bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhancement of Natural Killer Cell Interferon-Gamma Production By the Derivatives of the Natural Product Phyllanthusmins Via TLR-Mediated NF-Kb and STAT3 Signaling Pathways
Authors: Chen Luxi , Yi Long , Ren Yuning , Zhang Jianying , Kinghorn A. Douglas , Caligiuri Michael A. , Yu Jianhua 
Published: October 2019

Abstract
Natural killer (NK) cells are known to play a pivotal role in regulating immune defense against tumors and viruses. However, very few studies describe the effects of natural products or their derivatives on NK cell function. We previously showed that the natural product lignan phyllanthusmin C enhances human NK cell IFN-gamma production. In this study, we successfully synthesized dozens of phyllanthusmin derivatives and screened their activities to enhance IFN-gamma production by NK cells. We found that three of these derivatives possessed capacities to induce IFN-gamma production by NK cells. Among them, derivative No. 17 had the highest efficacy and was significantly superior (P < 0.05) to that of the original phyllanthusmin C. These three phyllanthusmin derivatives had no adverse effect on human NK cell survival or proliferation. All derivatives alone or in combination with IL-12 or IL-15 induced human NK cell interferon (IFN)-gamma in comparison with the corresponding vehicle control or cytokine alone (P < 0.05). These derivatives stimulated IFN-gamma production in both CD56bright and CD56dim human NK cell subsets (P < 0.01). Mechanistically, immunoblotting assays and chromatin immunoprecipitation (ChIP) analyses combined with promoter-reporter luciferase assays revealed that these phyllanthusmin derivatives induced the phosphorylation of NF-κB and STAT3, resulting in their increased binding on theIFN G promoter, which was dependent on the Toll-like receptor (TLR) 1 and the TLR3 signaling pathways, respectively. STAT3 knockdown with lentivirus shRNA and inhibition of NF-kB signaling with a specific inhibitor (TPCK) significantly attenuated IFN-gamma production induced by these phyllanthusmin derivatives in human NK cells (P < 0.05). Blockade of TLR1 or TLR3 with their corresponding neutralizing antibodies nearly abolished activation of NF-κB or STAT3 as well as IFN-γ induction by the phyllanthusmin derivatives (P < 0.05). In conclusion, we have successfully synthesized and screened phyllanthusmin derivatives and identified three compounds that induce IFN-gamma production in human NK cells, one of which had optimal potential. The induction of human NK cell IFN-gamma production by phyllanthusmin derivatives is mediated by TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering their potential for use as agents for cancer prevention or treatment.

Disclosures
No relevant conflicts of interest to declare.
Human Chorionic Villus Stem Cells May Contribute to Endogenous Factor VIII Production during Gestation
Authors: Miguelino Maricel G. , Kumar Priyadarsini , Powell Jerry S. , Wang Aijun 
Published: October 2019

Abstract
INTRODUCTION: The placenta is an organ that plays a vital role in providing nutrition, elimination of metabolic products and gas exchange for the growing fetus. Major organs that play these roles in an adult are undergoing development during gestation. The liver, a metabolic organ with a variety of endocrine and exocrine functions including the synthesis of coagulation proteins, is the major site of hematopoiesis at 11-15 weeks during gestation. Production of coagulation proteins during gestation remains unclear. We investigated the potential of very early gestation human chorionic villus mesenchymal stem cells (PMSCs) for Factor VIII (FVIII) mRNA and production of biologically active FVIII.
METHODOLOGY: PMSCs were isolated from human placenta at 9-13 weeks of gestational age by tissue explant culture and characterized via flow cytometry for mesenchymal stem cell (MSC) markers. Tri-lineage differentiation of PMSCs was investigated by inducing PMSCs in different culture conditions. Polymerase chain reaction (RT-PCR) was performed on cell lysates using FVIII primers. Chromogenic assay was performed on culture medium of PMSCs and HUVECs (negative control) after 72 hours in culture. Human plasma was used as positive control.
RESULTS: PMSCs isolated from preterm human placenta were plastic adherent, showed spindle-shaped morphology and demonstrated expression of MSC markers CD105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD31. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different culture conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs). PCR analysis revealed FVIII mRNA expression on PMSCs and was negative on HUVECs. Chromogenic assay showed ~0.4 U/ml on PMSCs and ~0.1 U/ml in HUVECs.
CONCLUSION: Here we report that early gestation PMSCs express and secrete biologically active FVIII. Further studies using different gestational ages of the placenta is needed to understand the potential contribution of chorionic villus to FVIII production during gestation.

