The role of NAMPT/SIRT1/FOXO3a pathway for the leukemic transformation in severe congenital neutropenia (CN) patients.

Severe congenital neutropenia 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 congenital neutropenia. 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 activation of SIRT1, an enzyme involved in deacetylation of several histone and non – histone proteins 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. FOXO3a transcription factor is a member of the family of forkhead proteins and has been shown to be regulated by SIRT1. Therefore, we were interested whether NAMPT/NAD+ -dependent activated SIRT1 affects FOXO3a levels. Indeed we demonstrated that overexpression of NAMPT leads to the upregulation of FOXO3a mRNA. In the HL-60 promyelocytic leukemia cell line and the 293T cells we show that FOXO3a is present in acetylated form and we found slight increase in FOXO3a acetylation after induction with oxidative stress stimuli such as H2O2. Further, endogenous SIRT1 interacts with endogenous FOXO3a and this interaction was slightly diminished when the 293T cells were treated with FK866, a specific inhibitor of NAMPT. Moreover, FOXO3a mediated activation of FOXO3a reporter construct was abrogated if SIRT1 was inhibited by specific SIRT1 siRNA. GADD45α, a protein involved in DNA damage repair, is a well known target of FOXO3a. We performed reporter gene assay using luciferase construct containing wild type GADD45α promoter and found that overexpression of FOXO3a leads to downregulation of GADD45α reporter. Interestingly, in G-CSF treated CN patients the high FOXO3a levels were associated with low GADD45α expression. Taken together our working hypothesis is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of FOXO3a protein thereby leading to FOXO3a activation, which in turn influence the downstream target genes with tumor suppression functions resulting in the leukemic transformation in CN patients.