bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using High-Throughput Sequencing of Red Blood Cells and Platelets to Identify Micrornas Associated with Hematopoiesis.
Authors: Doss Jennifer , Jima Dereje , Voora Deepak , Dave Sandeep , Chi Jen-Tsan Ashley 
Published: October 2019

Abstract
Abstract 2326
Human mature red blood cells (RBC) and platelets are both terminally differentiated cells lacking nuclei. However, these two cell types do possess a diverse and abundant set of microRNAs (miRNAs), a set of small, non-coding RNAs acting as post-transcriptional regulators. To identify novel microRNAs associated with differentiation of RBCs and platelets from common progenitors, we performed high-throughput sequencing of these differentiated cells types. In particular, these accessible cells may prove valuable to identify disease biomarkers.
We identified an unbiased set of both known and novel microRNAs by preparing small RNA libraries for application to the Illumina GAII high-throughput sequencing platform. We used a modified version of the probabilistic modeling algorithm, miRDeep (Friedländer 2008), to identify many novel and known microRNAs. Genomic loci that overlapped with miRNAs described in miRBase were identified as known miRNAs. The remaining genomic loci were identified as encoding candidate novel miRNAs. In RBCs we identified 253 predicted miRNA precursor loci, with 226 miRNA precursor loci annotated in miRBase (known miRNAs), whereas the remaining 27 precursor loci were identified as novel miRNAs. In platelets we identified 566 predicted miRNA precursor loci, with 488 known miRNAs and 78 novel miRNAs. Other small RNAs that did not pass miRDeep criteria were also analyzed. One of the most abundant RNA sequences in the RBC sample consisted of a distinct fragment of Ro-associated Y4 RNA (hY4). Y RNAs have been shown to be involved in chromosomal DNA replication, and Y1 and Y4 have been shown to be present in mature erythrocytes (O'Brien 1990). These distinct non-coding RNAs may possess a unique role in erythroid cell expansion.
In addition, we assessed dynamic changes in the expression level of several selected microRNAs during human erythropoiesis. We are currently investigating relevant targets and regulatory functions of these microRNAs during erythropoiesis and platelet development. This global analysis will enhance our understanding of events dictating red cell and platelet maintenance and development.

Disclosures:
No relevant conflicts of interest to declare.
Long Noncoding RNAs in Erythropoiesis and Megakaryopoiesis.
Authors: Paralkar Vikram R , Mishra Tejaswini , Luan Jing , Yao Yu , Konuthula Neeraja , Bodine David M. , Hardison Ross C , Weiss Mitchell J. 
Published: October 2019

Abstract
Abstract 2331
Lnc (long noncoding) RNAs are RNA transcripts greater than 200nt that regulate gene expression independent of protein coding potential. It is estimated that thousands of lncRNAs play vital roles in diverse cellular processes and are involved in numerous diseases, including cancer. We hypothesize that multiple lncRNAs regulate erythrocyte and megakaryocyte formation by modulating gene expression. To identify lncRNAs in erythro-megakaryopoiesis, we purified two biological replicates each of murine Ter119+ erythroblasts, CD41+ megakaryocytes and bipotential megakaryocyte-erythroid progenitors (MEPs) [Lin− Kit+, Sca1−, CD16/32−, CD34−]. We performed strand-specific, paired-end, 200nt-read-length deep sequencing (RNA-Seq) to a depth of ∼200 million reads per sample using the Illumina GAII platform. We used the Tophat and Cufflinks suite of bioinformatic tools to assemble and compare de-novo transcriptomes from these three cell types, producing a high-confidence set of 69,488 transcripts. We confirmed that the RNA-seq assemblies accurately reflect gene expression predicted from prior studies. For example, Ter119+ cells were highly enriched for key erythroid transcripts encoding globins, heme synthetic enzymes and specialized membrane proteins. Megakaryocytes expressed high levels of gene encoding lineage-specific integrins and platelet markers. MEPs expressed numerous progenitor genes including Gata2, Kit and Myc. Thus, the RNA-seq data are of high-quality and sufficient complexity to accurately represent erythroid, megakaryocytic and MEP transcriptomes. We used a series of Unix-based bioinformatic filtering tools to identify lncRNAs that are expressed in these transcriptomes. We identified 605 “stringent” lncRNAs, and 813 “potential noncoding” transcripts. 47% of the lncRNAs are novel unannotated transcripts, validating the use of de-novo RNA-Seq in unique cell populations for lncRNA discovery. Among the 605 “stringent” lncRNAs, 103 are erythroid-restricted, 133 are meg-restricted and 280 are MEP-restricted, consistent with reports that lncRNAs exhibit exquisitely cell-type specific expression. Current efforts are aimed at generating a more comprehensive map of lncRNA expression at specific stages of erythroid and megakaryocyte/platelet development, and performing high throughput functional screens to analyze currently identified lncRNAs. Our studies are beginning to define new layers of gene regulation in normal erythro-megakaryopoiesis and are relevant to the pathophysiology of related disorders including various anemias, myeloproliferative and myelodysplastic syndromes and leukemias.

