LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Author(s): Marneth, Anna E; Botezatu, Lacramioara; Hones, Judith M; Israel, Jimmy CL; Schutte, Judith; Vassen, Lothar; Lams, Robert F; Bergevoet, Saskia M; Groothuis, Laura; Mandoli, Amit; Martens, Joost HA; Huls, Gerwin; Jansen, Joop H; Duhrsen, Ulrich; Berg, Tobias; Moroy, Tarik; Wichmann, Christian; Lo, Mia-Chia; Zhang, Dong-Er; van der Reijden, Bert A; Khandanpour, Cyrus

Myelodysplastic syndrome (MDS) is a disease caused by an ineffective hematopoiesis. MDS can develop into acute myeloid leukemia (AML), an aggressive blood cancer of the myeloid lineage. AML patients have a poor prognosis despite treatment with intensive chemotherapy, hence alternative therapies are needed. Gfi1 (Growth factor independence 1) is a transcriptional repressor which, among other functions, recruits HDAC1 and 2 (histone deacetylase 1 and 2) to its target genes. Low Gfi1 levels (Gfi1-KD) in MDS and AML blasts are associated with poor patient survival and AML development in murine models of human AML. To understand how reduced Gfi1 levels contribute to AML, we analyzed the effect of Gfi1-KD on normal and malignant hematopoietic stem cells (HSCs) in mice. Gfi1-KD led to increased self-renewal in HSCs due to increased symmetric division. In addition, Gfi1-KD resulted in the numeric expansion of leukemic stem cells (LSCs). On a molecular level, expression of Gfi1 leads to the removal of acetyl groups at H3K9. Gfi1-KD therefore resulted in increased H3K9 acetylation at its target genes, causing increased target gene expression. A number of these target genes were oncogenes, explaining why Gfi1-KD promotes AML. We thus hypothesized that the administration of HAT (histone acetyltransferase) inhibitors could be beneficial for MDS/AML patients with reduced Gfi1 expression as it could reverse the increased acetylation of H3K9. Curcumin is a HAT inhibitor which is used as a spice and has so far no known toxic side effects. To study the effect of Curcumin on MDS/AML development, we crossed the well-established murine model of human MDS/AML, NUP98-HOXD13, with GFI1-WT or GFI1-KD mice. The different groups were treated with either curcumin or as a control with 5-Azacytidine (Aza), a DNA methyltransferase inhibitor used in the clinic for the treatment of MDS patients. Curcumin effectively prevented the development of AML in mice with low Gfi1 expression, while the control did not. Overall, our data suggest that Gfi1 regulates the epigenetic state of HSCs and LSCs and thus their stemness in a dose-dependent manner and that Curcumin could be used as a targeted therapy.

Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematopoetic disorders, which affect the myeloid lineages of hematopoiesis. Both are characterized by an accumulation of blast cells in the bone marrow (BM) that have the lost the ability to differentiate to mature cells. The proper differentiation of hematopoietic stem cells (HSCs) is regulated by transcription factors. Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of HSCs and the proper emergence and maturation of erythrocytes and platelets. Aim of the study was to identify I) do different level of Gfi1b influence onset and development of MDS and AML in human patients II) how does Gfi1b act in MDS/AML development on a molecular level. We correlated Gfi1b expression level in blast cells of patients with MDS and AML with the overall disease course. To get a better insight how does different Gfi1b level influence MDS/AML development, we used three different murine models of human AML with expression of different oncogenes (NUP98/HOXD13, MLL-AF9 and expression of a mutated K-Ras). In these models we either downregulated or conditionally knocked out Gfi1b expression. Finally, we performed ChIP Seq analysis as well as whole genome gene expression arrays to study the molecular functions of Gfi1b in AML development. Low expression or absence of Gfi1b expression was associated with an inferior outcome with regard to overall-survival as well as event-free survival of MDS/AML patients. Using the above murine models of MDS/AML, loss or low expression of Gfi1b accelerated AML development. Additionally we could show that loss of Gfi1b significantly enhanced number of functional leukemic stem cells. It is well known that Gfi1b has a function to recruit histone modifying enzymes to induce among other deacetylation of H3K9. ChIP seq data of Gfi1b deficient leukemic cells revealed that loss of Gfi1b led to a higher H3K9 acetylation of a number of target genes, among them a number of oncogenes. Among these target genes, we found MAPK as well as Reactive oxygen species (ROS) signalling, as one of the top hit in our data. Previously it was reported that loss of Gfi1b enhanced the ROS level in HSCs. In our case we also see an increased expression of ROS in Gfi1b deficient leukemic cells, a higher activity of the FOXO pathway as well as reduced p38 activity. The combination of these foundings contributes to the higher number of leukemic stem cells in Gfi1b deficient leukemic cells. To reduce the high level of ROS in leukemic stem cells we use with N-Acetylcystein (NAC). Use of NAC impeded growth of Gfi1b deficient cells in-vitro and in-vivo. Gfi1b act as a tumorsuppressor by restricting number of leukemic stem cells and treatment with NAC opens a potential targeted therapy for AML patients with low/absent expression of Gfi1b.

