Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.

FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrated

• by reduced QRT-PCR expression of several anti-oxidant enzymes leading to

• accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, and

• in vitro and in vivo rescue of the phenotype using ROS scavengers.

Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19^ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16^INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.