bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells
Authors: Yang Zhongfa , Drumea Karen , Cormier James , Wang Junling , Zhu Xuejun , Rosmarin Alan G. 
Published: October 2019

Abstract
Abstract 1284
GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs.

Disclosures:
No relevant conflicts of interest to declare.
Alcam Mediated Cellular Interaction Regulates Hematopoietic Stem Cell Repopulation and Self-Renewal
Authors: Jeannet Robin , Cai Qi , Liu Hongjun , Vu Hieu , Kuo Ya-Huei 
Published: October 2019

Abstract
Abstract 1291
Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. It has recently been shown that Alcam+ endosteal subset in the bone marrow contain hematopoietic niche cells able to support hematopoietic stem cell (HSC) activity. We examined Alcam mRNA levels and cell surface expression by quantitative RT-PCR and flow cytometry in various hematopoietic stem and progenitor subsets. We found that Alcam is highly expressed in long-term repopulating HSC (LT-HSC), multipotent progenitors (MPP), and granulocyte/macrophage progenitors (GMP). We use an Alcam null mouse allele to assess the function of Alcam in HSC differentiation and self-renewal. Clonogenic colony-forming progenitor serial-replating assay show that the replating potential of Alcam-deficient LT-HSCs is impaired. An in vitro single-cell differentiation assay of phenotypic LT-HSCs reveals that Alcam-deficiency leads to an enhanced granulocytic differentiation. In competitive repopulation transplantation, Alcam-deficient cells show a transient engraftment enhancement, however, the engraftment is significantly lower in secondary transplantation, suggesting that the self-renewal capacity of Alcam-deficient HSC is compromised. We performed a limiting-dilution transplantation assay and determined that the frequency of long-term repopulating cells in Alcam-deficient bone marrow is significantly reduced compared to wild type control. We further assessed the engraftment efficiency of phenotypically purified LT-HSCs. We show that the engraftment efficiency of Alcam-deleted LT-HSCs is significantly reduced compared to wild type LT-HSCs. Since Alcam-deleted HSCs are able to home efficiently to the bone marrow cavity, the engraftment defect is not due to inefficient homing upon transplantation. Collectively, These studies implicate Alcam mediated cell-cell interaction in the regulation of hematopoietic transplantation and recovery.

Disclosures:
No relevant conflicts of interest to declare.
Lack of the Transcription Factor NFAT (Nuclear Factor of Activated T cells) c2 in Hematopoietic Progenitor Cells Results in Profound Hematological Abnormalities in Mice
Authors: Arabanian Laleh S. , Haase Michael , Habermann Ivonne , von Bonin Malte , Waskow Claudia , Ordemann Rainer , Ehninger Gerhard , Kiani Alexander 
Published: October 2019

Abstract
Abstract 1296
Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow microenvironment in vivo is of considerable interest.
We have previously shown that aged mice lacking the transcription factor NFATc2 develop bone marrow hypoplasia, anemia, and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic progenitor cells (HPC) ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the bone marrow hematopoietic niche.
In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immunomagnetically purified NFATc2-deficient (ko) HPCs to lethally irradiated wildtype (wt) mice, and vice versa. After a posttransplantation period of 6–8 months, peripheral blood, bone marrow as well as spleen and liver of the transplanted animals were analyzed and compared to wt and ko mice transplanted with control cells.
Transplantation of NFATc2-deficient HPCs into wt recipients (ko → wt) induced similar hematological abnormalities as those occurring in non-transplanted ko mice or in ko mice transplanted with ko cells (ko → ko). Compared to wt mice transplanted with wt cells (wt → wt), ko → wt mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their bone marrow. Likewise, ko → wt mice developped clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in wt → wt control animals.
Our data demonstrate for the first time, that NFAT transcription factors directly regulate the intrinsic function of hematopoietic progenitor cells in vivo. The transcriptional targets for NFAT in these cells are yet unknown and are the focus of further investigations.

