Schematic representation of the Fli-1 drug-screening strategy. Fli-Lu: Multiple copies of the Fli-1 consensus-binding site (FB site) cloned upstream a minimal promoter immediately upstream the luciferase gene. MSCV-EWS/Fli-1: EWS/Fli-1 gene cloned into the MSCV retroviral vector. CMV-Lu: Luciferase gene driven by the CMV promoter. Fli-1–Lu was co-transfected with either MSCV-EWS/Fli-1 or CMV-Lu into 293T cells. Cells were treated with various drugs 34 h post transfection and screened for efficient downregulation of luciferase activity.

Effect of anti-Fli-1 drugs on the proliferation of the erythroleukemia cell line CB3. CB3 cells (1 × 10⁴) were plated in triplicate and treated with the indicated concentrations of (a) peruvoside (Per), (b) calcimycin (Cal), (c) CPT or vehicle DMSO control for 3 days (D1-3). Viable cells were counted on the indicated days using trypan blue exclusion assay. Fli-1 expression was analyzed 24 h post drug treatment. (d) CB3 cells were treated with the indicated concentrations of CPT for 24 h and subjected to northern blot analysis using a fli-1 cDNA probe.

Effect of anti-Fli-1 drugs on the proliferation of human erythroleukemia cell line HEL. HEL cells (1 × 10⁴) were plated in triplicate and treated with the indicated concentrations of (a) peruvoside (Per), (b) calcimycin (Cal), (c) CPT or the vehicle control DMSO for 4 days (D1-4). The viable cells were counted on the indicated days using trypan blue exclusion assay. At 24 h post drug treatment the leukemic cells were subjected to western blot analysis using an anti-Fli-1 antibody. (d) HEL cells were treated with the indicated concentrations of peruvoside for 24 h and subjected to northern blot analysis using a fli-1 cDNA probe.

Ectopic Fli-1 overexpression in erythroleukemic cells partially overcome growth inhibition induced by anti-Fli-1 drug treatment. (a) Increased levels of Fli-1 protein expression in the murine erythroleukemic cell line DP17-17, infected with retrovirus expressing Fli-1 (b–c) DP17-17 cell transduced with the vector control or Fli-1 expressing virus were treated with the indicated concentrations of (b) calcimycin (Cal), (c) CPT or the vehicle control DMSO for 3 days, and viable cells were counted on the indicated days using trypan blue exclusion assay.

Binding of Fli-1 to target gene DNA is inhibited by anti-Fli-1 drug treatment. (a) Nuclear extracts from drug treated (calcimycin 0.5 μ, CPT 0.1 μ) and untreated HEL cells were incubated with γ-³²P-labelled oligonucleotides containing a Fli-1-binding site, in the presence or absence of Fli-1 antibody and subjected to EMSA. Addition of the c-Myc antibody was used as a negative control (lane 5). Competition assays were performed in the presence of 100–fold excess unlabeled oligonucleotides (cold competitor). Arrows indicate the position of Fli-1-specific bands, Fli-1A and Fli-1B. (b) Inhibition of Fli-1 through anti-Fli-1 drug treatment results in protein upregulation of the Fli-1 target gene SHIP-1 in HEL cells, as indicated by western blot. (c) Reduced binding of Fli-1 to the SHIP-1 promoter (lane 1) in HEL cells treated with anti-Fli-1 drugs (calcimycin 0.5 μ, CPT 0.1 μ, peruvoside 0.1 μ), as determined by ChIP using 2 μg Fli-1 or rabbit IgG antibody. (d) ChIP quantitative-PCR using Fli-1 and rabbit IgG antibodies illustrating reduced Fli-1 chromatin occupancy on the SHIP-1 promoter in HEL cells treated with the indicated anti-Fli-1 drugs. The level of SHIP-1 enrichment is expressed as the percentage of input chromatin precipitated by Fli-1 or control IgG antibodies. (e) Similar to SHIP-1, inhibition of Fli-1 resulted in upregulation of Gata-1 protein expression in HEL and CB3 cells treated with the indicated dosages of anti-Fli-1 drugs or DMSO control. Treatment of CB3 and HEL cells with the indicated concentration of (f) calcimycin and (g) Can A inhibits the protein expression of PKCδ in a dose-dependent manner. (h) CB3 and HEL cells treated with calcimycin (0.5 μ) and Can A (1.0 μ) display reduced levels of phosphorylated Fli-1 protein, as detected by western blot analysis using an anti-threonine antibody. Anti-Fli-1 antibody was used to demonstrate total protein levels.

Inhibition of leukemogenesis by anti-Fli-1 drugs. (a) 6-week old mice, infected at birth with F-MuLV, were treated every other day with the indicated drugs (1 mg/kg), for a period of 2 weeks. At the end of drug treatment, mice were sacrificed, and characteristic indicators of leukemia development, spleen weights and hematocrit levels were measured. Treatment with representative drugs from groups I-III significantly delayed leukemia development, associated with increased hematocrit values and reduced spleen size. Statistical significance (P value) is shown for each group. (b) F-MuLV-infected mice treated at day 35 post-infection with calcimycin (Cal; 1 mg/kg), Can A (1 mg/kg) or DMSO and observed for the development of leukemia. All DMSO control mice died 70 days post viral infection; 5 days later (75 dpi) anti-Fli-1 drug-treated mice were killed for purposes of comparison.