Disclosures
Powell: CSL Behring: Employment.
Delayed Major Bleeding in Critically Ill Trauma Patients
Authors: Castellucci Lana A , Shaw Joseph , Le Gal Gregoire , Hawken Stephen , McIntyre Lauralyn , Carrier Marc 
Published: October 2019

Abstract
Introduction: Trauma is the leading cause of death in persons under the age of 40 years and hemorrhage is responsible for at least 40% of trauma related deaths within the first 24 hours following injury, with up to half of patients dying before arriving to hospital. During the last several decades, the focus of research in bleeding trauma patients has been targeted at the immediate post-injury period with little emphasis on the potential for delayed hemorrhage in survivors of the first 24 hours.
Methods: A retrospective review of critically ill trauma patients admitted at a tertiary care center between September 2010 and December 2011 was performed to determine the incidence of delayed major bleeding. Delayed major bleeding was defined as bleeding that occurred more than 24 hours after presentation to the emergency department. The HEME bleeding assessment tool was used to identify delayed major bleeding events. Cox proportional hazards multivariate analysis was performed to assess for risk factors associated with delayed major bleeding events.
Results: One hundred and fifty critically ill trauma patients were included and the incidence of delayed major bleeding was 44%. Predictors that were statistically significantly associated with delayed major bleeding included: male gender (hazard ratio (HR): 2.22; 95% confidence interval (CI):1.15-4.17), pre-injury use of the antiplatelet agents aspirin and/or clopidogrel (HR: 2.11; 95%CI: 1.07-4.18), presence of intracranial bleeding (HR: 3.05; 95%CI: 1.70-5.47), higher injury severity scores (HR: 1.12; 95%CI: 1.03-1.22), requirement of massive transfusion (HR: 2.53; 95%CI: 1.05-6.08), and low pH values (HR: 1.41; 95%CI: 1.08-1.84). Use of anticoagulant prophylaxis was not associated with delayed major bleeding.
Conclusions: The rate of delayed major bleeding in this cohort of critically ill trauma patients is high. Prospective studies are needed to confirm the reported associations of gender, antecedent antiplatelet drugs, intracranial bleeding, injury severity scores, massive transfusion, and acidosis with delayed major bleeding.

Disclosures
Carrier: LEO Pharma: Consultancy, Research Funding; Pfizer: Consultancy; Bayer: Consultancy; BMS: Research Funding.
Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells
Authors: Sun Lei 
Published: October 2019

Abstract
The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells.

Disclosures
No relevant conflicts of interest to declare.
Regulation of Hematopoietic Stem Cell Function By the Histone H2A Deubiquitinase Usp16
Authors: Gu Yue , Yang Wei , Jones Amanda , Liu Shanrun , Dai Qian , Swindle C Scott , Ryan Thomas , Townes Tim M. , Klug Christopher , Wang Hengbin 
Published: October 2019

Abstract
Epigenetic mechanism plays important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of Polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it was unclear which H2A deubiquitinase pairs with PRC1 to control H2A ubiquitination (ubH2A) level in vivo and regulates hematopoiesis. Here we investigated the function of Usp16 in mouse hematopoiesis. Deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and mouse lethality. Usp16 deletion was associated with a dramatic reduction of progenitor cell populations and unchanged HSC number, revealing a critical role for Usp16 in HSC differentiation. The HSC differentiation defect was correlated with a great reduction of G1 phase cell population. RNA-seq and RT-qPCR studies revealed that Usp16 regulates the expression of many genes associated with HSC differentiation and self-renewal including cell cycle regulator p21. Significantly, knockdown of p21 largely rescued the undifferentiated phenotype of Usp16 deleted HSCs. Usp16 binds to regions flanking transcription start site and knockdown of PRC1 subunits, which reduced ubH2A levels, largely rescued the altered gene expression pattern associated with Usp16 deletion. Therefore, these studies identified Usp16 as the H2A deubiquitinase that pairs with PRC1 to regulate hematopoiesis in vivo and revealed that Usp16 regulates HSC function by affecting HSC cell cycle progression.

