NAD⁺ starvation enhances the anti-MM activity of genotoxic stress and results in improved HDM-based programs’ efficacy. (A) Foci of DNA damage in KMS11 WT and NAPRT-KO (clone#8) cells were stained by immunofluorescence by γ-H2A.X marker (green) staining, in the presence of NA (1 μM), with or without FK866 (10 nM, 48 hours). Q-nuclear (red) was used as a nuclear reference. On the right of the panel, a graph of the quantification of foci is included. Original magnification 60× is shown and the scale bar is 20 μm. (B) WT and NAPRT-KO clone#8 cells were incubated with melphalan (20 μM). Cell death was measured after 48 hours of drug exposure and assessed by flow cytometry using AV/PI staining. (C) Specific viability of WT, NAPRT-KO, and NAPRT–added-back KO KMS11 cells (clone#8), in the presence of NA (1 μM), was assessed: increasing doses of FK866 (0.8-1-1.4 nM) were administered, and after 24 hours, the alkylating drug melphalan (20 μM) was either added or not for an additional 48 hours. Cell viability was ultimately assessed using the annexin V/PI method. Analysis of drug synergism performed with Combenefit (HSA model) is reported in the panel on the right. (D) Kaplan-Meyer curves of the PFS probability of transplant-eligible patients receiving HDM divided as FK866 sensitive (on the left, n = 126) or not sensitive (on the right, n = 216) in the CoMMpass data set, according to their NAPRT mRNA levels (first and fourth quartiles are represented in red and blue, respectively). Log-rank test is used to compute the P value. (E-F) Heat map showing FK866 activity signature expression in 20 patients with melphalan-exposed NDMM grouped using GSVA method: patients with gene expression in accordance with FK866 treatment are highlighted in red as “FK866 sensitive.” Overall response rate (ORR; includes complete response [CR]; very good partial response [VGPR]; stable disease [SD]; partial response [PR]; and progressive disease [PD]) was measured in profiled patients with MM with a focus on NAPRT expression: patients achieving at least PR after HDM carried overlapping profiling with FK866-sensitive cells with lower NAPRT mRNA levels than others. In panels A-B,D a representative experiment of 2 was shown as mean ± standard deviation (n = 3). Unpaired t test was used for panels A,F (∗P ≤ .05).