bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Duration of Erk1/2 Signaling Determines Neutrophil Versus Monocyte Cell Fates in Response to G-CSF and M-CSF
Authors: Hu Nan , Qiu Yaling , Dong Fan 
Published: October 2019

Abstract
Neutrophils and monocytes/macrophages are derived from hematopoietic stem cells that, through progressive commitment, give rise to granulocyte-monocyte progenitors that in turn develop into either neutrophils or monocytes/macrophages. Although it is well known that cell fate specification in the hematopoietic system depends on the expression of lineage specific transcription factors, the roles of cytokines in lineage commitment are less clear and two models have been proposed. According to the stochastic model, cell fate choice is stochastic and cytokines simply provide signals for the survival and proliferation of committed cells. The instructive model, on the other hand, proposes that cytokines stimulate intracellular signaling pathways that dictate cell fate decisions. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. Recent studies lend strong support to the roles of G-CSF and M-CSF in instructing lineage commitment. However, the signaling pathways that determine neutrophil versus monocyte cell fate following stimulation with G-CSF and M-CSF are unknown. Here we show that tyrosine (Y) 729 of the G-CSFR is involved in transducing signals that specify neutrophil cell fate. Substitution of Y729 with phenylalanine (F) results in monocytic differentiation in response to G-CSF in murine myeloid 32D and multipotent FDCP-mix A4 cells. G-CSF stimulated activation of Erk1/2 was prolonged in cells expressing G-CSFR Y729F mutant. Significantly, treatment of cells with Mek1/2 inhibitors U0126 or PD0325901 rescued neutrophilic differentiation. M-CSF has been shown to induce prolonged activation of Erk1/2, which is required for monocytic differentiation. Interestingly, the Mek1/2 inhibitors also promoted neutrophil cell fate at the expense of monocytic development in lineage marker negative (Lin-) primary bone marrow cells cultured in M-CSF. We further demonstrate that prolonged activation of Erk1/2 was associated with augmented activation of c-Fos and Egr1, both of which have previously been shown to promote monocytic development. Consistent with this, knockdown of c-Fos or Egr1 redirected 32D cells expressing G-CSFR Y729F mutant to develop into neutrophils in response to G-CSF. We propose that M-CSF stimulates more sustained activation of Erk1/2 than G-CSF does and that the duration of Erk1/2 signaling regulates neutrophil versus monocyte cell fate choices, likely through altering the activation statuses of c-Fos and Egr1.

Disclosures
No relevant conflicts of interest to declare.
Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice
Authors: Saito Yasuyuki , Ellegast Jana M. , Müller Rouven , Flavell Richard A. , Manz Markus G. 
Published: October 2019

Abstract
Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014).
To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice.
Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals.

Disclosures
No relevant conflicts of interest to declare.
Advanced Stage Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) in Children and Adolescents : Retrospective Study from Sfce and Ukccssg
Authors: Gaelle Roques , Shankar Ananth , Leblanc Thierry , Schmitt Claudine , Hayward Janis , Pacquement Helene , Lambilliotte Anne , Aladjidi Nathalie , gorde-Grosjean Stephanie , Martin Hewitt , Haouy Stephanie , Abbou Samuel , Plantaz Dominique , Plat Genevieve , Hall Georgina , Landman-Parker Judith 
Published: October 2019

Abstract
Background: Advanced stage nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) in children and adolescents is a very rare entity and therefore few data about their treatment and outcome are available.
Aim of the study: To describe the clinical characteristics and evolution of advanced stage NLPHL in children and adolescents
Methods:A retrospective analysis of clinical data of patients treated in the SFCE and UK CCLG centers
Patients and Results: 32 patients with NLPHL according to the REAL/WHO classification with disease stage ≥ IIB were recorded between 1998 and 2013. Two patients had composite NLPHL and B cell non-Hodgkin lymphoma at diagnosis. Median age was 12 years (range 4-17) and the majority were boys [n=28; 88%]. Stage distribution was: IIB =2 ( 6%), III =25 (71%) & stage IV =5 (15%). Twenty-seven patients had cervical node involvement while mediastinum involvement was seen in 11 (34% ) patients. B symptoms were seen in 2 and eight patients had ESR >30 mm. Sites of extra nodal localisation were bone, bone marrow, liver and lungs.Standard Hodgkin chemotherapy regimens (CT) were used in 26 patients (81%), combined with rituximab (R) in 5 children (3 to 6 doses) and along with 20Gy involved field radiotherapy in 6 children. NHL chemotherapy regimens were used in 5 patients; R-CHOP [n=3], LMB 01 regimen [n=1] and other regimen [n=1]. One patient received rituximab alone (4 doses) and 1 child had no treatment [watch and wait strategy]. Median follow up is 45 months (5 – 162).
Outcome: Overall survival is 96% and cumulative incidence of an event is 25%. Eight patients relapsed with a median delay of 15 months (5-73), and 3 had a second relapse 9,11 and 48 months respectively after first relapse. One patient treated with chemotherapy alone developed Ewing's sarcoma and died 2 years later. The only patient treated with rituximab alone relapsed at 22 months. Of note, no relapses were observed in patients who had chemotherapy and rituximab.
Conclusion: Our report shows that while advanced stage NLPHL in children and adolescents is rare, their clinical outcome is very good. Additionally, this report may inform the basis of future treatment of advanced stage NLPHL in children and adolescents.

