bluuebunny/crossref_metadata_2025_split huggingface dataset:
Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis
Authors: Hsu Jingmei , Lee Chung-Tsai , Gerber Scott , Yu Shuqian , Speck Nancy A. 
Published: October 2019

Abstract
Abstract 1300
Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies.

Disclosures:
No relevant conflicts of interest to declare.
RUNX1-Induced Silencing of Non-Muscle Myosin Iib (MYH10) Is Required for Megakaryocyte Polyploidization
Authors: Bluteau Dominique , Lordier Larissa , Antony-Debré Iléana , Jalil Abdelali , Legrand Céline , Rameau Philippe , Bluteau Olivier , Jouni Dima , Mercher Thomas , Leon Catherine , Gachet Christian , Debili Najet , Vainchenker William , Chang Yunhua , Raslova Hana 
Published: October 2019

Abstract
Abstract 1308
Megakaryocytes (MK) are unique mammalian cells that undergo polyploidization during differentiation, which leads to an increase in cell size and protein production that precedes platelet production. The molecular basis of MK polyploidization, denoted endomitosis, which is strongly altered in hematological malignancies and various other disease states, remain poorly understood. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis, associated with a contractile ring defect. Here, we demonstrate that the myosin non-muscle IIB heavy chain (MYH10) is expressed in immature human and mouse megakaryocytes and its expression is repressed during differentiation. In immature MK, MYH10 is specifically localized in the contractile ring while MYH9 is mainly present in the cytoplasm suggesting that they occupy two different functions during MK differentiation. Importantly, MYH10 down-modulation by shRNA or by addition of a chemical inhibitor blebbistatin, increases polyploidization by inhibiting the return of 4N cells to 2N. Conversely, re-expression of MYH10 in MKs prevents polyploidization and the transition of 2N cells to 4N cells. Furthermore, we demonstrated that RUNX1 directly repress the transcription of MYH10. In vitro and in vivo RUNX1 invalidation inhibits MK polyploidization and increases expression of MYH10. Accordingly, in patients with a germline mutation of RUNX1 (FPD/AML), MYH10 is still expressed in platelets. Altogether ours results demonstrated that the RUNX1-mediated silencing of MYH10 is needed for the switch from mitosis to endomitosis linking thus polyploidization with MK differentiation.

Disclosures:
No relevant conflicts of interest to declare.
Stepwise Signaling and Low Oxygen Promote Hematopoietic Progenitor Emergence From Human Pluripotent Stem Cells
Authors: Endo Hiroshi , Takayama Naoya , Koike Tomo , Nakauchi Hiromitsu , Eto Koji 
Published: October 2019

Abstract
Abstract 1317
Hematopoietic systems in mouse models have been well characterized based on the defined cell surface markers present on fetal and adult hematopoietic stem cells (HSCs) and their blood derivatives. In humans, by contrast, hematopoietic ontogeny and the subsequent hierarchy have not been determined. Human pluripotent stem cells (ES cells, ESCs and iPS cells, iPSCs) are embryo-type cells and a promising cell source for studying the ontogeny of blood cells within a differentiation system.
We previously established an in vitro co-culture method using C3H10T1/2 mesenchymal stromal cells, whereby vascular endothelial growth factor (VEGF) promotes the appearance of CD34+ hematopoietic progenitor cells (HPCs) from human ESCs or iPSCs (Takayama et al., Blood, 2008; Takayama et al., J Exp Med, 2010). Here we demonstrate that the use of a combination of low oxygen and growth factors is suitable for cells at specific developmental stages that include CD56+CD326- mesodermal progenitors during the early phase (days 0–4), CD34+CD56+CD90+CD105+CD43-KDR- hemangioblasts during the mid-phase (days 5–7), and CD43+ hematopoietic and KDR+ endothelial cells during the late phase (days 8–10).
During mid-phase, application of basic fibroblast growth factor (bFGF, 10 ng/ml) under 1% O2 significantly increased numbers of CD43+ cells by 5-fold, as compared to cells without bFGF under 21% O2. Administration of a MYC inhibitor (50 μM) to cells during the early-phase, and tumor growth factor beta (TGF-beta) receptor inhibitor (SB431542, 10 μM) during mid-phase, also stimulated generation of CD34+CD43+ HPCs (1.5-fold and 3-fold in increase respectively). Notably, although this protocol resulted in a prominent yield of mesodermal progenitors during the early phase and of HPCs during the late phase (which were 50% and 10% of all derivatives from human iPSCs, respectively), this signaling manipulation had the opposite effect at other stages. For example, bFGF or TGFbeta receptor inhibition significantly depressed HPCs during the early-phase.
The signaling modulation and low oxygen conditions in our co-culture system did not require factors known to affect ex vivo human CD34+ HSC / HPC expansion from cord blood cells, which contain stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, erythropoietin, interleukin (IL)-3 and IL-6.
Our novel culture protocol implicates new players in the stepwise development from a pluripotent state to blood cell generation. These players appear to be governed by circumstances resembling a developmental niche with lower oxygen conditions.