Disclosures:
No relevant conflicts of interest to declare.
A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.
Authors: Senyuk Vitalyi , Zhang Yunyuan , Liu Yang , Ming Ming , Chen Jianjun , Rowley Janet D. , Nucifora Giuseppina , Qian Zhijian 
Published: October 2019

Abstract
Abstract 2332
MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis.
EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9.
It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells.

Disclosures:
No relevant conflicts of interest to declare.
Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).
Authors: Arabanian Laleh S. , Fierro Fernando , Poitz David M. , Strasser Ruth H. , Bornhaeuser Martin , Ehninger Gerhard , Stölzel Friedrich , Illmer Thomas 
Published: October 2019

Abstract
Abstract 2350
CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood.
From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs.
However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200.
We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules.
Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs.

Disclosures:
No relevant conflicts of interest to declare.
Immunosuppressive Therapy with Antithymocyte Globulin and Cyclosporine for Fulminant Aplastic Anemia.
Authors: Yagasaki Hiroshi , Watanabe Ken-ichiro , Kudo Kazuko , Tsuchida Masahiro , Shichino Hiroyuki , Morimoto Akira , Kobayashi Ryoji , Yabe Hiromasa , Kikuchi Akira , Ohga Shouichi , Ito Etsuro , Ohara Akira , Nakahata Tatsutoshi , Kojima Seiji 
Published: October 2019

Abstract
Abstract 2364

Background:
Survival after immunosuppressive therapy (IST) for aplastic anemia (AA) depends on disease severity, which is classified according to polymorphic neutrophil (PMN) count as follows: nonsevere AA (nSAA; PMN > 0.5 × 109/L); severe AA (SAA; PMN 0.2 – 0.5 × 109/L); and very severe AA (vSAA; PMN < 0.2 × 109/L). Although patients with fulminant AA, defined as PMN = 0 for > 2 weeks, generally have poor outcome, no data are available for a large cohort. Therefore, we evaluated the outcome of children with fulminant AA who were enrolled in an AA-97 study and received ATG, cyclosporine, and granulocyte colony-stimulating factor (G-CSF).

Patients and Methods:
A total of 288 children newly diagnosed with AA (median age, 8 years) were enrolled into an AA-97 study between 1997 and 2006 and treated with a combination of horse antithymocyte globulin (Lymphoglobulin®) and cyclosporine. For patients with PMN < 0.2 × 109/L, G-CSF was added. Patients were classified into 3 groups according to disease severity as follows: fulminant (PMN 0, n = 35); vSAA (PMN 0 – 0.2 × 109/L, n = 129); and SAA (PMN 0.2 – 0.5 × 109/L, n = 124). Of the 35 patients with fulminant AA, 8 had a history of acute hepatitis at the time of diagnosis. One case was considered to be drug-induced AA. The remaining 26 patients had unknown etiology. We evaluated the response rate (RR) at 6 months, 5-year overall survival (OS), and 5-year failure free survival (FFS). Treatment failure was defined as death due to any cause, a requirement for a secondary therapy, relapse, clonal evolution to myelodysplastic syndrome/acute myelocytic leukemia, and paroxysmal nocturnal hemoglobinuria.

Results:
The children with fulminant AA showed a significantly lower RR than those with vSAA and SAA (40.0%, 62.8%, and 64.5%, respectively; p =.023). Of the 20 non-responders in the fulminant AA group, 11 were rescued by alternative donor stem cell transplantation, and 5 achieved a late response after 6 months. As a result, no significant difference was noted in 5-year OS and 5-year FFS among the children with fulminant AA, vSAA, and SAA (88.5%/48.2%, 95.8%/65.1%, and 96.8%/68.1%, respectively).

Conclusion:
In consideration of favorable OS, the choice of IST is indicated in children with fluminant AA who lack an HLA-matched family donor.

Disclosures:
No relevant conflicts of interest to declare.
Disseminated Mycobacterium Avium Infection Accelerates Hematopoietic Stem Cell Exhaustion,
Authors: King Katherine Y , Zheng Yayun , Goodell Margaret A. 
Published: October 2019

Abstract
Abstract 2365

Background:
Anemia, leucopenia, or pancytopenia is a common presenting sign in a number of chronic infectious diseases such as HIV and atypical mycobacterial infection. In order to understand the pathophysiology of pancytopenia as a complication of chronic infection, we have investigated the responses of hematopoietic stem cells (HSCs) to mycobacterial infection. We previously found that HSCs proliferate in the setting of a Mycobacterium avium infection. We hypothesized that sustained infection impairs self-renewal in HSCs and leads to premature exhaustion of the stem cell compartment.

Method:
Testing HSC self-renewal is nontrivial and requires serial bone marrow transplantation to measure HSC totipotency over time. Three successive rounds of bone marrow transplantation were conducted using HSCs from mice with M. avium infection.

