bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distinct c-Myb Regulation by HoxA9, Meis1 and Pbx1 in Haemopoietic and Leukaemic-Like Stem Cells.
Authors: Dassé Emilie , Volpe Giacomo , del Pozzo Walter , Frampton Jonathan , Dumon Stephanie 
Published: October 2019

Abstract
Abstract 1431
Poster Board I-454
The transcription factor c-Myb is an essential regulator of haemopoiesis and its expression is deregulated in several types of leukaemia. Although some c-Myb functions have been defined, the mechanisms involved in the control of its expression have yet to be elucidated. Previous studies have suggested that transcription initiation at the c-myb gene is constitutive, and that the level of mRNA is regulated by an elongation-blocking mechanism operating in its first intron. Here, we define and compare mechanisms influencing c-myb expression in haemopoietic stem cells (HSCs) versus leukaemic stem cell (LSC)-like cells. Using a nuclease sensitivity assay we have defined several potential regulatory elements in both HSC and LSC-like model cell lines. These hypersensitive sites are in the proximal promoter and the first intron, the latter correlating with the position of the putative transcription elongation regulatory region. Moreover, the hypersensitive sites are located in regions of sequence conservation and encompass a number of potential binding sites for homeodomain (HD)-containing proteins. In this study, we were able to demonstrate that the HD-containing transcription factors HoxA9 and Meis1, which are highly expressed in HSCs and whose co-expression in mouse bone marrow leads to rapid development of acute myeloid leukaemia (AML), are necessary but not sufficient for c-myb expression. In addition, we show that the pre-B-cell leukaemia transcription factor-1 (Pbx1), known to be a key binding partner of HD-containing factors, is indispensable in the regulation of c-myb expression. Comparing the effects of altered levels of HoxA9, Meis1 and Pbx1 in HSCs versus LSCs suggests that distinct mechanisms involving dimeric or trimeric complexes operate to regulate c-myb expression in these two stem cell types.

Disclosures
No relevant conflicts of interest to declare.
Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice.
Authors: Bersenev Alexey , Wu Chao , Balcerek Joanna , Tong Wei 
Published: October 2019

Abstract
Abstract 1437
Poster Board I-460
Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation.
Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2.

Disclosures
No relevant conflicts of interest to declare.
Stromal Microenvironment Regulates Progenitor Survival through Nitric Oxide Production.
Authors: Jouni Dima , Zhang Yanyan , Bouamar Hakim , Wittner Monika , Vainchenker William , Louache Fawzia 
Published: October 2019

Abstract
Abstract 1440
Poster Board I-463
Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium.
These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling.

Disclosures
Jouni: Région IDF: Employment. Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.
CREB Is Involved in the Regulation of C/EBPβ During 'emergency' Granulopoiesis.
Authors: Hirai Hideyo , Kamio Naoka , Matsusue Akiko , Ogino Shinpei , Kimura Nobuhiko , Satake Sakiko , Ashihara Eishi , Maekawa Taira , Tenen Daniel G. , Imanishi Jiro 
Published: October 2019

