Genetically engineered salvage-dependent cancer cells including H460Sal-dep, HCT116Sal-dep and U87Sal-dep were transduced with two different shRNAs against NAMPT followed by puromycin selection. A non-targeting shNTC was used a control. a, Top, intracellular measurement of NAD+ levels. Middle, representative images of clonogenic survival assay using crystal violet staining from one of two independent experiments. Both biological replicates showed similar results. Bottom, quantification of colony formation units. Cells were stained with crystal violet 15–18 days after seeding. Salvage-dependent cancer cells with NAMPT stably silenced grown for an extended duration of time (long-term depletion) were later silenced with shRNA against NMRK1. b, Immunoblotting for cleaved caspase-3 as a measure of cell death and to test for protein abundance of NAMPT, NMRK1 and NAPRT. Representative blots are from one of two independent experiments. Both biological replicates showed similar results. Actin was used as a loading control. Salvage-dependent cancer cells stably silenced for NAMPT and grown for an extended duration of time (long-term depletion) were later silenced for NMRK1 using siRNA. c, d, Relative NMRK1 and NAMPT, NMRK2 or NAPRT transcript levels as measured by qPCR. Salvage-dependent cancer cells stably silenced for NAMPT and grown for an extended duration of time (long-term depletion), were later silenced with NMRK1 shRNA. e, Schematic overview of the model illustrating NAD pathway addiction in cancer is driven by two separate mechanisms—one that gets shaped by gene amplification (left), and the other through epigenetic reprogramming (right). The model demonstrates tissue context-based amplifications of genes encoding key enzymes (NAPRT and NADSYN1) of the PH-pathway and subsequent tumour cell dependence that is absolute and not subjected to enzymatic bypass rewiring. By contrast, epigenetically determined dependence on the NAMPT driven salvage-pathway is subject to enzymatic bypass, requiring combination therapies. In all panels, for short-term depletion, cells were seeded 7–10 days after transduction/selection, and for long-term depletion, cells were seeded ≥30 days after transduction/selection. Data are representative of five (a, top, c), three (d) and two (a, middle, bottom) independent biological replicates from independent experiments. Data in scatter plots are mean ± s.d., with P values determined by one-way ANOVA with Tukey’s multiple comparisons test (a, c, d). For gel source data, see Supplementary Fig. 1. Source data