Figure 2.

ATR inhibition by VX-970 sensitizes multiple myeloma cells to melphalan. (A) Multiple myeloma (MM) cells were seeded in 96-well plates and treated for 72 hours (h) with DMSO (as a control, untreated [NT]) or increasing concentrations of VX-970 either alone or in combination with the indicated doses of melphalan. Cell viability was assessed using CellTiter-Glo assay. Results are presented as the mean percentage of viable cells in treated samples, relative to DMSO control cells averaged from a minimum of three independent experiments (mean ± standard error of the mean [SEM]), each with three repetitions per condition. Proliferation curves for each cell line were generated using GraphPad Prism. The dotted vertical line indicates the response of MM cells to increasing concentrations of VX-970 alone. Filled black dots indicate the cellular response to melphalan alone. Results of the statistical analysis are reported in the Online Supplementary Table S2. We could not rule out the potential appearance of general toxicity at the highest concentrations used. (B) For each cell line the Bliss synergy matrices and the relative drug synergy scores were calculated using the Combenefit software. The colored areas in the matrix are indicative of the degree of synergy between the drug combinations. (A-B) Different color codes (black, blue and red) are indicative of the concentrations of melphalan used to treat the cells depending on their sensitivity to the drug. (C) MM1.S and H929-melphalan sensitive and OPM2-melphalan resistant cell lines were treated with the indicated concentrations of melphalan either alone or in combination with increasing concentrations of VX-970 (0.075, 0.15 and 0.3 µM). After 48 h, cells were harvested, and immunoblotted for the indicated antibodies. The levels of cleaved PARP and caspase-3 served as indicators of apoptosis. GAPDH was used as loading control.