ALBERT

Description: The DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type–specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

Description: Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant (GFI136N, the form predisposing to AML) or by the more common form of GFI1 (GFI136S). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Description: Under normal conditions, humans maintain a blood content of 150-400 x 109 platelets per liter, whereas mice can reach 1000 x 109 platelets per liter. Thrombocytopenia occurs when the level of platelets becomes too low, a situation that increases the risk of spontaneous bleeding and hemorrhage. Although the number of platelets is continuously very high, they are produced by a rare cell population the megakaryocytes (MKs), which in turn are produced by megakaryocyte-erythrocyte precursors (MEPs) in the bone marrow. The first report of a potential role for the zinc finger transcription factor Gfi1b in megakarypoiesis and thrombopoiesis showed that the full knock-out of this gene in mice leads to a severe impairment of both erythropoiesis and megakaryopoiesis, translating into a severe thrombocytopenia and a lethality by day e15.5 (Saleque et al Genes Dev 2002). Because of this developmental arrest at mid-gestation, the function of Gfi1b in adult differentiated hematopoietic cells could not be analyzed. We have thus generated conditionally deficient mice carrying floxed Gfi1b alleles, to study the role of Gfi1b in adult hematopoiesis. To ablate Gfi1b expression, we crossed Gfi1b flox/flox mice with animals carrying two different Cre transgenes: a ROSA-Cre-ERT2 transgene that quickly deactivates Gfi1b in all cells upon treatment with tamoxifen, allowing to almost instantly measure effects on already differentiated MKs and ii) a PF4-Cre transgene that constitutively expresses the Cre specifically in MKs, excluding any adverse effects due to other cell types. With these mice, we could show that Gfi1b ablation leads to a strong proliferation and expansion of both MEPs, MK precursors and MKs, which was surprisingly associated with an almost complete loss of platelets (∼99.9% reduction compared to controls in PF4-Cre, Gfi1bflox/flox mice). Most striking was the strong increase in the number of MKs, even from an early stage of differentiation, when Gfi1b was deleted. It was not clear, however, if this expansion of MKs was caused directly by the loss of Gfi1b in early progenitors, or if it was a consequence of the severe thrombocytopenia that could stimulate megakaryopoiesis through a feedback loop. An in vivo kinetic study of platelet loss and MK proliferation in the ROSA-Cre-ERT2, Gfi1bflox/flox mice revealed that the expansion of MKs started as early as 3 days after administration of tamoxifen (over 4-fold increase compared to age matched controls). On the other hand, platelet counts started only to decrease noticeably 4 days after tamoxifen administration (about 400 x 109/L vs 1200 x109/L), although reticulated platelets started to decrease as early as day 2 after tamoxifen injection and almost completely disappeared by day 4 after tamoxifen treatment. This suggests an arrest in platelet release in the absence of Gfi1b. After 4 days of tamoxifen treatment, platelet counts decreased quickly to reach a minimum around day 7-8 (60 x 109/L vs 1350 x 109/L in controls). By this time, the number of MKs literally exploded to reach levels up to 15 fold higher than in wild type controls. These results indicate that the number of MKs increases prior to the decrease in circulating platelet when Gfi1b is deleted. However, the results may also suggest that a feedback loop could contribute to this phenomenon by boosting MK expansion upon platelet loss. A platelet lifespan analysis on the rare remaining platelets in PF4-Cre, Gfi1bflox/flox mice revealed that the low platelet level was not due to accelerated platelet clearance, confirming that the platelet loss was the result of an arrest in platelet release. Gfi1b deficient MKs are still polyploidy, but are significantly smaller and have a different nuclear cytoplasmic ratio than their wt counterparts. In addition, in contrast to normal wt MKs, Gfi1b deficient MKs were unable to properly spread or migrate on fibronectin or fibrinogen surfaces, showed lower F-actin content and an increased expression of the platelet glycoprotein IIb of IIb/IIIa complex (CD41/CD61) on their surface. These data suggest that Gfi1b controls the signaling of the platelet specific integrins to restrict MK proliferation, control MK size and their ability to produce platelets. Disclosures: No relevant conflicts of interest to declare.