Disclosures:
No relevant conflicts of interest to declare.
Tissue-Specific HIF-1 and HIF-2 Regulatory Elements and Their Roles in Erythropoietin Gene Expression
Authors: Yoon Donghoon , Song Jihyun , Kim Hyojin , Kim Bumjun , Shin Hyejin , Arnold Gregory , Elliott Steven , Prchal Josef T. 
Published: October 2019

Abstract
Abstract 1303
Studies of the regulation of erythropoiesis by hypoxic induction of erythropoietin (EPO) transcription led to the discovery of oligonucleotide binding motifs, hypoxia responsive elements (HREs) essential for hypoxic upregulation of EPO. HREs bind hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers of 3 α subunit isotypes; HIF-1α, HIF-2α, and HIF-3α, and a common β-subunit. EPO is expressed in diverse tissues, some regulated mainly by HIF-2 and others by HIF-1. Tissue-specific regulatory elements (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE) of the EPO gene spanning several kilobases were reported. Our in silico analysis of the human EPO genome found two additional canonical HIF-binding elements in the KIE and NRE regions, and comparative analysis of phylogenically conserved sequences of Epo genes refined these HIF-binding elements from several kbs to a few hundred nucleotides and further delineated them as mKIE, mNRE1, mNRE2, and mNRLE2. To further define the structure and function of these elements in vivo, we exposed mice to 8% O2 hypoxia and monitored hypoxic induction of Epo mRNA levels in the liver, kidney, brain, and spleen. Spleen, kidney, and brain had a peak induction of the Epo transcript at 3 hours of hypoxia treatment, while liver showed peak induction at 6 hours. At the end of the hypoxia treatment, the mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF-2α. The results from these studies are summarized below.
“+” denotes presence and “−” absence of binding of HIF-1 and HIF-2. “Norm” - normoxia, “Hyp” - hypoxia.
To further define the role of these elements in tissue-specific Epo expression, we cloned and mutated these elements with reporter constructs, and generated transgenic mouse strains. The reporter gene was expressed in renal tubules, the red pulp area of the spleen, and liver hepatocytes. By creating a designed point mutation to disrupt the function of KIE, we suppressed the hypoxia response in the kidney, while mutations of NRE1 and NRE2 abolished reporter expression in the liver. Studies of HREs in the brain are ongoing.
In conclusion, we have refined the previously reported HREs to a few hundred bases (particularly, KIE is a 6 nucleotide bearing motif) and now demonstrate tissue-specific differential hypoxia-induced binding of HIF-1 and HIF-2 and their functional roles in EPO induction. This provides a background to examine the molecular basis of these elements in patients with aberrant EPO expression.

Disclosures:
Elliott: Amgen, Inc: Employment.
A “Master Regulatory Cis-element” Governs the Hematopoietic Stem/Progenitor Cell Compartment, Vascular Integrity, and Cardiovascular Development
Authors: Johnson Kirby D , Ryu Myung-Jeom , Boyer Meghan E , Kelez Sunduz , Zhang Jing , Lee Youngsook , Bresnick Emery H 
Published: October 2019