Abstract
Abstract 1457
Poster Board I-480
Mobilization of sufficient numbers of granulocytes to the front line of infection is prerequisite for host defense. As granulocytes have a short half-life, the production of granulocytes in the bone marrow must be tightly regulated to meet emergency demands. Our previous findings suggested that granulopoiesis at steady state is largely dependent on CCAAT enhancer binding protein α (C/EBPα) transcription factor (Zhang D.E. et al., PNAS, 1996 and Zhang P. et al., Immunity, 2004), whereas the granulopoiesis during emergency such as infections is dependent upon C/EBPβ (Hirai H. et al., Nat Immunology, 2006). Indeed the transcripts of C/EBPβ in granulocyte precursors were upregulated in response to cytokine stimulation or infection. In order to elucidate the molecular switch between C/EBPα- and C/EBPβ-dependent granulopoiesis, we developed a novel lentivirus-based reporter system. The vector carries two independent expression units, a green fluorescent protein (GFP) driven by a promoter of interest and a mouse Thy1.1 gene under the control of a constitutively active phosphoglycerate kinase (PGK) promoter. The activity of a promoter can be monitored by the intensity of GFP in Thy1.1 positive cells. Using this system, the activity of the C/EBPβpromoter was evaluated in primary bone marrow cells. A series of deletion mutants of the promoter revealed the existence of two cyclic AMP responsive elements (CRE) in the positive responsive elements during GM-CSF induced differentiation. The transcripts of CRE binding (CREB) protein were detected at higher level in hematopoietic stem cells and common myeloid progenitors than other mature cells. When a dominant negative mutant of CREB (S133A), in which the serine residue at 133aa was mutated to alanine, was retrovirally transduced into bone marrow cells, the mRNA of C/EBPβ was reduced and the proliferation/differentiation of granulocyte precursors were significantly impaired. In contrast, a constitutively active form of CREB, CREBDIEDML, facilitated the transcription of C/EBPβ. In addition, CREB is phosphorylated and bound to the CRE in response to GM-CSF stimulation. These data suggest that CREB is involved in the regulation of granulopoiesis through upregulation of C/EBPβ.

Disclosures
No relevant conflicts of interest to declare.
Efficient Induction of Hematopoietic Progenitor/Stem Cell Differentiation by Lentiviral Gene Transduction of TAL1/SCL Into Human ES Cells.
Authors: Kurita Ryo , Miura Yoshie , Okada Michiyo , Kageyama Rui , Hiramoto Takafumi , Yokoo Tomoko , Narusawa Megumi , Suehiro Youko , Inoue Hiroyuki , Marumoto Tomotoshi , Takahashi Atsushi , Tani Kenzaburo 
Published: October 2019

Abstract
Abstract 1477
Poster Board I-500
Human embryonic stem (ES) cells differentiate into three lineages in vitro and in vivo as mouse ES cells. They are therefore highly promising source of various cells/tissues in the regenerative medicine. The current protocols, however, remain to be optimized for the induction of the cells/tissues required. We have recently reported that the lentiviral transduction of TAL1/SCL gene to ES cells derived from the common marmoset, a small nonhuman primate, enables efficient differentiation into hematopoietic progenitor cells even in the absence of stromal cells (Kurita et al. Stem Cells.24:2014-22, 2006). Such culture condition without any stromal cells is considered to facilitate clinical application of ES cell-derived cell/tissues therapy in the regenerative medicine. The present study addressed whether the strategy is also effective in human ES cells. First, we determined optimal culture conditions to induce multilineage hematopoietic differentiation in a human ES cell line, khES-1, kindly provided by Dr. Nakatsuji, Kyoto University, Japan, as assessed by the expression of Brachyury, Flk1 and CD34. We found that the addition of BMP4 and VEGF augmented hematopoietic differentiation of embryoid bodies, and determined optimal concentrations of the cytokines. We established four human ES cell lines stably expressing TAL1/SCL gene by lentiviral transduction. The TAL1/SCL transduction further increased the hematopoietic differentiation under the optimal culture condition as assessed by the expression of CD34, CD235a and CD133. We also observed increased number of hematopoietic progenitor cells derived from two of the TAL1/SCL expressing human ES cell lines by colony-forming assays. Hematopoietic differentiation of the TAL1/SCL expressing ES cells in vivo is also being investigated by transplantation into irradiated immune deficient mice. These results suggest that the combination of optimal culture conditions and lentiviral TAL1/SCL gene transduction is a highly effective strategy to obtain hematopoietic stem cells from human ES cells in the absence of any stromal cells.

