Search

Precision oncology hinges on linking tumour genotype with molecularly targeted drugs1; however, targeting the frequently dysregulated metabolic landscape of cancer has proven to be a major challenge2. Here we show that tissue context is the major determinant of dependence on the nicotinamide adenine dinucleotide (NAD) metabolic pathway in cancer. By analysing more than 7,000 tumours and 2,600 matched normal samples of 19 tissue types, coupled with mathematical modelling and extensive in vitro and in vivo analyses, we identify a simple and actionable set of 'rules'. If the rate-limiting enzyme of de novo NAD synthesis, NAPRT, is highly expressed in a normal tissue type, cancers that arise from that tissue will have a high frequency of NAPRT amplification and be completely and irreversibly dependent on NAPRT for survival. By contrast, tumours that arise from normal tissues that do not express NAPRT highly are entirely dependent on the NAD salvage pathway for survival. We identify the previously unknown enhancer that underlies this dependence. Amplification of NAPRT is shown to generate a pharmacologically actionable tumour cell dependence for survival. Dependence on another rate-limiting enzyme of the NAD synthesis pathway, NAMPT, as a result of enhancer remodelling is subject to resistance by NMRK1-dependent synthesis of NAD. These results identify a central role for tissue context in determining the choice of NAD biosynthetic pathway, explain the failure of NAMPT inhibitors, and pave the way for more effective treatments.

We identified nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B cell colony enhancing factor (PBEF), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD(+) amounts in myeloid cells, as well as plasma NAMPT and NAD(+) levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia. NAMPT administered both extracellularly and intracellularly induced granulocytic differentiation of CD34(+) hematopoietic progenitor cells and of the promyelocytic leukemia cell line HL-60. Treatment of healthy individuals with high doses of vitamin B3 (nicotinamide), a substrate of NAMPT, induced neutrophilic granulocyte differentiation. The molecular events triggered by NAMPT include NAD(+)-dependent sirtuin-1 activation, subsequent induction of CCAAT/enhancer binding protein-alpha and CCAAT/enhancer binding protein-beta, and, ultimately, upregulation of G-CSF synthesis and G-CSF receptor expression. G-CSF, in turn, further increases NAMPT levels. These results reveal a decisive role of the NAD(+) metabolic pathway in G-CSF-triggered myelopoiesis.

A novel family of transcription factors responsible for regulation of various aspects of NAD synthesis in a broad range of bacteria was identified by comparative genomics approach. Regulators of this family (here termed NrtR for Nudix-related transcriptional regulators), currently annotated as ADP-ribose pyrophosphatases from the Nudix family, are composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding HTH-like domain. NrtR regulons were reconstructed in diverse bacterial genomes by identification and comparative analysis of NrtR-binding sites upstream of genes involved in NAD biosynthetic pathways. The candidate NrtR-binding DNA motifs showed significant variability between microbial lineages, although the common consensus sequence could be traced for most of them. Bioinformatics predictions were experimentally validated by gel mobility shift assays for two NrtR family representatives. ADP-ribose, the product of glycohydrolytic cleavage of NAD, was found to suppress the in vitro binding of NrtR proteins to their DNA target sites. In addition to a major role in the direct regulation of NAD homeostasis, some members of NrtR family appear to have been recruited for the regulation of other metabolic pathways, including sugar pentoses utilization and biogenesis of phosphoribosyl pyrophosphate. This work and the accompanying study of NiaR regulon demonstrate significant variability of regulatory strategies for control of NAD metabolic pathway in bacteria.

NMNAT-1 and PARP-1, two key enzymes in the NAD(+) metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD(+) to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD(+) production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD(+) in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.