Abstract
Abstract 1304
The expression and activities of master regulatory proteins are exquisitely controlled to ensure the proper development of complex organisms. As a master regulator can orchestrate multiple developmental processes, presumably mechanisms involving a large ensemble of cis-elements and trans-acting factors are deployed to establish this control. GATA-2, a master regulator of hematopoiesis expressed in hematopoietic, endothelial, neuronal, and fat cells is likely subject to such complex regulation. GATA-2 occupies dispersed sites of the Gata2 locus, highlighting a positive autoregulatory mechanism. As GATA-1 levels rise during erythropoiesis, GATA-1 displaces GATA-2 from the Gata2 locus, instigating repression. “GATA switches” occur at five dispersed Gata2 sites (−77, −3.9, −2.8, −1.8 and +9.5 kb). Targeted deletion of GATA factor switch sites −2.8 and −1.8 revealed that these sites individually contribute to maximal Gata2 expression, but are otherwise dispensable for embryogenesis and steady-state hematopoiesis. Herein, we describe the targeted deletion of an intronic GATA switch site (+9.5) comprised of an E-box GATA factor composite cis-element. Strikingly, whereas the Gata2 gene knockout results in lethal anemia by E10.5, the +9.5 mutation yielded embryonic lethality around E13.5. The +9.5 mutation greatly reduced Gata2 expression in fetal livers, heart and endothelial cells, but not in the brain. Consequently, by E12.5 mutant mice exhibited severely reduced numbers of hematopoietic stem/progenitor cells (HSPCs) in fetal livers as assayed by cell sorting and colony assays. However, mutant mice were not overtly anemic and Ter119+ cells were abundant in the mutant fetal livers, accounting for the delay in lethality compared to the Gata2 knockout. Additionally, +9.5 mutant embryos displayed vascular/cardiovascular malformations and severe hemorrhaging. This work reveals that, unlike the −2.8 and −1.8 GATA switch sites, the +9.5 composite element functions as a “master regulatory cis-element” required for HSPC development, vascular integrity, and cardiovascular development. Genomic analyses identified a large cohort of loci containing conserved intronic composite elements resembling the +9.5 element, with heavy representation at genes critical for HSPC and vascular development/function, as well as novel genes of unknown function.

Disclosures:
No relevant conflicts of interest to declare.
Thymoglobuline Is As Effective As Lymphoglobuline in Japanese Patients with Aplastic Anemia Possessing Increased Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Deficient Cells
Authors: Yamazaki Hirohito , Saito Chizuru , Sugimori Naomi , Hosokawa Kohei , Kiyu Yu , Mochizuki Kanako , Takamatsu Hiroyuki , Nakao Shinji 
Published: October 2019

Abstract
Abstract 1339

Background:
Thymoglobuline (TG), a rabbit anti-thymocyte globulin (ATG) preparation which has more potent immunosuppressive effects than horse ATG Lymphoglobuline (LG), has been shown to be as effective as LG as both first and second line immunosuppressive therapy (IST) for aplastic anemia (AA) by several retrospective studies. However, recent prospective studies from the U.S. and Europe have shown a lower effectiveness of TG compared to horse anti-thymocyte globulin (ATG). The conflicting results may be due to the heterogeneity in the pathophysiology of AA or in the ethnic backgrounds of the patients evaluated. Small populations of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient (GPI-AP−) blood cells are detectable in approximately 60% of patients with newly-diagnosed AA, and are associated with a good response to LG (Blood, 107: 1308, 2006). TG may have comparable effects on LG if the study subjects are limited to AA patients possessing GPI-AP− blood cells. Objectives/Methods: To evaluate the effectiveness of TG in Japanese patients with AA, we retrospectively analyzed the outcomes of TG therapy in 57 patients (25 males and 32 females) with AA (40 with severe AA and 17 with non-severe AA) aged from 17 to 84 (median 58) years. Peripheral blood obtained before therapy was examined for the presence of GPI-AP− cells using flow cytometry with liquid fluorescent aerolysin, which can accurately determine the percentage of GPI-AP− granulocytes, erythrocytes, and monocytes at percentages of less than 0.01%. The cut-off values were defined as >0.003% for granulocytes, >0.005% for erythrocytes, and >0.01% for monocytes based on the analytical results of 57 healthy individuals. Results: The cumulative rate of response after 6 months of TG therapy was 59%. Significant increases in the GPI-AP− cell percentages in at least one of the three lineages were detected in 38 (67%) of the 57 patients before therapy. The response rate in patients with increased GPI-AP− cells (PNH+ patients, 69%) was significantly higher than that in patients without GPI-AP− cells (PNH− patients, 35%, P=0.016). Seventeen percent of PNH+ patients achieved complete remission (CR) within 1 year, while only 12% of PNH− patients achieved a CR. 97% of PNH+ and 84% of PNH− patients were alive at the median time of 1 year after the therapy. The response rate in PNH+ patients was comparable to that in PNH+ patients treated with LG (70%) in our recent prospective study. Conclusions: TG is therefore considered to be as effective as LG in Japanese adult patients with AA, particularly in PNH+ AA patients where immune pathophysiology is definitely involved. Alternative therapies may thus need to be considered for PNH− patients.

