Combinatorial treatment with CFZ and AGI-6780 acts through the inhibition of the NAMPT/SIRT/IDH2 pathway. (A) Schematic representation of the NAMPT/SIRT3/IDH2 pathway and inhibitors. (B) KMS-27 cells treated with DMSO, AGI-6780 (5 µM), CFZ (3 nM), or a combination of the 2 drugs were analyzed for NF-κB activity 6 hours posttreatment. NF-κB activity was detected in total extracts measuring the DNA-binding capability of NF-κB on its target sequence (see “Materials and methods”). Data represent the percentage of NF-κB binding activity normalized vs DMSO samples and are the means ± SD of 3 independent experiments. (C) KMS-27 cells UT, treated with DMSO, CFZ (2.5 nM), AGI-6780 (5 µM), or a combination of the 2 drugs were analyzed for NAMPT mRNA expression levels 24 hours posttreatment. Data are the means ± SD of 3 independent experiments. (D-E) KMS-27 cells were left UT, treated with DMSO or FK866 (10 nM), for 48 hours; vehicle or CFZ (2.5 nM) were added for additional 48 hours. Cells were analyzed for (D) IDH2 activity 6 hours posttreatment with CFZ and for (E) cell viability by TMRM staining-flow cytometry 6 and 48 hpt with CFZ. Data are the means ± SD of 3 independent experiments. (F-G) KMS-27 cells UT, treated with DMSO, 1.25 nM CFZ, 10 µM AGK7, or a combination of the 2 drugs were analyzed for (F) IDH2 activity 6 hours posttreatment and for (G) cell viability measured by TMRM staining-flow cytometry 6 and 48 hpt. Data are the means ± SD of 3 independent experiments (*P < .05; **P < .01; ***P < .001).