Disclosures:
No relevant conflicts of interest to declare.
Deletion of Bcl-X in the Megakaryocyte Compartment Results in Profound Thrombocytopenia Caused by Impaired Platelet Production and Survival.
Authors: James Chloé , Josefsson Emma C. , White Michael J. , Henley Katya J. , Kile Benjamin T. 
Published: October 2019

Abstract
Abstract 1495
Poster Board I-518
Recent studies have suggested a role for the intrinsic apoptosis pathway in megakaryocytic differentiation and platelet shedding. The intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- cell death proteins. We recently demonstrated that platelet life span is controlled by an intrinsic cell death pathway, whereby the anti-apoptotic protein Bcl-xL constrains the pro-apoptotic activity of Bak to maintain platelet survival. As Bcl-xL is expressed in megakaryocytes, we investigated whether this protein is required for megakaryocyte survival, differentiation and/or platelet shedding. We specifically deleted the Bcl-x gene in the megakaryocyte lineage by crossing mice carrying a floxed allele of Bcl-x with mice carrying a platelet factor 4-regulated Cre transgene. Bcl-xfl/flCre+ mice were profoundly thrombocytopenic (26 ± 5 × 103/μl, n=14) compared with Bcl-xfl/flCre− animals (1157 ± 202 × 103/μl, n=13). Platelet life span in these mice was reduced to only 5 hours, as compared to 5 days in wild type littermates. This result confirmed that Bcl-xL is absolutely required for platelet survival. To determine whether Bcl-x deletion has an impact on platelet production, we analyzed the megakaryocyte compartment in Bcl-xfl/flCre+ and Bcl-xfl/flCre− mice. We observed that the number of megakaryocyte progenitors, and number of megakaryocytes in the bone marrow were increased in Bcl-xfl/flCre+ mice (23 ± 9 megakaryocyte progenitors vs 11 ± 5, and 51 ± 9 megakaryocytes vs 12 ± 1). This result suggested that Bcl-xL is not required for the survival of megakaryocytes or their progenitors. To determine whether Bcl-xL is required for the last step of megakaryocyte differentiation, i.e. platelet shedding, we cultured fetal liver cells with thrombopoietin. Large megakaryocytes were isolated after 3 days of differentiation on discontinuous bovine serum albumin gradient. They were cultured for 3 more days in the same media and the percentage of megakaryocytes displaying proplatelets was determined each day. Interestingly, Bcl-xfl/flCre+ megakaryocytes died much more quickly than Bcl-xfl/flCre− megakaryocytes, and almost none of those that survived were able to form proplatelets. Our study indicates that Bcl-xL is not only essential for platelet survival, but it is also required for the survival of mature megakaryocytes at the stage of platelet shedding.

Disclosures:
No relevant conflicts of interest to declare.
Generation of Functional Lymphoid (Natural Killer) Cells From Human ESC-Derived Hemangioblasts.
Authors: Kimbrel Erin A. , Lu Shi-Jiang , Lanza Robert 
Published: October 2019

Abstract
Abstract 1502
Poster Board I-525
Studies with human and mouse embryonic stem cells (ESCs) have shown that a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture. We have developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which will be important for their productive use in regenerative medicine. Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown. Natural killer (NK) cells, which are part of the innate immune system, provide rapid, non-specific responses against viral infection and are involved in tumor cell detection and elimination. Interplay between various activating and inhibitory signals control the three main functions of NK cells, which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, we have been able to produce mature CD56low/−CD16+ NK cells and found that their production does not require the use of stromal feeder layers. The differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 12-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population. An additional 14-17 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture. Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential.

Disclosures:
Kimbrel: Stem Cell & Regenerative Medicine International: Employment. Lu: Stem Cell & Regenerative Medicine International: Employment. Lanza: Stem Cell & Regenerative Medicine International/Advanced Cell Technology: Employment.
Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.
Authors: Ye Min , Zhang Hong , Zhang Pu , Tenen Daniel G. 
Published: October 2019

Abstract
Abstract 1506
Poster Board I-529
During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes.

