bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secondary Malignancies in Mantle Cell Lymphoma
Authors: Talakokkla Sandhya , Mahatara Renuka , Welch Christine A , Nagarajan Arun 
Published: October 2019

Abstract
Background: Mantle cell lymphoma is a B cell lymphoma (CD5 +) which represents 5-10 % of Non-Hodgkin's lymphoma. Most patients have advanced disease at presentation and thus carry a poor prognosis. This type of uncommon malignant lymphoma has a distinct and recurring cytogenetic abnormality involving t(11, 14) q(13, 32). Although extra nodal involvement is common, not many cases of MCL having concomitant other malignancies are reported. We hypothesized that patients with MCL have chronic immunosuppression, comparable to chronic lymphocytic leukemia patients, and therefore are at risk for developing secondary malignancies similar to CLL patients. The aim of our study is to report the retrospective analysis of patients diagnosed with MCL and the associated secondary malignancies before or after the diagnosis of MCL.
Patients and methods: The records of 41 patients who presented with MCL to "The David Lee Cancer Center" at CAMC, WVU with MCL from 2000 - 2015 were retrospectively reviewed. The number of other malignancies presenting before or after the diagnosis of MCL were analyzed.
Results: The population with MCL was represented by 41, all Caucasian patients, which were 75.6% male (n = 31) and had an average age of 68.6 ± 11.3 years. Mean follow up for all patients was 23.9 ± 32.43 months. A total of 14.6% (n=6), 83.3% males, experienced a second primary. Within the second primaries 50.0% were GI cancers which included pancreatic cancer and cholangiocarcinoma while 16.7% were prostate cancer, diffuse large B-cell lymphoma or adenocarcinoma of lungs. The time to second primaries varied with 50% of the cases being diagnosed simultaneously with MCL, while 33.3% were diagnosed prior to MCL at an average time of 34.5 months, and 16.7% were diagnosed post MCL diagnosis at an average time of 18 months.
Conclusion:
Patients with MCL are typically CD5 positive, CD10 negative, and CD23 negative which make it different from other lymphomas like small cell lymphoma, B-cell CLL that are CD 23 positive. Rare cases of MCL may be CD5 negative or CD23 Positive. We hypothesized that MCL patients have an increased risk for developing secondary cancers due to their disease biology and from underlying chronic immunosuppression. Patients with MCL have twice the risk of developing secondary malignancies and an increased frequency of certain types of cancers such as cholangiocarcinoma & pancreatic cancer.

Disclosures
No relevant conflicts of interest to declare.
Therapeutic Efficacy of Anfibatide in a Murine Model of Thrombotic Thrombocytopenic Purpura
Authors: Zheng Liang , Mao Yingying , Li Mandy , Dai Xiangrong , Li Benjamin , Zheng X. Long 
Published: October 2019

Abstract
Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 activity. Plasma infusion or exchange is the mainstay of treatment. However, complications associated with the administration of blood products and with the placement of a central venous line remain to be a major concern. To address the unmet clinical need, we test hypothesis that anfibatide, a novel snake venom-derived glycoprotein (GP) Ib antagonist, may be efficacious in treating TTP. Here, we show that purified anfibatide from snake venom inhibited murine platelet agglutination induced by botrocetin in the presence of recombinant murine VWF in a concentration-dependent manner. At 6-9 micro grams per ml (final concentrations), anfibatide nearly completely abolished botrocetin-induced murine platelet agglutination (Fig. 1A). As expected, anfibatide at the same concentrations showed little or no inhibitory effect on the protease-activating receptor 4 (PAR4) agonist (AYPGKF)-induced murine platelet aggregation. In vivo, an intravenously infused radiolabeled anfibatide exhibited a half-life of 5-7 hours depending on the initial loading dose. This information provided pharmacokinetic basis for therapeutic strategy for TTP in the murine model. An infusion of purified anfibatide at the dose of 60-90 ng per gram of body weight twice daily significantly reduced the rate of thrombocytopenia in Adamts13-/- mice triggered by intravenous infusion of a bacterial toxin, i.e. shigatoxin-2 (Stx2) (250 pg per gram body weight). As shown, 5/5 (100%) Adamts13-/- mice after being challenged with Stx2 and but treated with normal saline (control) exhibited severe thrombocytopenia (defined by 50% reduction of platelet counts from the baseline). Similarly, treatment of Adamts13-/- mice with anfibatide at the dose of 30 ng per gram body weight twice daily had little or no effect in preventing thrombocytopenia. However, treatment of the same mice with anfibatide at doses of 60-90 ng per gram body weight twice daily resulted in significantly reduced rate of severe thrombocytopenia to 11%-37.5% (Fig. 1B). Our ongoing effort is to determine the efficacy of anfibatide in treatment of acquired TTP associated with inhibitors. We conclude that anfibatide when given at the optimal doses and interval can efficiently prevent Stx2-induced TTP syndrome in the murine model. These findings support a rationale for further development of anfibatide as a novel therapeutic for TTP in humans.

