Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD⁺ / SIRT1 Mediated Deacetylation of p53 in Myeloid Cells.

Abstract 3589

Poster Board III-526

Recently we found that NAMPT, a protein involved in biosynthesis of NAD⁺ was significantly increased in Severe Congenital Neutropenia (CN) patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD⁺ levels correlated with the elevated levels of SIRT1, a NAD⁺-dependent deacetylase involved in the deacetylation of histone and non-histone proteins. CN is considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. SIRT1 mediated deacetylation of p53 at the lysine 382 has been shown to attenuate the transcriptional activity of p53. We asked if inactivation of p53 by NAMPT/ NAD⁺ / SIRT1 dependent deacetylation plays a role in the leukemic transformation in patients suffering from CN. Here we can demonstrate that presence of NAMPT or NAD⁺ enhances the SIRT1 mediated deacetylation of acetylated lysine peptide in vitro. Further in 293T cells overexpression of NAMPT induces the SIRT1 mediated deacetylation of p53. Using the promyelocytic cell line NB4 we show that endogenous p53 interacts with SIRT1 and the presence of NAMPT leads to decreased acetylation of p53 at the lysine 382. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. Acetylation of p53 is considered to be important for its tumor suppressor function and we asked if inhibition of NAMPT using FK866 increases the acetylation of p53. Indeed we demonstrate that the acetylation level of p53 is enhanced when NB4 cells were treated with FK866 and increased acetylation of p53 correlates with the decreased interaction between p53 and SIRT1. Treatment of NB4 with NAMPT further leads to the decreased expression of p53 target gene cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein. p21 is a well known target of p53 and is involved in cell cycle arrest after the cell undergoes stress. Using p21 promoter-luciferase construct we show that SIRT1 inhibits the p53 mediated activation of p21 promoter and this inhibition was further enhanced in the presence of NAMPT or NAD⁺. Taken together our working hypothesis is that NAMPT/NAD⁺ activated SIRT1 mediates deacetylation of p53 protein thereby leading to p53 inactivation and downregulation of downstream target genes with tumor suppression functions (e.g. p21) which might possibly resulting in the leukemic transformation in CN patients.