Disclosures:
No relevant conflicts of interest to declare.
CD56(+)CD146(+) Cells in Normal Human Bone Marrow-Stroma Proliferated to Form Tumors in severe Combined Immunodeficiency Mice
Authors: Shirasaki Ryosuke , Tashiro Haruko , Oka Yoko , Yamamoto Tadashi , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki 
Published: October 2019

Abstract
Abstract 1328

Background and Aims:
We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and in vivo. Recently, we also reported that myelogenous leukemia-derived myofibroblasts formed blastomas in non-obese diabetes severe combined immunodeficiency (NOD/SCID) mice, in which the CD56-positive cell-fraction was selectively proliferated (16th EHA). To ascertain whether normal human bone marrow-stroma-derived cells also behave like leukemia-derived cells, stromal cells were injected to NOD/SCID mice, and tumor-formation was observed.

Materials and Methods:
Bone marrow cells were collected from informed normal individuals, whose adherent cells were separated and were cultured for one month to eliminate monocyte/macrophage, vascular cell, and pericyte to prepare stromal myofibroblast-rich fraction. Cells were injected to NOD/SCID mice intra-venously, peritoneally, and subcutaneously (3 × 106/mice). When the tumor formation was observed or mice were dead, they were sacrificed and the engrafted cells were analyzed.

Results and Discussion:
Between at day 70 and 90 after injection mice were dead. Autopsy findings revealed tumor formation at subcutaneous injection sites, and cells were also infiltrated to the liver and ascitic fluid. These cells expressed CD56, CD146, and Nestin strongly, but not other lineage-specific markers including CD133; however, the ratio of CD56(+)CD146(+) fraction in the injected stromal cells was below 0.1%. When the tumor cells were cultured in vitro, they exhibited spindle-shaped appearance, and doubling time was 12 to 16 hours. They expressed c-Myc, Klf4, Nanog, Sox2, and other molecules that are expressed in an immature somatic stem cell. These observations indicate that CD56(+)CD146(+) cell-fraction in bone marrow-stroma is very immature and highly proliferative. We are now analyzing biological characteristics of this specific cell-fraction, and determining the contribution to normal hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Regulatory Potential of miRNAs on CXCL12 Expression in Human Mesenchymal Stem Cells (MSC)
Authors: Illmer Thomas , Heder Carolin , Stölzel Friedrich , Fierro Fernando , Ehninger Gerhard 
Published: October 2019

Abstract
Abstract 1332
CXCL12 is the major CXC chemokine produced by various cell types of the hematopoietic niche. It is well known for it‘s regulatory potential in physiological processes like stem cell mobilization and homing. In addition it participates in regulation of proliferation and survival of cells with expression of it‘s receptor CXCR4. Thus, variable CXCL12 levels can influence a multitude of mechanisms associated with stem cell migration, homing and proliferation, e.g. during stem cell mobilisation CXCL12 levels are down regulated in MSC and osteoblasts thereby enabling conversion of CD34 positive cells into the circulation. The regulatory mechanisms of this process are however incompletely understood. In a systematic approach we investigated whether miRNAs are able to mediate CXCL12 regulation in human MSC. We transfected the immortalized human MSC cell line SCP-1 with a library consisting of 702 different pre-miRNAs. Subsequently, CXCL12 level determination by ELISA revealed a total of 27 miRNAs downregulating CXCL12 and 34 miRNAs which upregulated CXCL-12. Since the most important effect of miRNAs is the interaction with the 3 ‘UTR of target genes we further validated our candidate miRNAs with a luciferase assay using the 3 ‘UTR of CXCL12 transcript variant 2. Here, 8 miRNAs (miR-23, 130b, 135a, 200b, 200 c, 216, 222 and 602) were shown to interact significantly with CXCL12. Next, expression of candidate miRNAs was determined in 46 human resting primary MSC samples which proved strongest expression of miR-23 in MSC, miR-130b and 222 are expressed at lower but detectable levels, whereas the other candidate miRNAs showed no expression in human MSC. Subsequently, transfection of pre-miR-23 molecules in primary human MSC reduced both CXCL12 protein values and the attraction of migrating cells in a transwell assay.
In conclusion, using a large scale library transfection approach we identified miR-23 as an important regulator of CXCL-12 In human MSC.