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B-related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B-related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b-null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b-null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content.

The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit+ cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.

Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36s)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted. Copyright (C) 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

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.

Several types of cancer are characterized by global hypomethylation accompanied by regional hypermethylation and overexpression of DNA methyltransferase (cytosine-5) 1 (Dnmt1). In addition to the established role of Dnmt1 as maintenance methyltransferase, it has been suggested that Dnmt1 might also methylate certain target sites de novo. We created a transgenic mouse model to investigate whether the overexpression of the somatic form of Dnmt1, Dnmt1s, is sufficient to cause erroneous methylation and disease. Because ubiquitous Dnmt1 overexpression has been reported to be embryonic lethal, we designed a CAG promoter-driven Cre-loxP conditional transgene containing a floxed EGFP sequence followed by the Dnmt1s coding sequence. The EGFP sequence is excised and transgenic Dnmt1s expression is activated at specific time points or in specific tissues depending on the Cre deleter strain used for cross-ins. Pronucleus injections with the Dnmt1s transgene construct resulted in six founder lines as verified by PCR, Southern blot and EGFP fluorescence. We performed cross-ins with a CMV-Cre deleter strain to clarify if ubiquitous overexpression of Dnmt1s alone causes the previously observed embryonic lethality. Unexpectedly, these cross-ins yielded viable recombined offspring that ubiquitously overexpressed Dnmt1 mRNA at tissue-dependent levels of up to 229-fold. However, the recombined offspring did not significantly overexpress Dnmt1 protein and showed no apparent signs of disease or pathological phenotype. Here, we describe the establishment of our Dnmt1s-transgenic mouse model and propose possibilities for the absence of transgenic protein.

Growth factor independence 1b (GFI1B) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b, we used conditionally deficient mice that harbor floxed Gfi1b alleles and inducible (Mx-Cre, Cre-ERT) or erythroid specific (EpoR-Cre) Cre expressing transgenes. In contrast to the germline knockout, EpoR-Cre mediated erythroid specific ablation of Gfi1b allows full gestation, but causes perinatal lethality with very few mice surviving to adulthood. Both the embryonic deletion of Gfi1b by EpoR-Cre and the deletion in adult mice by Mx-Cre or Cre-ERT leads to reduced numbers of erythroid precursors, perturbed and delayed erythroid maturation, anemia and extramedullary erythropoiesis. Global expression analyses showed that the Hba-x, Hbb-bh1 and Hbb-y embryonic globin genes were upregulated in Gfi1b deficient TER119+ fetal liver cells over the gestation period from day 12.5-17.5 p.c. and an increased level of Hbb-bh1 and Hbb-y embryonic globin gene expression was even maintained in adult Gfi1b deficient mice. While the expression of Bcl11a, a regulator of embryonic globin expression was not affected by Gfi1b deficiency, the expression of Gata1 was reduced and the expression of Sox6, also involved in globin switch, was almost entirely lost when Gfi1b was absent. These findings establish Gfi1b as a regulator of embryonic globin expression and embryonic and adult erythroid maturation.

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

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.

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.

The transcriptional repressor Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells. Gfi1 deficient mice are severely neutropenic and accumulate ill-defined CD11b(+)GR1(int) myeloid cells. Here we show that Gfi1 expression levels determine mono- or granulocytic lineage choice in precursor cells. In addition, we identify CD48 as a cell surface marker which enables a better definition of monocytes and granulocytes in mouse bone marrow. Using the CD48/Gr1/Gfi1 marker combination we can show that the CD11b(+)GR1(int) cells accumulating in Gfi1 deficient mice are monocytes and not granulocyte precursors. Expression of CD48, Gr1 and Gfi1 define different bone marrow subpopulations that are either committed to the granulocytic lineage, or bipotential precursors of granulocytes or monocytes. Finally, a comparison of genes differentially expressed between murine Gfi1 high granulocytic precursors and mature granulocytes with gene expression changes from human myeloblasts versus neutrophils show a strong resemblance of human and mouse differentiation pathways. This underlines the value of the markers CD48 and Gfi1 identified here to study human and murine granulo-monocytic differentiation.

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-1(ΔPOZ) 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-1(ΔPOZ) 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.

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.

Abstract 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.