Disclosures
No relevant conflicts of interest to declare.
Heme Oxygenase-1 Gene Therapy in a Murine Model of Sickle Cell Disease.
Authors: Belcher Belcher D. , Vineyard Julie V. , Bruzzone Carol M , Chen Chunsheng , Beckman Joan D , Ngyuen Julia , Steer Clifford J , Vercellotti Gregory M. 
Published: October 2019

Abstract
Abstract 1527
Poster Board I-550
We previously demonstrated that increases in heme oxygenase-1 (HO-1) and its products, carbon monoxide and biliverdin, inhibit inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin promoter driven Sleeping Beauty transposase plasmid with a wild type rat hmox-1 gene (wt-HO-1) transposable element (289 amino acids) was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had 3-fold increases in HO-1 activity in their livers, similar to hemin-treated mice. mRNA transcription of the transposable element was confirmed by qRT-PCR RFLP analysis. HO-1 overexpressing mice had marked activation of the phospho-p38 MAPK and phospho-Akt proteins and decreased NF-kB p65 in liver nuclear extracts. Hypoxia-induced stasis, a characteristic of SCD but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice. None of these effects were seen in SCD mice injected with Sleeping Beauty containing a nonsense rat hmox-1 gene (ns-HO-1) that encodes for amino acids 1-241 with little or no enzymatic activity. Immunohistochemistry of HO-1 in livers demonstrated perinuclear HO-1 staining in both hepatocytes and sinusoidal Kupffer cells in wt- and ns-HO-1-treated mice. We conclude HO-1 gene therapy targeted to the liver is beneficial in SCD by activating cytoprotective signaling pathways and inhibiting vascular stasis at sites distal to transgene expression.

Disclosures
No relevant conflicts of interest to declare.
Chemo- and Radiotherapy Induce Common Apoptotic Mediators in Hodgkin's Lymphoma.
Authors: Frazzi Raffaele , Nicoli Davide , Iori Mauro , Merli Francesco 
Published: October 2019

Abstract
Abstract 1555
Poster Board I-578
Current therapeutic regimen for classical Hodgkin lymphoma (HL) includes the combined ABVD chemotherapy often followed by radiotherapy. Albeit HL is, in many cases, a curable malignancy, several issues still pose serious threats to the patients. For instance late toxicities, chemoresistance insurgence and relapses represent open and unresolved challenges. We are currently investigating the molecular response of lymphoid cells to doxorubicin treatment and ionizing radiation (IR) in an experimental model represented by HL-derived cell lines. We show the expression and modulation of the stress-inducible protein clusterin/XIP8 (clusterin) in lymphoid cells. The nuclear isoform (having a MW of about 55kDa) is up-regulated upon doxorubicin treatment starting at a concentration of 0.5 micromolar. Interestingly enough, also interferon-gamma determines clusterin and p53 up-regulation starting at 50 nanograms/ml even though just a modest cytostatic effect is observed with this cytokine. High doses of IR increase intracellular clusterin levels, extending to lymphomas a phenomenon first described in the breast carcinoma cell line MCF-7. p53 and Bax are also up-regulated by 12Gy IR at the different time points studied, consistently with the activation of the intrinsic apoptotic pathway. A long-lasting, intracellular clusterin up-regulation observed in surviving cells following irradiation (up to 72h) suggests that this protein is part of the IR-triggered adaptive response. The three cell lines tested here (L-428, KM-H2 and L-540) feature different caspase-3 activity levels. By means of the caspase-3 inhibitor Ac-DEVD-CHO we show here that, in the caspase-3 deficient KM-H2, clusterin is up-regulated by doxorubicin independently of the inhibitor. On the contrary, L-540 shows a different isoforms' expression profile when treated with doxorubicin with or without Ac-DEVD-CHO. This suggests that a linkage exists between caspase-3 activity and clusterin expression.

Disclosures
No relevant conflicts of interest to declare.