Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD⁺ / SIRT1 Mediated Deacetylation of FOXO3a in Myeloid Cells.

Abstract 1352

Poster Board I-374

Severe congenital neutropenia (CN) is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 10/l. G-CSF treatment increases blood neutrophil numbers in more than 90% of individuals with CN. CN is also considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. Recently we found that NAMPT, a protein involved in biosynthesis of NAD⁺ was significantly increased in 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, an enzyme involved in deacetylation of several histone and non -histone proteins (e.g. FOXO3a) by utilising NAD⁺. In search of the downstream factors regulated by G-CSF/ NAMPT /SIRT1 pathway, we found elevated levels of FOXO3a protein in CN patients treated with G-CSF compared to healthy controls or patients with other types of neutropenia. Therefore, we were interested whether NAMPT/ NAD⁺ -dependent activated SIRT1 affects FOXO3a levels. Indeed we observed that overexpression of NAMPT leads to the upregulation of FOXO3a protein in 293T cells and endogenous FOXO3a interacts with endogenous SIRT1 in both 293T cells and promyelocytic cell line NB4. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. As acetylation of FOXO3a is considered to be important for its tumor suppressor function we asked if inhibition of NAMPT using FK866 increases the acetylation of FOXO3a. Indeed we show that acetylation level of FOXO3a is enhanced when 293T and NB4 cells were treated with FK866. Furthermore, increased acetylation of FOXO3a correlates with the decreased interaction between FOXO3a and SIRT1 in 293T cells after treatment with FK866. GADD45, a protein involved in DNA damage repair, is a well known target of FOXO3a. We found that in CD33⁺ myeloid progenitor cells from G-CSF treated CN patients with high NAMPT / SIRT1 / FOXO3a levels also had low GADD45 mRNA levels. We performed reporter gene assay using luciferase construct containing wild type GADD45 promoter and observed that SIRT1 enhances the FOXO3a mediated downregulation of GADD45 promoter and interestingly NAD⁺ further augments this effect. Taken together we hypothesized that NAMPT/NAD⁺ activated SIRT1 mediates deacetylation and activation of FOXO3a protein, leading to downregulation of target genes (e.g. GADD45) with tumor suppression functions which might possibly be involved in the leukemic transformation in CN patients.