FACS analysis of BM and spleen cells from representative diseased Mx-Cre Mll-Cbp^STOP mice. Single-cell suspensions were prepared from the BM and spleens of diseased Mx-Cre Mll-Cbp^STOP mice and stained with a combination of Mac-1 and Gr-1 antibodies (A) or B220 and CD3 antibodies (B). The staining antibodies are indicated adjacent to each axis.

MLL-CBP induced myeloid hyperplasia. (A) We analyzed BM cells isolated from Mx-Cre Mll-Cbp^STOP mice and control littermates at 10 days following the last pI-pC treatment. A representative FACS profile of BM cells stained with Mac-1 and Gr-1 antibodies is shown. (B) A representative FACS profile of BM cells at 29 days following the last pI-pC treatment.

MLL-CBP alters the genetic programs of GMP. The top 50 genes most closely associated with MLL-CBP expression—either upregulated (red) or downregulated (blue)—are shown. Each column represents a sample and each row a gene. Samples 1 and 2 represent Mx-cre GMP cells, samples 3 and 4 represent Mll-Cbp^STOP GMP cells, and samples 5–7 represent Mx-Cre Mll-Cbp^STOP GMP cells.

GMPs were substantially increased in Mx-Cre Mll-Cbp^STOP mice. (A) Representative FACS-staining profiles of HSCs and progenitors from the BM of Mx-Cre Mll-Cbp^STOP mice (3 weeks post-pI-pC treatments; gates are indicated in Materials and methods). Similar results were obtained from five Mx-Cre Mll-Cbp^STOP mice and their control littermates. The FACS profile for a leukemic Mx-Cre Mll-Cbp^STOP mouse is also shown for comparison and indicates an even greater expansion of GMP compared to the preleukemic phase of mice expressing MLL-CBP. (B) Bar graph showing the absolute numbers of HSC, CLP, CMP, GMP, and MEP cells in the BM of Mx-Cre Mll-Cbp^STOP mice compared to controls. Values represent the average from five mice.

Enhanced replating efficiency of MLL-CBP-expressing GMP. (A) RT–PCR analysis for the Mll-Cbp transcript in HSC, CLP, CMP, GMP, and MEP cells. MLL-CBP is expressed in all of these cell populations. (B) Purified HSC, CMP, GMP cells were serially replated in methylcellulose culture. Bar graphs show the number of colonies obtained after each round of serial replating (the mean and s.d. of triplicate cultures are indicated). Similar results were obtained from two experiments. (C) Morphology of a typical tertiary colony derived from the MLL-CBP-expressing HSC, CMP, or GMP cultures. Cytospins of these colonies revealed the presence of myeloblasts and terminally differentiated macrophages. (D) Percent of donor-derived mature myeloid cells (% Ly5.2+Gr-1+) as assessed by FACS of peripheral blood (PB). Percentage values represent the average from 6–8 mice and representative PB smears from a recipient mouse that was reconstituted with MLL-CBP-expressing HSC, which show mature neutrophils, monocytes, and lymphocytes (magnification × 60).

Hox a9 is required for mediating the effects of MLL-CBP. (A) MLL-CBP upregulated Hox a9 gene expression. As measured by real-time quantitative PCR, Hox a9 expression was increased in HSC, CLP, CMP, GMP, and MEP cells expressing MLL-CBP when compared to controls. Relative transcript levels were internally normalized to GAPDH levels. Values represent the average of at least two independent experiments performed in triplicate. (B) Effects of shRNA-HoxA9 constructs on BM cells expressing MLL-CBP. Three different shRNA constructs directed against Hox a9 were used (lanes 3–5). Empty retroviral vector and an shRNA construct directed against GFP were used as controls (lanes 1 and 2, respectively) (C) Effects of shRNA-Hox a9 on the size of the colony formation. (D) Effects of Hox A9 overexpression on the in vitro growth and replating efficiency of BM progenitors. Total cell numbers were counted at the specified days after the initial plating.

Mice expressing MLL-CBP progress to a fatal myeloid disease. (A) Kaplan–Meier survival plot for Mx-Cre Mll-Cbp^STOP mice (n=15) that received a single dose of sublethal γ-irradiation. One Mll-Cbp^STOP mouse (n=10) died from irradiation-induced BM failure. None of the Mx-Cre mice (n=11) developed disease. (B) Kaplan–Meier survival plot for ENU-treated Mx-Cre Mll-Cbp^STOP mice (n=22). Two Mx-Cre mice (n=13) died from T-cell lymphomas. ENU-induced T-cell lymphoma in normal mice has been reported (Breuer et al, 1989). (C) Representative PB smear from an irradiated Mx-Cre Mll-Cbp^STOP mouse #24065, which shows leukocytosis consisting predominantly of mature neutrophils and/or monocytes with immature forms present (mag × 60). (D) BM cytospin from the same animal demonstrated predominantly neutrophilic and monocytic cells of which immature forms/blasts were >20% (magnification × 60). (E) The spleen from the same animal showed an infiltrate of poorly–moderately differentiated myeloid cells (magnification × 60). (F) Representative PB smear from an ENU-treated Mx-Cre Mll-Cbp^STOP mouse #565, which shows moderate neutrophilic leukocytosis. (G) BM cytospins from the same animal showed well-differentiated myelopoiesis without increased immature forms/blasts. (H) Spleen from the same animal showed marked red-pulp expansion with megakaryocytes, prominent erythropoiesis, and well-differentiated myelopoiesis. (I) Splenomegaly in a leukemic, irradiated Mx-Cre Mll-Cbp^STOP mouse (right) compared to an age-matched wild-type control (left). (J) A representative spectral karyotype of leukemic cells from an irradiated Mx-Cre Mll-Cbp^STOP animal. Tumor displays a t(18;X) as indicated by the arrows.

Generation of mice with a conditional Mll-Cbp knockin allele. (A) The most common MLL-CBP fusion protein associated with the human t(11;16) and its recapitulation in the Mx-Cre Mll-Cbp^STOP mice. AT: AT hooks; DNMT: DNA methyltransferase homology domain; CH1, 2, 3: zinc-finger domains 1, 2, 3; Bromo: bromodomain; HAT: histone acetyltransferase domain; Gln-rich: glutamine-rich domain. (B) The endogenous Mll allele, the targeting vector, the predicted Mll-Cbp^STOP (M-C^stop) knockin allele resulting from homologous recombination, and the activated Mll-Cbp allele following deletion of the stop cassette by Cre recombinase. (RI) EcoRI; (B) BamHI. (C) Southern blot analysis of EcoRI-digested genomic DNA from wild-type and targeted (ES #22, 26, 75, 112) embryonic stem (ES) cells, probed with a 5′ external probe. (D) Southern blot analysis of EcoRI-digested genomic DNA from the BM of Mx-Cre Mll-Cbp^STOP mice and control littermates. Excision of the STOP cassette is indicated by the presence of the 3.5-kb band. (E) Southern blot analysis showing deletion of the STOP cassette in different tissues of Mx-Cre Mll-Cbp^STOP mice following pI-pC treatments. BM: bone marrow; Liv: liver; Sp: spleen; Thy: thymus. (F) RT–PCR analysis confirming the presence of the Mll-Cbp transcript in different tissues of Mx-Cre Mll-Cbp^STOP mice following pI-pC treatments. WT: Mx-Cre BM.