NAPRT activity is crucial for redox homeostasis and oxidative metabolism of MM cells thus influencing the anti-MM activity of NAD⁺-lowering agents. (A) KMS11 cells expressing inducible Cas9 were used to generate different clones of NAPRT-KO cells. WB analysis of wild type (WT) and indicated 4 different NAPRT-KO clones of KMS11 cells confirmed specific KO. Whole-cell lysates were collected and probed with NAPRT antibody. GAPDH was used as a loading control. (B) Antioxidant enzymes (GR and GPX) activities and MDA levels were assessed in WT and in 2 different KMS11 NAPRT-KO clones in presence of 1 μM NA, with or without increasing concentrations of FK866 (1.5-2.5 nM) for 24 hours. (C) Representative WB images of KO cells (clone#8 and #34) expressing NAPRT addbacks or KO cells. (D) indicated antioxidant enzymes activities and MDA levels were assessed in NAPRT added-back and in KO cells, in presence of 1 μM NA, with or without increasing concentrations of FK866 (1.5-2.5 nM) for 24 hours. (E) Mitochondrial complexes (I, II, III, and IV) activities normalized to specific control (expressed as percentage, %) measured in KMS11 WT and NAPRT-KO cells (clone#8 and clone#34), in the presence of 1 μM NA with or without increasing concentrations of FK866 (1.5-2.5 nM) for 48 hours. Data are presented as mean ± standard deviation (n = 6). (F) Specific viability of KMS11 WT and NAPRT-KO cells (clone#8 and clone#34) in the presence of 1 μM NA, was assessed. Increasing doses of FK866 (0.8-1-1.4 nM) were administered, and after 24 hours, the oxidative phosphorylation (OXPHOS) inhibitor IACS010759 (0.6 μM) was either added for an additional 48 hours. Cell viability was finally measured using an MTS-based assay. In the right panel, synergism of the same experiment was analyzed by Combenefit software, using the Lowe method. A representative experiment of 2 was shown as mean ± standard deviation (n = 3). ∗∗P < .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; unpaired t test. cl., clone.