Fig. 7
Response of human MLL-fusion AML cell lines to BH3 mimetics. a EC50s of daunorubicin and BH3 mimetics. The indicated cell lines were treated for 24 h with drug (0–10 μM) and viability was determined by CellTiter-Glo assay on a microplate reader. Values were calculated using GraphPad Prism by log-transforming the data and fitting it to a nonlinear regression. Data were determined from three independent experiments, each performed in triplicate. b Immunoblot analysis to detect indicated BCL-2 protein family members. Positive controls were lysates from the cell lines RS4;11 (for BFL-1) and U266 (for all other proteins). HSP 70 and ACTIN were used as loading controls. c Treatment of cell lines with MDM-2 inhibitor RG-7388 (0–10 μM) to determine p53 status [60]. Viability (PI-negative) was determined by flow cytometry. Data represent means ± SEM, determined from 3 independent experiments, each performed in triplicate. RS4;11 and p53−/− RS4;11 cell lines were employed as controls. The data indicate that p53 is functional in MOLM-13 and MV4;11 but not in THP-1. d–h Combination responses of THP-1 (left), Molm-13 (middle) and MV4;11 (right) human MLL-fusion AMLs to (d) daunorubicin plus ABT-737, (e) daunorubicin plus ABT-199, (f) daunorubicin plus S63845, (g) daunorubicin plus ABT-199 + S63845, or (h) ABT-199 plus S63845. Drug concentrations are indicated on x and y-axes of the matrices. All lines were tested with 0–50 nM daunorubicin but the concentration range for the BH3 mimetics was varied according to the EC50 of the line under test. Cell viability was determined after 24 h by CellTiter-Glo assay and normalised to the viability of untreated samples. The sum of Bliss scores is shown below each matrix. The data represent means determined from three independent experiments. Also shown in (h) is the response to ABT-199 across the dose range as a single agent and in combination with S63845 (50 nM for THP-1 and Molm-13; 12.5 nM for MV4;11); data shown represent means ± SEM from three independent experiments