Disclosures
No relevant conflicts of interest to declare.
B-Lymphoid Transcription Factors Restrict Glycolytic Energy Supply for Oncogenic Signaling
Authors: Chan Lai N , Chen Zhengshan , Braas Daniel , Geng Huimin , Hurtz Christian , Shojaee Seyedmehdi , Cazzaniga Valeria , Ng Carina , Ernst Thomas , Hochhaus Andreas , Kornblau Steven M , Cazzaniga Giovanni , Liu Grace , Milne Thomas , Koeffler H. Phillip , Armstrong Scott A. , Dickins Ross Alexander , Yamamoto Keith R , Graeber Thomas G , Muschen Markus 
Published: October 2019

Abstract
Oncogenic lesions in hematopoietic progenitor cells give rise to B-cell or myeloid malignancies. While often transformed by the same oncogenes, B-cell and myeloid leukemias markedly differ in biological and clinical characteristics. Our metabolic analyses revealed that B-cell-unlike myeloid-leukemia cells are massively restricted in their glycolytic capacity. Low glycolytic reserves in B cells resulted in a state of chronic energy depletion and engaged the energy sensor LKB1-AMPK. Myeloid cells strongly activated glucose transport through insulin receptor (INSR)-AKT signaling and lacked activity of LKB1-AMPK, reflecting energy abundance. Conversely, B-cells lacked INSR-AKT signaling and were critically dependent on LKB1-AMPK-mediated glucose uptake. Cre-mediated deletion of Lkb1 caused acute glycolytic exhaustion and cell death in B-lineage but increased glycolysis, energy levels and proliferation in myeloid leukemia. C/EBPa-mediated conversion of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion and restored glycolysis, energy levels and survival of B→myeloid reprogrammed cells. In >80% of B-lineage leukemia cases, we found genetic lesions of transcription factors (e.g. deletion of PAX5, IKZF1, rearrangement of MLL) that caused a B→myeloid lineage shift. While previously of unknown functional significance, these lesions relieved B-cell-specific transcriptional repression of molecules that mediate glucose uptake and utilization (INSR, GLUT1, HK2, G6PD) and amplified glycolytic energy supply for transforming oncogenes. Likewise, glucocorticoid receptor (NR3C1)-mediated inhibition of glucose uptake and glycolysis was strictly dependent on a B-lymphoid transcriptional program. B→myeloid lineage conversion abolished NR3C1 expression and activity, which provides a mechanistic explanation for the empiric finding that glucocorticoids are highly active in the treatment of B-cell-but not myeloid malignancies. In conclusion, B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation and reveals LKB1-AMPK as a novel target for the treatment of human B-lineage leukemia.

Disclosures
No relevant conflicts of interest to declare.
Incidence of Allergic Reactions to Pegaspargase (PEG) Administered Intramuscularly Versus Intravenously (IM vs. IV) in Children and Young Adults with High Risk B-Lymphoblastic Leukemia (HR B-ALL): Results of Children's Oncology Group (COG) Studies AALL0232/AALL1131
Authors: Salzer Wanda , Burke Michael J. , Larsen Eric C. , Chen Si , Gore Lia , Hilden Joanne M. , Loh Mignon L. , Raetz Elizabeth , Winick Naomi J. , Carroll William L. , Devidas Meenakshi , Hunger Stephen P 
Published: October 2019

