title: Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.
abstract: Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.
authors.affiliation: Department of Translational Oncology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Translational Oncology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Mandy
authors.initials: M
authors.lastname: Kwong
authors.affiliation: Department of Bioinformatics and Computational Biology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Daemen
authors.affiliation: Department of Translational Oncology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Marcia
authors.initials: M
authors.lastname: Belvin
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Translational Oncology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Georgia
authors.initials: G
authors.lastname: Hatzivassiliou
authors.affiliation: Department of Translational Oncology, Genentech Inc. South San Francisco, California, United States of America.
authors.forename: Thomas
authors.identifier: http://orcid.org/0000-0002-9161-8070
authors.initials: T
authors.lastname: O'Brien
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D009243:NAD / Q000378:metabolism*; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D005973:Glutamine; D009243:NAD; D054409:Nicotinamide Phosphoribosyltransferase