Results:
HSCs from infected mice were significantly less capable of sustaining trilineage peripheral blood production after serial transplantation compared to control HSCs. These findings demonstrate that self-renewal of HSCs is compromised during chronic infection. It follows that the total number of HSCs should decline with time during sustained infection. To test this, serial monthly infection with M. avium was conducted over an 8-month period. Repeatedly infected animals showed a gradual decrease in the number of committed lymphoid and myeloid progenitors and a significant depletion of HSCs in the bone marrow by 6 months following initial infection. Transcriptional profiling of HSCs from M. avium-infected animals by RNAseq revealed previously uncharacterized potential regulators of HSC self-renewal.

Conclusion:
Our results demonstrate that HSC self-renewal is compromised during chronic infection, leading to exhaustion of the stem cell compartment. These findings elucidate both the basis for and possible therapeutic approaches towards hematologic complications in patients with atypical mycobacterial infection, tuberculosis, HIV, and other chronic inflammatory conditions.

Disclosures:
No relevant conflicts of interest to declare.
ATM and 53BP1 Function in Nonoverlapping Pathways to Suppress T Lineage Lymphomagenesis.
Authors: Rybanska Ivana , Chou Jeremy , Prakash Mansi , Huso David , Franco Sonia 
Published: October 2019

Abstract
Abstract 2374

Background:
In response to DNA double-strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that promotes their detection, signaling and repair. Among DDR factors, the Ataxia-Telangiectasia Mutated (ATM) kinase and the 53BP1 adaptor localize to chromatin surrounding DSBs, where 53BP1 is thought to transduce a subset of ATM-dependent reactions. In addition, 53BP1 may have ATM-independent functions via its interactions with the MRE11/RAD50/NBS1 (MRN) complex and others to protect DNA ends from degradation and to promote synapsis of distant DSBs. Both ATM and 53BP1 are tumor suppressors in humans and mice, but whether the combined mutation may aggravate the DSB repair defect and/or accelerate oncogenesis in vivo is currently unknown.

Objectives:
1) To generate a compound mouse model deficient for both ATM and 53BP1 and investigate their genetic interaction during growth and development; 2) To determine whether 53BP1 plays ATM-independent functions in DSB repair and cell cycle checkpoint activation in primary thymocytes; 3) To determine the effect of 53BP1 deficiency on the penetrance and latency of T cell lymphomas in ATM−/− mice.

Design/Methods:
We have generated 53BP1/ATM double knock out (DKO) mice and assessed the kinetics of DSB signaling and repair in primary thymocytes and mature B cells. We have also followed cohorts of DKO and control mice for tumor development and evaluated mechanisms of tumorigenesis using a combination of molecular, biochemical and cytogenetic approaches.

Results:
When compared to ATM−/− mice, DKO mice showed no differences in organismal growth and development, or in lymphoid differentiation. However, DKO mice developed T cell lymphomas with higher penetrance and at younger age than ATM−/− mice, and their survival was markedly reduced (p=0.0001; log-rank test). Like ATM−/− lymphomas, DKO lymphomas contained clonal translocations with breakpoints at the TCRα/δ locus and amplification of upstream DNA sequences, indicating that 53BP1 deficiency markedly accelerates the acquisition of oncogenic translocations in an ATM−/− background. In primary cells, loss of 53BP1 further increases the frequency of ionizing irradiation (IR)-induced chromosomal aberrations in ATM−/− cells. We will present a model for how 53BP1 actions at DSBs suppress genomic instability in ATM-deficient thymocytes, including its effects on checkpoint activation and DSB repair.

Conclusion:
Our studies in a mouse model of T cell lymphomagenesis suggest that mutations in 53BP1 and ATM may be nonmutually exclusive in human lymphomas. The combined mutation may define a subgroup of patients with more aggressive hematological malignancies, and may have implications for their response to therapy with DNA damaging agents.

Disclosures:
No relevant conflicts of interest to declare.
PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.
Authors: Bera Rabindranath , Liang Der-Cherng , Chiu Ming-Chun , Huang Ying-Jung , Liang Sung-Tzu , Shih Lee-Yung 
Published: October 2019

Abstract
Abstract 2393
Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells.

Disclosures:
No relevant conflicts of interest to declare.
Interaction of c-Myb with p300 Is Required for the Induction of Acute Myeloid Leukemia by Human AML Oncogenes, and Represents a Potential Therapeutic Target.
Authors: Pattabiraman Diwakar R , Shakhbazov Konstantin , Barbier Valerie , McGirr Crystal , Krishnan Keerthana , Mukhopadhyay Pamela , Hawthorne Paula , Trezise Ann , Grimmond Sean , Papathanasiou Peter , Perkins Andrew C. , Levesque Jean-Pierre , Alexander Warren S. , Winkler Ingrid G , Gonda Thomas J 
Published: October 2019

Abstract
Abstract 2402
The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2).

These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach.
We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5).

Disclosures:
No relevant conflicts of interest to declare.