Abstract
Introduction: Asparaginase is a vital component of multi-agent chemotherapy for patients with HR B-ALL, but rates of allergic reactions following administration of PEG range from 3-24%. Similar or increased rates of allergic reactions have been reported following IV vs. IM PEG when administered on varying chemotherapy backbones. The COG trials recently changed from IM to IV administration of PEG. We report the rate of allergic reactions/anaphylaxis (Grade 3-4) following IM vs. IV PEG on sequential trials for newly diagnosed patients with HR B-ALL.
Methods: Patients 1-30 years of age with newly diagnosed HR B-ALL were eligible for the sequential COG studies, AALL0232 and AALL1131. We limited this analysis to non-Down Syndrome patients who received highly similar chemotherapy regimens prior to maintenance therapy, including a 4-drug Induction with 14 days of dexamethasone or 28 days of prednisone, the COG augmented Berlin-Frankfurt-Munster (BFM) Consolidation, High Dose Methotrexate during Interim Maintenance, and Delayed Intensification (DI). Patients received a total of 5 doses of PEG (2,500 units/m2/dose) administered IM (AALL0232) or IV (AALL1131) during these phases of therapy: Induction (n=1), Consolidation (n=2), DI (n=2). Chi square analysis of the rates of Grade 3-4 allergic reactions/anaphylaxis (Common Terminology Criteria for Adverse Events v4.0) following IM vs. IV PEG was performed.
Results: Grade 3-4 allergic reactions/anaphylaxis rates in patients receiving IM vs. IV PEG (Table) were 0.54% vs. 0.22% during Induction (p=0.10), 14.4% vs. 12.64% during Consolidation (p=0.18), and 2.1% vs. 0.97% during DI (p=0.07).
Conclusion: Rates of Grade 3-4 allergic reactions/anaphylaxis were similar following IM vs. IV administration of PEG in patients with newly diagnosed HR B-ALL when treated on sequential COG studies, further establishing the safety of delivering IV PEG.
Table 1. Allergic Reactions Following IM vs IV PEG Allergic reaction/Anaphylaxis (Grade 3-4) AALL0232(IM PEG) AALL1131(IV PEG) p-value(Chi square test) Induction 15/2771 (0.54%) 4/1797 (0.22%) 0.10 Consolidation 342/2369 (14.4%) 114/902 (12.64%) 0.18 DI 22/1060 (2.1%) 7/715 (0.97%) 0.07

Disclosures
Hunger: Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership.
Whole Exome Sequencing of FLT3-ITD Sorafenib-Resistant Acute Myeloid Leukaemia
Authors: Lam Stephen Sze Yuen , Cher Chae Yin , Ng Nelson K. L. , Man Cheuk Him , Leung Anskar Y.H. 
Published: October 2019

Abstract
Acute myeloid leukaemia (AML) is a heterogeneous disease characterised by an abnormal increase in myeloblasts. Current treatment protocol employs the standard "7+3" chemotherapy regimen complemented with haematopoietic stem cell transplantation. However overall cure rate remains at 30-40%. Targeted therapy, especially the use of tyrosine kinase inhibitors, has been the main focus in the pursuit of an alternative solution. Internal tandem duplication at the juxatmembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) was found in around 30% of AML cases, conferring unfavourable treatment outcome and prognosis. Sorafenib, and other FLT3 inhibitors, successfully induced remission or partial remission in most of the relapsed/refractory FLT3-ITD AML patients (CR/CRi/nCR = 82.4%, n=34 in our sorafenib monotherapy cohort). Although consolidation with combination of sorafenib and chemotherapy or hypomethylating agents was reported to significantly extend the remission period, all patients relapsed with drug resistance. Mutations at the tyrosine kinase domain (TKD) were reported to alter the binding affinity of the drugs to the receptor and hence generate resistance. However, they were found only in around 25% of the patients while other non-mutational mechanisms of resistance have also been reported. Their relative importance in patients remains uncertain. We hypothesised that there is an emergence of drug-resistant clones acquiring new mutations or pathways to develop the resistance.
We have performed whole exome sequencing (HiSeq pair-end sequencing of 101 bp) for sorafenib-sensitive and sorafenib-resistant samples obtained from 8 FLT3-ITD relapsed/refractory AML patients before sorafenib treatment and at relapse with drug resistance. Genomic content of the bulk population was observed to change drastically among the 8 patients, with disappearance of single nucleotide polymorphisms (SNPs) and emergence of new SNPs, suggesting the emergence of drug-resistant clones. Emergence of TKD D835Y mutation was observed in 3 patients with variant allele frequencies (VAF) at 11%, 44% and 50% that were undetectable in the sorafenib-sensitive samples. Mutations at other reported sites of TKD were not found. Filtering of the generated list of single nucleotide changes and insertion/deletions (indels) to sort out emerging non-synonymous mutations with high VAF at resistant samples but undetectable in the sensitive samples was done. They include SLC15A1, CDC27, NADKD1, DGAT2, UBXN11, TFR2 and TTBK1, potentially implicated in sorafenib resistance. They are involved in different pathways, including membrane transporter, cell cycle regulation and energy metabolism. Further functional validation of their role in sorafenib resistance is required to understand the mechanisms involved.