Disclosures
Li: 3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong: Employment. Dai:4Zhaoke Pharmaceutical Co. Limited, Hefei, Anhui, China: Employment. Li:3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong, China: Employment.
Exosome-Transferred APOC3-Ncrna Complex Mediates Iron Regulation in Beta-Thalassemia Major
Authors: Peng Ching-Tien 
Published: October 2019

Background: Exosomes are small membrane vesicles (50-90 mm in diameter) containing bioactive proteins and genetic materials that may be transferred to accept cells, resulting in potent biological effects in the circulatory system. Exosome-transferred APOC3-ncRNA may be a crucial function in iron regulation. However, the role in β-thalassemia major is remains unclear.
Aim: The aim of this study was to investigate how Exosome-transferred APOC3-ncRNA adapts to iron regulation in β-thalassemia major.
Design: Using Proteomics, RNA-sequencing and lncRNA Q-PCR array, we demonstrated expression of exosomes-transferred APOC3-ncRNAs in plasma of β-thalassemia major patients (n = 40). Identification of exosome by Dynamic light scattering (DLS), Flow cytometry and western blots. Lentiviral infection, ncRNAs transfection and in vitro studies revealed that knockdown or overexpression obviously increased or ameliorated exosomes-induced iron regulation. Bioinformatics analysis, luciferase assay and in vitro studies revealed that ncRNAs functioned as a repressing mediator and formed a feedback loop with ncRNAs and target gene to mediate iron regulation.
Results: We observed that ncRNA is differential expressed in the exosomes of β-thalassemia major. We analyzed the effects of ex vivo-derived exosomes for iron regulation by monocytes/hepatocytes model. In vitro model showed that exosomes were internalized by THP-1/HuH-7 cells, and regulates the IRP1/FPN/HAMP. In turn, Exosome-transferred APOC3-ncRNA mediates inflammatory cytokine expression and iron regulation via p38/pAKT/TRAF6/NF-kB pathway. Knockdown of APOC3-ncRNA reversed IRP1/FPN/HAMP expression.
Conclusions: This is the first example of extracellular APOC3/ncRNA regulating gene expression via circulatory exosomes-transferred to accept cells. Increased Exosome-transferred APOC3-ncRNA expression is one of the key factors that might have contributed to abnormal iron regulation in β-thalassemia major patients.

Disclosures
No relevant conflicts of interest to declare.
Single Cell RNA seq for Analysis of Cell Fate Decisions
Authors: Grimes H. Leighton , Harinder Singh , Olsson Andre , Salomonis Nathan , Aronow Bruce J. , Chaudhry Viren 
Published: October 2019

Abstract
Single-cell RNA-Seq has the potential to become a dominant approach in probing diverse and complex developmental compartments. Its unbiased and comprehensive nature could enable developmental ordering of cellular and regulatory gene hierarchies without prior knowledge. To test general utility we performed single-cell RNA-seq of murine hematopoietic progenitors focusing on the myeloid developmental hierarchy. Using novel unsupervised clustering analysis, ICDS, we correctly ordered known hierarchical states as well as revealed rare intermediates. Regulatory state analysis suggested that the transcription factors Gfi1 and Irf8 function antagonistically to control homeostatic neutrophil and macrophage production, respectively. This prediction was validated by complementary genetic and genomic experiments in granulocyte-macrophage progenitors. Using knock-in reporters for Gfi1 and Irf8 and clonogenic analyses coupled with single-cell RNA-seq we distinguished regulatory states of bi-potential progenitors from their lineage specifying or committed progeny. Thus single-cell RNA-Seq is a powerful developmental tool to characterize hierarchical and rare cellular states along with the regulators that control their dynamics.