Disclosures:
No relevant conflicts of interest to declare.
PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia
Authors: Bach Christian , Staber Philipp B. , Ye Min , Zhang Pu , Friedman Alan D. , Delwel Ruud , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 1353
The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells.
To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells.
Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes.
Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo.
The first two authors contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.
PML/RARα -Regulated MicroRNA 181b Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia
Authors: Braeuer-Hartmann Daniela , Hartmann Jens-Uwe , Gerloff Dennis , Katzerke Christiane , Falzacappa Maria Vittoria Verga , Pellicci Pier Guiseppe , Mueller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard 
Published: October 2019

Abstract
Abstract 1361
In the acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. Additionally, we showed the transcriptional induction of miR-181b by the APL-associated PML-RARα oncogene which is released after treatment with ATRA. The overexpression of miR-181b by microRNA mimics leads to an inhibition of ATRA induced granulocytic differentiation. Furthermore, the downregulation of miR-181b by locked nucleic acids (LNAs) causes the induction of granulocytic transcription factor C/EBPβ. In a PML/RARα knock in mouse model we could show the enhanced expression of miR-181b. Also, APL patient samples show a significant enhanced expression of miR-181b in comparison to AML patient samples with normal karyotype. In addition, the miR-181b expression was strongly reduced in APL patient samples after ATRA treatment. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181b. In functional studies we showed that enforced expression of miR-181b reduces the protein level of RASSF1A by binding to the 3'UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. In conclusion, we identified miR-181b as a new player in the PML/RARα associated APL. Moreover, we firstly described the miR-181b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated miRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia.

Disclosures:
No relevant conflicts of interest to declare.
PDGF-RTK Inhibition Restores CD95/Fas-Mediated Apoptosis in Leukemic T-LGL Via Promoting ASM Translocation, Ceramide Production and CD95 Signal Amplification
Authors: Yang Jun , Li Yongping , Loughran Thomas P. 
Published: October 2019

Abstract
Abstract 1374
Human memory T cells are protected from Fas/CD95-induced apoptosis. The mechanisms involved in blocking of CD95 signal transduction revealed the presences of anti-apoptotic factors. T-LGL leukemic cells are terminal effector memory T cells (Temra [CD45RA+/CD62L−]) featuring over-expression of Fas/Fas ligand, constitutive activation of PDGF-RTK and its downstream Src family kinase (SFK), PI3K/PKB/AKT/ERK1/2 signal cascade, as well as high expression levels of c-FLIP and Mcl-1. Inhibition of the downstream targets of PDGF-RTK pathway, such as SFK and PI3K induce significant level of apoptosis in leukemic LGL. However, direct targeting of PDGF-RTK showed a limited efficacy in induction of cell death in vitro. Loss of Fas sensitivity is the key pathological aberration in leukemic LGL, and restoration of CD95/Fas-mediated apoptosis in LGL leukemic cells remains a challenge. Therefore, reprogramming LGL leukemic cells to become sensitive to the already existing over-expressed Fas/Fas L in patients can be a potential therapeutic strategy. In the current study, we observed that inhibition of PDGF-RTK activity with inhibitors, such as AG1296 or imatinib myselate (Gleevec) restored CD95/Fas sensitivity in T-LGL leukemic cells. Our data suggest that the mechanism of PDGF-RTK inhibition restores Fas sensitivity in T-LGL leukemic cells via induction of acid sphingomyelinase (ASM) cell membrane translocation and activation, promoting ceramide production and Fas receptor aggregation in the cell membrane lipid rafts of leukemic cells. We also observed that PDGF-RTK inhibition significantly downregulates Mcl-1 expression in T-LGL leukemic cells. These observations suggest a possible role for future clinical application of TKIs in clinical management of this disease.

Disclosures:
No relevant conflicts of interest to declare.