Description: The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

Description: Chronic myeloid leukemia (CML) is one of the most frequent leukemic diseases and invariably associated with a reciprocal t(9;22) translocation which creates a juxtaposition of the BCR and ABL genes to form the p230-, p190- or p210BCR-ABL constitutively active tyrosine kinases. This constitutive tyrosine kinase activity is the cause of CML and other leukemic diseases. CML is very efficiently treated with small molecule tyrosine kinase inhibitors, but CML is a stem cell initiated disease and surprisingly the leukemic stem cells cannot be eradicated by tyrosine kinase inhibitor treatment, resulting in a high risk of the development of therapy resistance. To design more successful therapies, it is primordial to understand the molecular mechanisms underlying this disease. Here we show that growth factor independent 1b (Gfi1b), also translocated in CML, is strongly induced in peripheral blood mononuclear cells (PBMCs) in patients with chronic- or acute myeloid leukemia (AML) as well as in patients suffering from myeloprolifarative syndrome (MPS) or B-lymphoblastic leukemia (B-ALL). Gfi1b is a transcriptional repressor essential for erythroid- and megacaryocytic cells, but also expressed in hematopoietic stem cells, early myeloid precursors and mature myeloid cells in peripheral blood. Interestingly, the expression of Gfi1b is further induced by long term treatment with tyrosine kinase inhibitors like Glivec, although the patients are phenotypically in remission. Additionally we describe a new splice variant of Gfi1b which is overrepresented in CML, AML and B-ALL, but not in MPS. Using a mouse model system we demonstrate, that Gfi1b strongly accelerates p210BCR-ABL induced leukemogenesis. We further show that Gfi1b inhibits the activity of PU.1, a key molecule in hematopoiesis and repressor of leukemogenesis. Our findings bear considerable significance with regard to the role of Gfi1b as an oncogene in human leukemia and to its possible value as a new target for leukemia therapy.

