Specific binding of GFP-M&M to MYB-binding sites, recruitment of AML1-ETO, and repression of MYB-responsive promoters by AML1-ETO in the presence of GFP-M&M. (A) In vitro recruitment of AML1-ETO to MYB-binding sites. Nuclear extracts from Cos cells transfected with Myb, GFP-M&M, and AML1-ETO as indicated were analyzed by an electrophoretic mobility-shift assay (EMSA). Competition experiments with specific MYB and nonspecific oligonucleotides demonstrated the specificity of M&M binding. The supershift of M&M cotransfected with AML1-ETO evidenced the recruitment of AML1-ETO to MYB-binding sites. (B) Recruitment of AML1-ETO in vivo. KCL22 cells transfected with FLAG-AML1-ETO and GFP or GFP-M&M were crosslinked with formaldehyde, lysed, sonicated, and immunoprecipitated with anti-Flag or with unspecific mouse IgG. Immunoprecipitated chromatin was analyzed by PCR for the KIT promoter region and the p14^ARF promoter region. One representative of two experiments with similar results is shown. (C) Expression of FLAG-AML1-ETO and GFP/GFP-M&M in the transfected cells. The expression level of FLAG-AML1-ETO and GFP/GFP-M&M in transfected cells was detected by Western blot. (D) KCL22 cells were transiently transfected with a MYB-responsive luciferase construct and AML1, AML1-ETO, GFP-ΔM&M, and GFP-M&M (as indicated). Firefly luciferase values were standardized to expression of a cotransfected Renilla luciferase vector construct. Results are shown as means and SEM of three independent experiments. (E) Primary murine bone marrow cells were transduced with GFP or GFP-M&M and analyzed for the expression of KIT. The results of one of two independent experiments are shown.

GFP-M&M induces apoptosis in AML1-ETO-containing cells. 32D cells were transfected with AML1-ETO, GFP-M&M, or both and subsequently transfected cells were sorted by flow cytometry. The transfected cell population was analyzed in a TUNEL assay. (A) Fluorescence-activated cell sorter (FACS) analysis for BrdUrd-positive apoptotic cells is shown. Apoptosis in pcDNA3.1-transfected control cells is indicated in the open histograms. (B) The percentages of apoptotic cells within the transfected 32D cell populations.

Construction of an AML1-ETO-redirecting chimeric protein. (A) Model of antileukemic activity of a chimeric protein (M&M) consisting of the MYB DNA-binding domain and the MEF AML1-binding domain. (B) Construction of an expression vector for the chimeric M&M protein and a deletion mutant lacking DNA-binding activity. (C) Expression analysis of the proteins encoded by the various constructs. Immunoblotting of Cos cell lysates after transfection with GFP, GFP-ΔM&M, and GFP-M&M. Western blot analysis was performed by using an anti-GFP antibody.

GFP-M&M represses colony growth in cells expressing AML1-ETO. (A) 32D cells were transfected with GFP-pcDNA3.1 as a control or AML1-ETO and GFP-M&M as indicated, and a total of 1 × 10⁵ cells were seeded in colony assays. Pictures of representative colonies were taken on day 10. (B) Transfected 32D cells (as in A) were seeded in colony assays. The repression of colony growth compared with the control transfection with GFP-pcDNA3.1 (set as 1) is shown. (C) Analysis of the importance of DNA binding of GFP-M&M for colony growth. AML1-ETO alone or in combination with either GFP-M&M or GFP-ΔM&M was transfected into 32D cells, which were subsequently plated in colony assays. (D) Human leukemic Kasumi-1 cells that naturally express AML1-ETO were transfected with GFP or GFP-M&M. Cells were subsequently plated in colony assays.