AZD4785 potentiates bortezomib-induced anti-MM activity. MM.1S cells were treated with AZD4785 (0.3-3 μM) or bortezomib (1.25-2.5 nM) as a single agent or in combination. Modulation of cell proliferation at 72 hours (A) and apoptosis and cell cycle progression at 48 hours (B) was tested on MM cells using MTS, annexin V/proteasome inhibitor (PI), and PI staining, respectively. Average of triplicate experiments ± standard deviation is shown. (C) MM cells were treated with AZD4785 (3 μM) for 48 hours in the presence or absence of bortezomib (2.5 nM) added for the last 24 hours of treatment; cell lysates were subjected to western blot using anti-KRAS, –p-ERK, –p-MEK, –p-CRAF, –p-AKT, -PARP, -cdk2, and –glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. (D) MM cells were treated with either AZD4785 (3 μM) for 48 hours in the presence or absence of bortezomib (2.5 nM) for the last 24 hours of treatment and subjected to cytofluorimetric analysis of mtROS production (Mitosox). (E) MM cells were treated with AZD4785 (3 μM) for 48 hours in the presence of absence of bortezomib (2.5 nM) for the last 4 hours of treatment and then exposed tumor necrosis factor-α (TNF-α; 10 ng/mL) for the last 20 minutes. Nuclear lysates were extracted and subjected to evaluation of p65/NF-κB activation.