Description: Abstract 350 Gfi1b is hematopoietic transcription factor most highly expressed in hematopoietic stem cells, megakaryocyte-erythroid precursors, megakaryocytes and throughout erythroid development. Gfi1b deficiency is lethal in mice around 13.5 dpc caused by a failure to produce functional erythrocytes, megakaryocytes and platelets, which causes severe hemorrhaging. Since this lethality has hampered further analysis of the function of Gfi1b, we used Cre-recombinase inducible conditional Gfi1b knock-out mice (Gfi1bfl/fl). The pIpC induced knock-out of Gfi1b in Gfi1bfl/flMxCre mice leads to a pronounced drop in peripheral blood platelet numbers and induces a strong extramedullary erythropoiesis in the spleen. We sorted Ter119+ bone marrow cells from wt and pIpC induced Gfi1bfl/flMxCre mice for a genome wide expression array analysis and found a significant increase in the expression levels of platelet/coagulation related genes such as PF4, vWF, F2r or Ppbp as well as of the fetal globin genes Hba-x, Hbb-ey and Hbb-ßh1, suggesting that Gfi1 regulates globin gene expression or globin switching. It remained unclear whether the disturbed erythropoiesis in Gfi1b deficient mice was caused by a bone marrow failure, or was a reaction to the anemia caused by internal bleedings as a result of low platelet counts. To clarify this and to avoid deletion of Gfi1b in megakaryocytes, we crossed Gfi1bfl/fl mice with EpoR-EGFP-Cre mice allowing a Gfi1b deletion specifically in erythroid cells at the pro-erythroblast stage. Gfi1bfl/flEpoR-EGFP-Cre embryos were paler than wt littermates, but in contrary to complete knock-outs showed no internal bleedings and had normal platelet counts. In addition, EpoR-EGFP-Cre embryos showed a mild block in terminal erythroid differentiation and a pronounced hyper-proliferation at the Ter119-,CD71+, cKit+ proerythroblast stage where Cre expression is activated. Gfi1bfl/flEpoR-Cre cells showed a strong increase of fetal Hbb-ßH1 globin gene expression and a pronounced decrease of the expression of the adult globin genes Hba, Hbb, as well as of Gata1, Foxo3a and Nfe2l2 but not Gata2. Gene expression-array analysis of fetal liver cells from wt and Gfi1bfl/flEpoR-Cre embryos from day 14.5 dpc showed that besides fetal globin genes, many genes where up-regulated that normally decrease in expression during the development between the embryonic stages 11.5 dpc to 14.5 dpc. These findings confirm that Gfi1b is required for the regulation of globin gene expression during or at the transition from embryonic/fetal to adult stages. Interestingly, Gfi1bfl/flEpoR-Cre mice were viable very likely because these animals have normal platelet counts and do not suffer from hemorrhaging like constitutive Gfi1b deficient mice. However, this also suggested that the block in erythroid development is tolerable, or that it can be overcome during maturation of the embryo. Q-PCR analysis on mRNA from sorted erythroid cells from wt and adult Gfi1bfl/flEpoR-Cre mice showed a highly increased expression of the fetal globin genes Hbb-ßh1, Hbb-ey and Hba-x but only a slight decrease of Gata1 expression, a mild increase in Nfe2l2 expression and no significant expression of Gata2 compared to age matched wild type controls. A recently published study of genome wide in vivo DNA binding of ten major hematopoietic transcription factors (Wilson et al., Cell Stem Cell, 2010) showed Gfi1b binding to hypersensitive site 2 in the globin locus control region (LCR) where also the Gfi1b interaction partner Gata1 and Nfe2 bind. From these data we conclude, that Gfi1b is required to regulate the expression of fetal globin genes during the switch from embryonic/fetal to adult stages and thereafter during adult globin expression and exerts this function by directly binding to regulatory sites in the globin locus. Since the re-expression of fetal globin genes in adult stages is a therapeutic approach for ß-thalassemia, the function of Gfi1b and its regulatory mechanisms could point to new therapeutic strategies for this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3217 Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells and is required for normal multi-lineage blood cell development. Mutations affecting the function of Gfi1 are implicated in leukemia, lymphoma, auto-immune disease and neutropenia. Gfi1 deficient mice are severely neutropenic and accumulate an aberrant CD11b+GR1int myeloid population, which was assumed to contain precursors either arrested in granulocytic differentiation or redirected into the monocytic lineage. To further analyze the function of Gfi1 during granulopoiesis, we used bone marrow cells from Gfi1-GFP knock-in mice, which enable to follow Gfi1 expression by simply measuring green fluorescence. By this, we found that the CD11b+GR1lo bone marrow fraction that contains monocytes separates into two populations expressing low or high Gfi1. We FACS-sorted these two subsets (CD11b+GR1loGfi1hi and CD11b+GR1loGfi1lo) and observed that, 99% of CD11b+GR1loGfi1lo cells resembled monocytes, while over 90% of the CD11b+GR1loGfi1hi cells were myelocytes or metamyelocytes. After three days in culture in the presence of GM-CSF, almost all CD11b+GR1loGfi1lo cells developed into adhering macrophages, while 73% of the CD11b+GR1loGfi1hi cells developed into mature neutrophils, suggesting that Gfi1 expression drives myeloid precursors into the granulocytic lineage. We determined the genome-wide expression patterns of these two populations and as a control also of CD11b+GR1+ granulocytes and the aberrant CD11b+GR1int population found in Gfi1−/− mice (termed: CD11b+GR1loGfi1−/−). Unsupervised clustering analysis showed that CD11b+GR1loGfi1−/− cells are similar to CD11b+GR1loGfi1lo monocytes, while CD11b+GR1loGfi1hi cells are more closely related to mature granulocytes (CD11b+GR1hi). Gene-Set enrichment analysis (GSEA) revealed that genes associated with cell cycle progression were up regulated in CD11b+GR1loGfi1hi, while genes controlling the humoral immune response and chemokine receptor signaling activity were downregulated. Promoter analysis of differentially expressed genes showed for CD11b+GR1loGfi1hi an over-representation of binding sites for E2F, a regulator of cell cycle genes, and for CD11b+GR1loGfi1lo cells an over-representation of binding sites for STAT5, a transcription factor required for monocyte development. The comparison of CD11b+GR1loGfi1hi cells with granulocytes also showed a higher expression of cell cycle associated genes and a lower expression of humoral immune response genes. This indicated that CD11b+GR1loGfi1hi cells show features of faster cycling, less differentiated precursor cells compared to monocytes (CD11b+GR1loGfi1lo) and granulocytes (CD11b+GR1hi). It is thus likely that CD11b+GR1loGfi1hi monocyte bone marrow subset contains the precursors committed to granulocytic differentiation. The unsupervised clustering analysis also revealed that CD48, a 40–45 kD cell surface glycoprotein, is inversely correlated to the Gfi1 expression and might enable to differentiate between CD11b+GR1loGfi1hi and CD11b+GR1loGfi1lo cells and to find a definition for precursors committed for granulopoiesis. We found that CD48 can replace Gfi1/GFP as well as CD11b in a flow cytometric analysis and using GR1, CD48 staining only, we were able to clearly separate granulocytic precursors from other monocytic cells and mature granulocytes as shown by Wright-Giemsa staining and in vitro culture of sorted cells in the presence of M-CSF or GM-CSF. GR1loCD48hi cells gave rise to adherent macrophages in the presence of M-CSF but generated monocytes and granulocytes in the presence of GM-CSF, while GR1loCD48lo cells can only develop into granulocytes in the presence of GM-CSF. Thus CD48 allowed the separation of bipotential precursors from other precursors that are fully committed to granulopoiesis. Analysis of Gfi1−/− bone marrow cells using the GR1, CD48 markers clearly showed that the aberrant CD11b+Gr1int population represents regular monocytes that accumulated at the expense of granulocytes. This suggests that Gfi1 regulates cell-fate decision in bipotential granulocytic-monocytic precursors, which can be precisely defined by expression of GR1 and CD48. This allows for the first time a more precise definition of monocytic-, granulocytic and bipotential precursors in mice on a phenotypic basis. Disclosures: Duehrsen: Alexion: Honoraria.

