NAPRT silencing reduces intracellular NAD+ content and sensitizes MM cells to NAMPT-i in vitro and in a xenograft mouse model. (A) In RPMI 8226 cells, NAPRT depletion was achieved using 2 different shRNA (sh2 and sh5) specific for NAPRT or scrambled control. NAPRT silencing was validated using qPCR (left panel) and WB (right panel) analyses. (B) Isogenic RPMI 8226 cells as in panel A were treated for 48 hours with FK866 in the presence or absence of NA and their combinations. Then, intracellular NAD+ level was determined by cyclic enzymatic assay, expressed in nmol and normalized for cell mass (mg of total proteins). (C) Cell viability of scramble, shNAPRT 2, and shNAPRT 5 RPMI 8226 cells treated with FK866 in the presence or absence of NA (2 μM), 2-HNA (1 mM), or their combos for 72 hours was measured with MTS assay and presented as a percentage of control (specific control). (D) Schematic representation of the in vivo experiment. (E) Female NOD/SCID J mice (8 weeks of age) were injected subcutaneously in both flanks with MM1S cells transduced with shNAPRT2 or scramble (4.5 × 106 viable cells). After the detection of tumors, mice from both groups were randomized and treated with either vehicle dimethyl sulfoxide (DMSO; scramble, n = 5; shNAPRT 2, n = 6) or FK866 (30 mg/kg; scramble and shNAPRT 2, n = 6) administered intraperitoneally twice a day for 18 days. Tumor volume was evaluated by caliper measurement. A significant delay in tumor growth was observed after treatment in shNAPRT2 cell–xenografted mice compared with scramble (∗∗∗P = .0008). Data represent the mean tumor volume ± standard deviation. (F) Kaplan-Meier survival curve of xenograft mice bearing MM1S scramble and shNAPRT 2 tumors. Mice carrying NAPRT-silenced tumors showed increased survival after FK866 treatment compared with mice bearing control tumors (∗P = .011). n indicates the number of tumors per treatment group. Data were analyzed by 2-tailed Student t test for panels A-B,E or by log-rank Mantel-Cox test for panel F. For panels A-C, data are representative of at least 2 independent experiments. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; unpaired t test. CTR, control; NOD/SCID, nonobese diabetic severe combined immunodeficiency; ns, not significant; qPCR, quantitative polymerase chain reaction.
