Smac mimetic and dexamethasone cooperate to induce cell death in primary ALL blasts but not in nonmalignant cells of the lymphohematopoietic system. (A) Primary leukemic blasts from children with ALL before the onset of chemotherapy were treated for 48 hours with indicated concentrations of dexamethasone and/or BV6. Cell death was determined by forward/side scatter analysis and flow cytometry. The percentage of specific cell death was calculated as follows: 100 × [experimental cell death (%) − spontaneous cell death (%)]/[100% − spontaneous cell death (%)]. Mean of 1 experiment performed in triplicate is shown. (B) One fragment each of the human ALL-SCID4 was subcutaneously transplanted into the flank of NSG mice. Treatment started on day 21 after tumor transplantation with 6 mg/kg BV6 intravenously every fourth day and/or 1 mg/kg dexamethasone orally 5 days/week. Mice were killed when tumors reached a volume exceeding 1.5 cm³. Because of complete remissions in the dexamethasone groups, treatment was stopped at day 33, and only BV6 therapy was continued until day 75. Tumor growth and body weight were monitored twice per week. Tumor volumes were calculated by [length × (width)2] × 0.5, and mean values per group were determined. (Left): Mean tumor volume with standard error of the mean; statistical comparisons between dexamethasone- and dexamethasone/BV6-treated groups were performed with the Mann-Whitney U test; *P < .05. (Right): Tumors of dexamethasone- and dexamethasone/BV6-treated groups were taken at day 75 and photographed. (C) PBLs of 2 distinct donors were treated for 48 hours with indicated concentrations of BV6 and/or 200 µM dexamethasone. Cell death was determined by forward/side scatter analysis and flow cytometry. Mean of 1 experiment performed in triplicate is shown. (D) MSCs of 2 distinct donors were treated for 48 hours with 200 µM dexamethasone and/or 7 µM BV6. As positive control (PC), MSCs were treated with 10 µM Actinomycin D. Cell death was determined by PI uptake and flow cytometry. Mean of 1 experiment performed in triplicate is shown. (E) Clonogenicity of human CD34⁺ hematopoietic cells treated with 300 µM dexamethasone and/or 7 µM BV6 was assessed by colony-forming assay. The percentage of colony-forming unit-granulomonocyte, colony-forming unit-multipotent, and burst-forming unit-erythroid relative to untreated controls with mean and SD of 3 experiments performed in triplicate are shown. (F) Monocyte-derived iDCs were generated by cultivation for 7 days with GM-CSF/IL4. iDCs were treated for 72 hours with 5 µM BV6 and/or 300 µM dexamethasone. As PC, iDCs were treated with 300 ng/mL TNFα as a potent trigger of iDCs maturation. Maturation was analyzed by fluorescence-activated cell sorter for cell surface expression of CD86 and is shown relative to untreated controls with mean and SD of 3 different experiments performed with iDCs from 3 different donors.