Disclosures
No relevant conflicts of interest to declare.
PML-Rara Drives Acute Promyelocytic Leukemia Genesis By Enhanceosome Depletion Leading to 3D Chromatin Reorganization
Authors: Wang Ping , Tang Zhonghui , Zhang Hui , Ruan Yijun 
Published: October 2019

Abstract
Dynamic changes in chromatin looping structure are identified to reflect interactions of enhancers to target promoters. Such changes modulate hematopoietic lineage differentiation and are known to be mediated by some critical transcription factor, such as GATA1 and MYC. How chromatin topology changes in cancer such as leukemia is poorly understood on a global genomic view. Moreover, how oncogenic transcription factors, such as PML-RARA, AML1-ETO and others, manipulate gene regulation by globally altering the topology of chromatin, which contribute to leukemogenesis in acute myeloid leukemia (AML) remains to be described. Here we identified PML-RARA involved chromosomal interactions spanning hundreds of kilobases between promoters and distal regulatory elements. The RNAPII mediated chromatin connectivity between transcriptionally active genes and its distal regulatory elements were lost in the reorganized chromatin structure caused by PML-RARA, while the chromatin topological associated domain mediated by CTCF remained stable. PML-RARA mediated chromatin loops eliminated the occupancy of myeloid specific transcription factors (such as PU.1, IRF1 and CEBPB et al.) as well as coordinately general transcriptional factors (such as P300 and RNAPII) at the distal regulatory elements and respective promoters. Finally we show that interacting loci designated as super enhancers become depleted by PML-RARA looping to suppress myeloid differentiation regulomes. These distinct results based on 3D genome architecture uncover novel modes of how PML-RARA disrupts the regulome of myeloid differentiation to contribute to leukemogenesis. This study provides new insights and accessible tools to delineate the aberrant regulome originated from the chaotic gene regulatory interactions leading to cancer genesis.

Disclosures
No relevant conflicts of interest to declare.
Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy
Authors: Takam Kamga Paul , Dal Collo Giada , Cassaro Adriana , Adamo Annalisa , Gatti Alessandro , Carusone Roberta , Di Trapani Mariano , Midolo Martina , Resci Federica , Bonifacio Massimiliano , Krampera Mauro 
Published: October 2019

Abstract
Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents.
Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments.
Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively.
Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin.
Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes.

Disclosures
Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.
Expression of MPL in Leukemia Stem Cells and Its Role in Stemness Maintainance
Authors: Li Huan , Rao Qing , Yu Pei , Chen Shuying , Li Zheng , Xing Haiyan , Wang Min , Wang Jianxiang 
Published: October 2019

Abstract
Osteoblast cells play an important role in bone marrow niche. The interaction between osteoblast and leukemia cells promotes leukemia development, which is mediated by some cytokines including TPO. It has become evident that TPO-MPL signaling is essential for the quiescence and self-renewal of hematopoietic stem cells, however, its expression pattern and the role in leukemia stem cells have not been reported. This study was aimed to determine the expression of MPL in acute myeloid leukemia (AML) and investigate the role of MPL in the leukemia stem cells' quiescence, drug resistance and self renewal.
The expression levels of CD34, CD38 and MPL were detected by flow cytometry in bone marrow cells from 57 newly diagnoses AML patients. The correlation between MPL and CD34, CD38 expression in AML patients were analyzed. The results showed that expression of MPL in AML patients was higher significantly than that in 13 normal donors (P<0.05). Expression of MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P<0.01). MPL was higher expressed in CD34+ cells than that in CD34- cells significantly (P<0.0001). We also detected the expression of MPL in different populations of leukemia cells in AML1-ETO9a mouse leukemia model established in our lab. We found that the ratios of MPL positive cells in Lin-c-kit+ and Lin-c-kit+sca-1+ populations were significantly higher than that in total leukemia cells. In addition, in chemotherapy treated AML1-ETO9a mice, the proportion of Lin-c-kit+MPL+ leukemia cells were increased 23.5 folds than that in untreated leukemia mice, which indicates that MPL+Lin-c-kit+ LSCs population could be enriched by chemotherapy. Furthermore, MPL+ and MPL- cells in Lin-c-kit+ leukemia population were sorted by flow cytometry and the colony formation and quiescence state were determined. The results showed that MPL+Lin-c-kit+ cells produced significantly more colonies in the second round of colony formation (p<0.05) than MPL-Lin-c-kit+ cells. The G0 phase accumulation of MPL+Lin-c-kit+ cells was significantly higher than that of MPL-Lin-c-kit+ cells (p<0.01). Above results indicate that MPL+ leukemia cells display more clonogenic potential and maintain quiescence.
These data demonstrate that as a receptor of TPO, MPL is highly expressed in leukemia stem cells and MPL positive leukemia stem cells could be enriched by chemotherapy. MPL positive leukemia stem cells exhibit more clonogenic potential, quiescence and drug resistance. It suggests TPO-MPL mediated interaction of osteoblast and leukemia cells take a role in the stemness of leukemia stem cells.

