) Expression of Lef-1. Western blot analysis of cellular extracts from NIH 3T3 cells transfected with the different constructs (left image, the molecular mass is indicated); TaqMan real-time RQ-PCR with cDNA (triplicates) of BM cells from Lef-1 WT (n = 2) and Lef-1 CA leukemic mice (n = 2) compared with the endogenous Lef-1 mRNA expression level in a healthy EGFP control animal (middle); and quantification of Lef-1 expression level by RQ-PCR in BM cells freshly infected with Lef-1 WT, Lef-1 CA, and empty vector control using highly purified EGFP⁺ populations (right). (C–E) Flow cytometry analysis of PB samples of (C) a Lef-1 WT (no. 8) and (D) a Lef-1 CA (no. 10) –engrafted mouse, respectively, compared with a control EGFP animal (E). Representative dot plots with the proportion of positive stained cells within the EGFP⁺ compartment compared with the EGFP⁻ compartment are shown. (F) Accumulation of mature myeloid cells in Lef1-transplanted animals. Wright-Giemsa–stained cytospins from PB of a Lef-1 CA–transduced mouse (no. 10; left image, GFP⁺ compartment; middle image, GFP⁻ compartment) and a control animal (right image). Bar, 10 μm. (G) Percentage of lymphoid and myeloid cells in PB in transduced animals. Mean values ± SEM are indicated (n [WT] = 20, n [CA] = 12, n [EGFP] = 8; *, P < 0.02; **, P < 0.001). Significant differences between Lef-1 WT and Lef-1 CA animals are indicated when present. (H) Lymphoid/myeloid ratio in PB of transduced mice. Lymphoid cells included B220⁺, CD4⁺, and CD8⁺ cells, and myeloid cells included Mac1⁺/Gr1⁺, Mac1⁺/Gr1⁻, and Mac1⁻/Gr1⁺ cells. Individual mice are indicated by different colors and were analyzed at one or multiple time points after transplantation. The median is presented as a black bar (*, P < 0.02; **, P < 0.002).

Lef-1 overexpression induces acute leukemia. (A) Survival curve of mice transplanted with BM cells expressing Lef-1 WT, Lef-1 CA, and EGFP as well as survival of secondary recipient mice transplanted with BM from diseased primary mice. (B) Blood smears (BS) and cytospin preparations from PB and BM of a representative animal with AML (no. 8, 1–3) and ALL (no. 13, 4–6). Slides were stained with Wright-Giemsa. Bars: 1, 60 μm; 4, 40 μm; 2 and 5, 30 μm; 3 and 6, 20 μm. (C) Immunophenotype analysis of diseased mice. Representative dot plots from the BM and SP of a mouse with myeloproliferative disease (MPD, 1–4, no. 7), with AML (5–8, no. 11) and ALL (9–12, no. 14). The Mac1/Gr1 and CD43/B220 costainings were gated on GFP⁺ cells. The percentage of positive-stained cells is indicated. (D) Histochemical and immunohistological analysis of diseased mice. Panels 1–3, B-ALL (mouse no. 4); panels 1 and 2, tumor cells infiltrating lymph nodes staining positive for TdT and negative for CD3; residual normal T cells in the lymph node are CD3⁺ (2, right side), whereas infiltrated leukemic blast cells are TdT⁺, but CD3⁻ (1; left side and insets 1 and 2, SP). Panels 4–8: primary AML (4 and 5, mouse no. 9; 6–8, mouse no. 10). Panels 9 and 10: diseased secondary recipient of mouse number 10. H&E, hematoxylin and eosin; MPO, myeloperoxidase; CAE, N-acetyl-chloroacetate esterase; TdT, terminal deoxynucleotidyl transferase; LN, lymph node; LV, liver. Bars: 8, 200 μm; 1, 2, and 10, 100 μm; 3, 6, and 7, 50 μm; 4, 5, 9, and insets 1 and 2, 25 μm.

Lef-1–induced acute leukemias are propagated by a LSC with lymphoid characteristics. (A) Morphology of sorted BM populations of a representative AML (1–3, no. 10) and an ALL case (3–6, no. 14). B⁺M⁻, B220⁺/Mac1⁻; BM⁺⁺, B220⁺Mac1⁺; B⁻M⁺, B220⁻Mac1⁺. Bars, 20 μm. (B) Multiplex PCR for DJ_H loci rearrangement in diseased mice. Line 2, germline control (GL, myeloid cell line 32D); line 3, splenic B220 cells. (C) Seeding efficiency of B⁺MG⁻, BMG⁺⁺⁺, and B⁻MG⁺ populations from BM cell cultures of leukemic mice (n = 3; mouse nos. 4, 10, and 11; Table S1). Shown are the mean values ± SEM. (D) B⁺M⁻ cells from a cell population generated from a single B⁺M⁻ cell (mouse no. 4) developed into BM²⁺ and B⁻M⁺ populations. (E) Multiplex PCR for DJ_H rearrangement for highly purified B⁺M⁻, BM²⁺, and B⁻M⁺ cells generated from a single B⁺M⁻ cell in vitro. CL, cell line.

Frequency of leukemia-propagating cells and transcriptional profile analysis. (A) Highly purified B⁺MG⁻, BMG⁺⁺⁺, B⁻MG⁺, and triple negative B⁻MG⁻ cell populations from a primary mouse with AML (no. 10) were injected into cohorts of secondary mice. The frequency of leukemia-propagating cells in each of the populations is indicated. For details see Table S3, available at http://www.jem.org/cgi/content/full/jem.20071875/DC1. (B) Analysis of mRNA expression of regulatory factors in BM cells of diseased mice by semiquantitative RT-PCR. BM bulk (BM ctrl), Mac1⁺ BM cells (BM Mac1⁺), and B220⁺ splenic cells (SP B220⁺) of a nontransplanted mouse served as controls. (C) Analysis of C/ebpα expression in subpopulations generated from a B220⁺Mac1⁻ single cell originating from an ALL-diseased mouse single cell. HPRT, hypoxanthine guanine phoshoribosyltransferase.