Description: Inflammatory responses are complex and comprise multiple mediators including cytokines such as TNF-alpha (TNF-α) and IL-1beta. These cytokines are synthesized and secreted in response to signaling by plasma membrane receptors of the Toll-like receptor (TLR) family. A central downstream element of TLR-dependent signaling is the transcription factor NF-kappaB (NF-κB), which plays a pivotal role in controlling the proper sequence of events during an inflammatory response. In unstimulated cells, NF-κB is bound to inhibitory IkappaB (IκB) proteins and remains sequestered in the cytoplasm. Stimulation of TLRs triggers a signaling cascade that leads to phosphorylation and proteasomal degradation of IκB, resulting in the translocation of NF-κB to the nucleus, where it acts as a transcriptional activator of target genes. To keep the innate immune system under control, the TLR signaling cascade is under a tight control of many positive and negative regulators. We have previously shown that the transcription factor Growth Factor Independence 1 (Gfi1) represents a novel factor limiting the inflammatory immune response including TNF-α. Gfi1-deficient (Gfi1−/−) mice show a very strong systemic response to the TLR4 ligand and endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here, we investigated the molecular mechanism of this exaggerated TNF-α production in the absence of Gfi1. It is known that endotoxin stimulation results in the activation of the transcription factor NF-κB through TLR4, leading to TNF-α production. This activation also resulted in rapid and de novo expression of Gfi1 in the nucleus in a time- and dose-dependent manner. The expression of Gfi1 was not due to feedback regulation from secreted TNF, since TNF-deficient macrophages were also able to upregulate Gfi1 mRNA following LPS stimulation. As expected, LPS stimulation of Gfi1−/− macrophages resulted in significantly higher levels of TNF-α mRNA, and secreted TNF-α cytokine. Strikingly and in contrast to most known negative regulators of TLRs, Gfi1 did not affect the activity or the expression levels of the cytoplasmic components of TLR signaling pathway. Additionally, NF-κB phosphorylation and nuclear translocation post- LPS treatment were intact in both Gfi1−/− and Gfi1+/+ macrophages. Immunoprecipitation analysis from cells endogenously expressing Gfi1 and NF-κB or over-expressing these two proteins post transfection, clearly revealed a direct interaction between Gfi1 and the p65 subunit of NF-κB. Immunofluorescence staining of macrophages post-LPS treatment confirmed direct interaction of these two proteins in the nucleus at the endogenous level. Gfi1 represses transcription by binding to DNA recognition sequences in target gene promoters. Thus, aiming to investigate the effect of Gfi1 expression on NF-κB nuclear signaling, we found that LPS treatment enhances NF-κB DNA binding activity in Gfi1−/− macrophages as compared to Gfi1+/+ cells. Furthermore, over expression of Gfi1 protein resulted in negative regulation of NF-κB mediated gene activation in a dose-dependent manner. Chromatin immune precipitation with anti-p65 antibodies from LPS stimulated Gfi1+/+ and Gfi1−/− macrophages revealed enhanced NF-κB promoter occupancy at the TNF gene in Gfi1−/− macrophages as compared to Gfi1+/+ cells. In conclusion, our findings reveal a novel function for Gfi1 in the innate immune response by directly antagonizing NF-κB function. This molecular perceptive of TNF-α regulation during inflammation may provide an attractive strategy for therapeutic intervention in chronic inflammatory diseases and certain cancers.

Description: T cells originate from early T lineage precursors that have entered the thymus and differentiate through well-defined steps. Mice deficient for the BTB/POZ domain of zinc finger protein-1 (Miz-1) almost entirely lack early T lineage precursors and have a CD4−CD8− to CD4+CD8+ block causing a strong reduction in thymic cellularity. Miz-1ΔPOZ pro-T cells cannot differentiate in vitro and are unable to relay signals from the interleukin-7R (IL-7R). Both STAT5 phosphorylation and Bcl-2 up-regulation are perturbed. The high expression levels of SOCS1 found in Miz-1ΔPOZ cells probably cause these alterations. Moreover, Miz-1 can bind to the SOCS1 promoter, suggesting that Miz-1 deficiency causes a deregulation of SOCS1. Transgenic overexpression of Bcl-2 or inhibition of SOCS1 restored pro-T cell numbers and their ability to differentiate, supporting the hypothesis that Miz-1 is required for the regulation of the IL-7/IL-7R/STAT5/Bcl-2 signaling pathway by monitoring the expression levels of SOCS1.

Description: Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.