NAPRT deficiency is associated with increased oxidative stress in MM cells. (A) Kaplan-Meier curves showing the prognostic impact of low NAPRT levels in terms of OS, using the CoMMpass data set. Log-rank test is used to compute the P value (log-rank test). First and fourth quartiles of NAPRT expression are represented in red and blue, respectively. (B) PROGENy (pathway responsive genes for activity inference) was used to infer pathway activities from CoMMpass RNA-sequencing data, comparing patients with MM with low NAPRT expression (first quartile, red) with those with high expression (fourth quartile, blue). Normalized enrichment scores (NES) for each pathway were displayed. High NES indicate high enrichment. (C) Oxidative stress-related genes mRNA levels were evaluated by qRT-PCR using the 2-ΔΔCt method, with normalization to GAPDH as a housekeeping gene, in scramble and NAPRT-silenced (sh2) RPMI 8226 cells. (D) Detection of human reactive oxygen species (hROS) in scramble and NAPRT-silenced RPMI 8226 cells using CellROX Deep Red and 2',7'-dichlorofluorescin diacetate (DCFDA; for H₂O₂ detection) fluorescent probes. (E) Time-dependent detection of indicated hROS productions after FK866 treatment (10 nM) in control and NAPRT-silenced MM cells. Hydrogen peroxide (H₂O₂) and cytosolic (cO₂⁻) were detected by flow cytometry using DCFDA and dihydroethidium fluorescent probes, respectively. (F) Scramble and shNAPRT #2 RPMI 8226 cells were incubated with or without exogenous catalase (CAT; H₂O₂ scavenger, 1000 U/mL) with or without increasing concentrations of FK866 (3-10 nM). Cell viability was measured after 96 hours of drug exposure and assessed by flow cytometry using annexin V (AV)/propidium iodide (PI) staining. (G-K) Oxidative stress markers malondialdehyde (MDA) (G), activities of antioxidant enzymes (glutathione peroxidase [GPX], glutathione reductase [GR]) (H-I), and GSH:GSSG and NADPH:NADP⁺ ratios (J-K) were assessed in control and NAPRT-silenced (sh2) RPMI 8226 cells in presence of 1 μM NA, with or without increased concentrations of FK866 (2-3 nM) for 24 hours. Panels C-G represent means (±SD) of duplicate experiments; panels H-L show representative experiments as mean ± SD (n = 6). ∗P ≤ .05, ∗∗P < .01; ∗∗∗P ≤ .001; unpaired t test. ns, not significant.