Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase 1 . It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells [2][3][4] . Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemiainitiating cells (LICs) that drive the recurrence of CML [5][6][7][8] . Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a 1/1 and Foxo3a 2/2 mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-b is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-b inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-b inhibitor impaired their colonyforming ability in vitro. Our results demonstrate a critical role for the TGF-b-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.Although tyrosine kinase inhibitor (TKI) therapy of CML patients efficiently induces the death of leukaemia cells [5][6][7][8] , LICs in these patients can survive this therapy. To understand the molecular mechanisms maintaining CML LICs, we characterized LICs in vivo using a mouse model for CML-like myeloproliferative disease (MPD) 9 . Consistent with previous reports 10-13 , we found that a rare c-Kit 1 Lineage 2 (Lin 2 )Sca-1 1 (KLS 1 ) population of CML cells (that is, bearing markers of normal haematopoietic stem cells (HSCs)) induced efficient CML development in recipient mice (Supplementary Fig. 1). In contrast, neither c-Kit 1 Lin 2 Sca-1 2 (KLS 2 ) cells (which correspond to normal progenitors), nor other CML cell populations expressing differentiation markers, induced CML.We and others have shown that Foxo transcription factors, which are important downstream targets of PI3K-Akt signalling, are essential for the maintenance of self-renewal capacity in normal HSCs [14][15][16] . When a growth factor binds to the appropriate receptor, Akt is activated and phosphorylates Foxo proteins, resulting in their nuclear export and subsequent degradation in the cytoplasm. In the absence of growth factor stimulation, Foxo proteins are retained in an active state in the nucleus and induce their transcriptional targets. In CML cell lines, BCR-ABL is thought to activate PI3K-Akt signalling that leads to nuclear export of Foxo factors and suppression of their transcriptional activity...

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1–/– phenotype. Here we demonstrate that cells derived from Bmi1–/– mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1–/– mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.