Disclosures:
No relevant conflicts of interest to declare.
Ionizing Radiation Induces Hematopoietic Stem Cell Senescence and Long-Term Bone Marrow Suppression in a p16Ink4a/Arf-Independent manner
Authors: Shao Lijian , Feng Wei , Li Hongliang , Wang Yong , Sharpless Norman , Zhou Daohong 
Published: October 2019

Abstract
Abstract 1345
Many patients receiving chemotherapy and/or ionizing radiation (IR) develop residual (or long-term) bone marrow (BM) injury that can not only limit the success of cancer treatment but also adversely affect their quality of life. Although residual BM injury has been largely attributed to the induction of hematopoietic stem cell (HSC) senescence, neither the molecular mechanisms by which chemotherapy and/or IR induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate the injury. The Ink4a-Arf locus encodes two important tumor suppressors, p16Ink4a (p16) and Arf. Both of them have been implicated in mediating the induction of cellular senscence in a variety of cells including HSCs. Therefore, we examined the role of p16 and/or Arf in IR-induced HSC senescence and long-term BM suppression using a total body irradiation (TBI) mouse model. The results from our studies show that exposure of wild-type (WT) mice to a sublethal dose (6 Gy) of TBI induces HSC senescence and long-term BM suppression. The induction of HSC senescence is not associated with a reduction in telemore length in HSCs and their progeny, but is associated with significant increases in the production of reactive oxygen species (ROS), the expression of p16 and Arf mRNA, and the activity of senescence-associated β-galacotosidase (SA-β-gal) in HSCs. However, genetical deletion of Ink4a and/or Arf has no effect on TBI-induced HSC senescence, as HSCs from the Ink4a and/or Arf knockout mice after exposure to TBI exhibit similar changes as those seen in the cells from irradiated WT mice in comparison with the cells from un-irradiated mice with correspondent genotypes. In addition, TBI-induced long-term BM suppression is also not attenuated by the deletion of the Ink4a and/or Arf genes. These findings suggest that IR induces HSC senescence and long-term BM suppression in a p16Ink4a/Arf-independent manner.

Disclosures:
No relevant conflicts of interest to declare.
Runx1 Is Required for Cbfb-MYH11 Activity During Primitive Hematopoiesis
Authors: Hyde R. Katherine , Zhao Ling , Alemu Lemlem , Liu Pu Paul 
Published: October 2019

Abstract
Abstract 1357
Inversion of chromosome 16 (Inv(16)) is found in nearly all patients with acute myeloid leukemia (AML) subtype M4 with eosinophilia. Inv(16) results in the fusion of the transcription factor gene CBFB, and the MYH11 gene, which encodes Smooth Muscle Myosin Heavy Chain (SMMHC). It has been proposed that the Inv(16) fusion protein, CBFβ-SMMHC, initiates leukemogenesis through repression of the transcription factor RUNX1. However, we recently found that CBFβ-SMMHC also has activities that are independent of Runx1 repression. Mice expressing a knockin allele of Cbfb-MYH11 (Cbfb+/MYH11) show a severe differentiation defect during primitive hematopoiesis. Interestingly, this severe defect is not seen in mice homozygous for a null allele of Runx1 (Runx1−/−). We also found genes deregulated uniquely in Cbfb+/MYH11 embryos were also expressed in leukemic cells from mice and Inv(16) patients. These findings imply that CBFβ-SMMHC's Runx1 repression independent activities play a role during leukemogenesis.
There are two potential models to describe these observations: a Runx1-independent model and a Runx1-dependent model. In the first model, Cbfb-MYH11 initiates leukemia through a novel mechanism that does not involve Runx1. In the second model, Cbfb-MYH11 cooperates with Runx1, but rather than repressing Runx1 activity, Cbfb-MYH11 alters it. To test these two models, we generated mice that express a conditional allele of Cbfb-MYH11 on a Runx1−/− background. Surprisingly, we found a complete rescue of the Cbfb-MYH11 induced primitive hematopoietic defect: the primitive blood from Runx1−/−; Cbfb+/MYH11 embryos was indistinguishable from Runx1−/−; Cbfb+/+ embryos. Preliminary data showed that Cbfb-MYH11 expression in the embryos was not affected by the loss of Runx1, indicating that Runx1 is not regulating Cbfb-MYH11 expression, but is required for its activity. Interestingly, another allele of Runx1 in which Runx1 is fused to the β-galactosidase gene, lacZ (Runx1lzd) and that has been shown to have dominant negative activities (Nancy Speck, personal communication) also rescued the primitive blood defect in the Cbfb+/MYH11 embryos. This demonstrates the Cbfb-MYH11 requires Runx1 activity for its primitive hematopoietic differentiation defect. Based on this finding, we hypothesize that Cbfb-MYH11 may have a similar requirement for Runx1 activity during leukemogenesis. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb+/MYH11; Runx1+/+ littermates.

