Echinomycin induces long-lasting remission in syngeneic hosts transplanted with relapsed Mll^PTD/WTFlt3^ITD/WT AML. (A) Relapsed and untreated AML cells were equally sensitive to echinomycin. The untreated and relapsed AML cells were thawed and allowed to recover overnight in vitro. After treatment with vehicle control or different concentrations of echinomycin for 48 hours, the drugs were washed away, and the cells were placed in a CFU assay or a MTT assay to determine viability. The drug doses are 0, 50, 150, 450, and 1350 pM. Data shown are means ± SD of colony numbers plated in triplicate or assessment of overall viability by MTT assay in triplicate. The data are representative of 2 independent experiments. (B-E) Therapeutic efficacy of a low dose of echinomycin in vivo for relapsed AML. (B) Diagram of experimental design. One million CD45.2⁺ relapsed AML cells were injected intravenously into each of 20 sublethally irradiated CD45.1⁺ recipients. On day 8, when the CD45.2⁺ AML cells were detectable in all recipient mice, 2 5-dose cycles of echinomycin treatments were initiated. Arrows indicate dosing and arrowheads indicate date of flow cytometry of blood cells. (C) Flow cytometry of PBL for CD45.2⁺ leukemia cells in the blood of vehicle-treated (top 2 rows) or echinomycin-treated (bottom 2 rows) mice at 23 days after transplantation. Each histogram represents 1 mouse, and data show reduction in the number of leukemic cells in the blood following 1 cycle of echinomycin treatment. (D) Complete remission in a significant fraction of Echinomycin-treated mice shown in [c] harboring relapsed AML. The lower panel shows percent of leukemic cells in the 10 vehicle-treated mice throughout their life span and the point at which death was recorded in all 10, while the upper panel shows percent of leukemic cells in the 10 Echinomycin-treated mice over a 12-week period. Of the 10 Echinomycin-treated mice harboring relapsed AML, 4 had a complete remission at 12 weeks (as defined by lack of leukemia cell in the PBLs) and never relapsed over the 300 days of observation or serial transplantation. No mice in the vehicle-treated group experienced remission. Similar results were observed in another independent experiment involving 5 mice per group. (E) Echinomycin treatment significantly increased the life span of mice with transplanted relapsed AML. Data shown are Kaplan-Meier survival curves of 2 independent experiments.