Disclosures
No relevant conflicts of interest to declare.
RUNX1 Is a Candidate Transcriptional Effector in Juvenile Myelomonocytic Leukemia
Authors: Dorantes Acosta Elisa , Huang Hui , Garcia Sara P. , Stieglitz Elliot , Loh Mignon L. , Yuan G C , Cantor Alan B. 
Published: October 2019

Abstract
Juvenile Myelomonocytic Leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm. Mutations leading to constitutive activation of the non-receptor tyrosine phosphatase Shp2 (PTPN11) and/or RAS signaling are driver events. Yet, the key downstream transcriptional effectors of this aberrant signaling have not been defined. We previously found that Shp2 dephosphorylates the hematopoietic transcription factor RUNX1, leading to its activation. Overexpression of a mutant RUNX1 molecule that mimics constitutive RUNX1 tyrosine dephosphorylation in murine hematopoietic stem/progenitor cells led to massive expansion of myelomonocytic cells and growth under low cytokine conditions. Likewise, RUNX1 is known to be activated by extracellular signal-related kinase (ERK) mediated phosphorylation, which is downstream of activated RAS. To further investigate the role of RUNX1 in JMML, we analyzed gene expression datasets derived from CD34+ cells from JMML patients versus healthy controls. Thirty-four genes were down regulated and 13 were up regulated in the JMML versus control CD34+ cells using a cut-off of a log2-fold change > 0 and false-discovery rate (FDR) adjusted p-value ≤ 0.1. H3K27Ac and K3K4Me1 peaks located between -50 kb 5' of the transcriptional start site (TSS) to +50 kb 3' of the transcriptional end site (TES) of the differentially expressed genes were used to identify putative enhancer elements. DNA-binding factor motif enrichment analysis was then performed at these regions. This revealed a significant enrichment for RUNX DNA consensus binding sequences in the putative enhancer elements (p-value 8.05e-03). There was also significant enrichment for ETS family transcription factors, including Spi1 (PU.1) (p-values 1.94e-06 to 1.21e-03) and for Serum Response Factor (SRF) (p-value 1.11e-04). ETS factors frequently physically and functionally cooperate with RUNX transcription factors. Analysis of the proximal promoters showed only enrichment for ETS factors (p-value 2.42e-04 to 4.02e-03) and BATF::JUN (4.64e-03). Examination of ChIP-seq datasets for RUNX1 and PU.1 in CD34+ cells from healthy controls demonstrated that many of these putative sites are occupied by RUNX1, and some with PU.1. Overall, this data supports a model in which RUNX1 (and likely ETS factors) are important transcriptional effectors of aberrant cell signaling in JMML.

Disclosures
Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.
NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation
Authors: Xu Jie , Zhang Wu , Yan Xiaojing , Zhao Chen , Zhu Jiang , Chen Zhu , Chen Sai-Juan , Hu Jiong 
Published: October 2019

Abstract
NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor.

Disclosures
No relevant conflicts of interest to declare.
DNA Epigenome Editing Using Crispr-Cas Suntag-Directed DNMT3A
Authors: Huang Yung-Hsin , Jianzhong Su , Lei Yong , Gundry Michael C , Zhang Xiaotian , Jeong Mira , Li Wei , Goodell Margaret A 
Published: October 2019

Abstract
DNA methylation, an epigenetic modification, has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. To overcome this barrier, we utilized nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltranferase 3A (DNMT3A) (CRISPR-Cas SunTag-directed DNMT3A) to amplify local DNMT3A concentration and to methylate genomic sites of interest. Here, we demonstrated that CRISPR-Cas SunTag-directed DNMT3A not only dramatically increased CpG methylation but also, to our surprise, CpH (H =A or C or T) methylation at the HOXA5 lociin human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, CRISPR-Cas SunTag-directed DNMT3A was capable of methylating 4.5 kb genomic regions, surpassing previous targeted methylation tools whose activity is limited to 200bp. Using reduced representation bisulfite sequencing (RRBS) and RNA-seq, we concluded that CRISPR-Cas SunTag-directed DNMT3A methylated regions of interest without affecting global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship beteween DNA methylation and gene expression.

Disclosures
No relevant conflicts of interest to declare.