Disclosures:
No relevant conflicts of interest to declare.
Anthracycline Treatment Induces Extracellular Calreticulin in Acute Myeloid Leukemia
Authors: Mans Sarah , Schaubitzer Kerstin , Shan Deborah , Mueller Beatrice U , Pabst Thomas 
Published: October 2019

Abstract
Abstract 1376
The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is blocked by the mRNA binding protein calreticulin (CRT) thereby affecting myeloid differentiation. We previously reported that the unfolded protein response (UPR) is activated in 24.5% of AML patients, involving the up-regulation of endoplasmatic reticulum (ER) stressors, such as CRT. In addition, anthracycline treatment was shown to trigger the UPR and to specifically induce CRT cell membrane surface expression in some solid tumor types. This ecto-CRT is specifically targeting dendritic cells and initiates immunogenic cell death of cancer cells by acting as an “eat-me” signal. However, UPR induced translocation of CRT in leukemogenesis has hardly been investigated so far.
This study aimed at analyzing anthracycline induced CRT exposure and extracellular release of CRT in leukemic cells as well as at elucidating the candidate pathways involved such as the UPR transducers PERK, ATF6 and IRE1.
In leukemic cell lines, we observed that anthracycline treatment was inducing CRT expression on the cell membrane surface within 2 hours. This effect was caused by intracellular translocation, but not de novo synthesis. Exposure of CRT was decreased by Brefeldin A, which blocks the vesicle transport from the ER to the Golgi apparatus within 2 hours of treatment, suggesting that exocytosis is critical for CRT delocalization. By specific inhibition of the PERK pathway of the UPR using Salubrinal, we observed reduced CRT expression on the cell membrane. In contrast, blocking the ATF6 or the IRE1 pathway did not affect CRT exposure.
Mean CRT levels on the cell membrane of blasts from 53 AML patients at diagnosis were significantly (21-fold; p<0.0005) increased compared to CRT levels of myeloid cells from healthy volunteers (n=75). Using an ELISA assay investigating serum from 104 AML patients at diagnosis and 86 healthy volunteers, we found that extracellular CRT was significantly (2.2-fold; p<0.0001) increased in AML patients compared to healthy controls. We observed a more favorable outcome in AML patients with increased CRT expression on the cell membrane, as well as in patients with higher levels of extracellular CRT in the serum. Finally, we identified a strong correlation between CRT serum levels and CRT expression on the cell surface of leukemic cells. Ongoing studies aim at elucidating the mechanisms of CRT secretion from the cell membrane to the serum.
In conclusion, CRT translocation to the cell membrane and increasing levels of secreted CRT in the serum represent early events of anthracycline treatment in leukemic cells, and both are associated with a more favorable outcome in AML patients.

Disclosures:
No relevant conflicts of interest to declare.
Mechanisms of Drug Resistance Against JAK2 Inhibitors
Authors: Azam Mohammad 
Published: October 2019

Abstract 1426
The discovery of activating somatic mutations of JAK2 kinase as a contributing factor in the pathogenesis of myeloproliferative neoplasms (MPNs) —polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF)—prompted the development of JAK2-targeted therapies for these diseases1–3. Therapy with JAK2 inhibitors (INCB018424 and TG101348) induces rapid and marked reductions in spleen size and can lead to remarkable improvements in constitutional symptoms and overall quality of life suggesting that therapeutic targeting of JAK2 holds great promise for the treatment of JAK2 mediated disorders. It is likely that these two clinical agents will soon be approved by the FDA for the treatment of these diseases. Given our experience in development of resistance in tyrosine kinase inhibitor therapy (TKI) especially in the context of BCR/ABL targeting by Gleevec, we anticipate that resistance to JAK2 inhibitors will develop as well. To understand the mechanism of resistance against JAK2 targeted therapies, we performed mutagenesis studies and in vitro screening for resistance as previously reported for selection of resistance mutation in BCR/ABL (Azam et. al. (2003), Cell and Azam et. al. (2006), PNAS.).
We sequenced the resistant clones isolated from INCB018424 resistant screening and identified 125 different amino substitutions spanning across FERM, SH2, pseudokinase and kinase domains. Frequency of resistant mutations demonstrated several major hot-spots clustered in the FERM, pseudokinase and kinase domain. We observed sixty different mutations in the kinase domain that clustered into eight major hot-spots with several minor-spots in regulatory regions including the hinge region, catalytic-loop, activation-loop and in the helical region of c-lobe. Structural modeling of these mutations suggests that the JAK2 kinase, like ABL kinase, is subject to auto-inhibited regulatory mechanisms and selects for drug resistant mutations from allosteric domains (FERM, SH2 and Pseudokinase domain). We anticipate that these results will be valuable in diagnosis of clinical resistance and in the identification and design of next generation small molecule JAK2 inhibitors

Disclosures:
No relevant conflicts of interest to declare.