Disclosures
No relevant conflicts of interest to declare.
Chemoresistant Leukemia-Initiating Cell Expansion Is Inhibited By Targeting Oncogenic Self-Renewal
Authors: Perry John M. , Roy Anuradha , Lu Xiuling , He Xi C. , Tao Fang , Zhao Meng , Qian Pengxu , Li Linheng 
Published: October 2019

Abstract
Leukemia is often maintained by a relatively rare subset of tumor-initiating cells, which may be resistant to traditional chemotherapy. Targeting these leukemic stem cells is expected to eliminate or reduce acquired resistance to chemotherapy and disease recurrence, but this goal has been elusive. It's also not clear whether eliminating the 'root' of cancer is sufficient or whether targeting both 'root' and 'branch' (i.e. LSCs and their bulk progeny) is necessary. To test this, we established and characterized a mouse model with clearly defined populations of rare leukemic stem cells (LSCs), abundant leukemic blast cell progeny, and normal hematopoietic stem/progenitor cells (HSPCs). This was achieved by simultaneously activating the Wnt/β-catenin and PI3K/Akt signaling pathways in HSPCs to drive stem cell self-renewal-resulting in successive expansion of HSPCs, LSCs, and leukemic blasts. Functional analysis showed a 1,300-fold increase in leukemia-initiating activity of sorted LSCs compared to bulk blast cells or sorted HSPCs. Since Akt activates β-catenin by phosphorylation at serine 552 and thus represents a molecular link mediating the synergistic, self-renewal promoting activity of these pathways, we sought to specifically target pS552-β-catenin. Unexpectedly, high-throughput screening (HTS) and subsequent validation assays found that doxorubicin (DXR) inhibits pS552-β-catenin with minimal effects on total β-catenin.
DXR exhibits the broadest spectrum of anti-cancer activity known and has been employed as a standard chemotherapeutic agent for decades, but severe side-effects limit its use. We found that while standard chemotherapeutic treatment reduced the bulk leukemic blast cells as expected, it also induced pS552-β-catenin uniquely in LSCs and stimulated LSC expansion. Using long-circulating nanoparticles (NanoDXR) to deliver very low, sustained doses of DXR, we repurposed DXR as a targeted therapy for pS552-β-catenin inhibition rather than a broadly cytotoxic agent. In vivo, this treatment reduced pS552-β-catenin levels in LSCs, prevented LSC expansion, essentially eliminated LSC tumorigenic activity in transplant recipients, and was accompanied by recovery of HSPCs and substantially increased median survival from 44.5 days to 139 days.
Our data reveals a dynamic relationship between rare LSCs and bulk leukemic blast cells in their response to cytotoxic chemotherapy and show how oncogenic self-renewal in chemoresistant LSCs can be targeted while sparing normal HSPCs. In distinguishing the unique properties of LSCs and their progeny, we find that both populations must be differentially targeted at both the 'root' and 'branch' of leukemia and can be effectively reduced or eliminated while allowing for recovery of normal HSPCs.

Disclosures
No relevant conflicts of interest to declare.