Disclosures
No relevant conflicts of interest to declare.
Systematic Coronary Risk Evaluation (SCORE) Chart Identify Chronic Myeloid Leukemia Patients at Risk of Cardiovascular Diseases during Nilotinib Treatment
Authors: Breccia Massimo , Molica Matteo , Zacheo Irene , Alimena Giuliana 
Published: October 2019

Abstract
Nilotinib is currently approved for the treatment of chronic myeloid leukemia (CML) in chronic (CP) and accelerated phase (AP) after failure of imatinib and in newly diagnosed patients. Atherosclerotic events were retrospectively reported in patients with baseline cardiovascular risk factors during nilotinib treatment. We estimated the risk of developing atherosclerotic events in patients treated with second or first line nilotinib, with a median follow-up of 48 months, by retrospectively applying the SCORE chart proposed by the European Society of Cardiology (ESC) and evaluating risk factors at baseline (diabetes, obesity, smoking and hypertension). Overall, we enrolled in the study 82 CP patients treated frontline (42 patients, at the dose of 300 mg BID) or after failure of other tyrosine kinase inhibitors (40 patients, treated with 400 mg BID). The SCORE chart is based on the stratification of sex (male vs female), age (from 40 to 65 years), smoker vs non-smoker, systolic pressure (from 120 to 180 mm Hg) and cholesterol (measured in mmol/l, from 150 to 300 mg/dl). For statistical purposes we considered patients subdivided in low, moderate, high and very high risk. There were 48 males and 34 females, median age 51 years (range 22-84). According to WHO classification, 42 patients were classified as normal weight (BMI < 25), 26 patients were overweight (BMI 26- <30) and 14 were obese (BMI > 30). Retrospective classification according to the SCORE chart revealed that 27 patients (33%) were in the low risk category, 30 patients (36%) in the moderate risk category and 24 patients (29%) in the high risk category.
As regards risk factors, we revealed that 17 patients (20.7%) had a concomitant type II controlled diabetes (without organ damage), 23 patients (28%) were smokers, 29 patients (35%) were receiving concomitant drugs for hypertension, 15 patients (18%) had concomitant dyslipidaemia. Overall, the cumulative incidence of atherosclerotic events at 48 months was 8.5% (95% CI: 4.55-14.07): none of the low-risk patients according to the SCORE chart experienced atherosclerotic events compared to 10% in the moderate risk and 29% in the high risk category (p=0.002). Atherosclerotic-free survival was 100%, 89% and 69% in the low, moderate and high-risk population, respectively (p=0.001). SCORE chart evaluation at disease baseline could be a valid tool to identify patients at high risk of atherosclerotic events during nilotinib treatment.

Disclosures
Breccia: novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy.
NADPH Oxidase 2 Is Critical for Heterotypic Neutrophil-Platelet Interactions during Vascular Inflammation
Authors: Li Jing , Kim Kyungho , Andrews Robert K. , Cho Jaehyung 
Published: October 2019

Abstract
Intravital microscopic studies have shown that neutrophil-platelet interactions on activated endothelial cells are the major determinant of vascular occlusion under thromboinflammatory conditions. Heterotypic neutrophil-platelet interactions are mainly mediated by two crucial receptors and counter receptors, P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. Although the major receptors and counter receptors are well identified, it remains unclear how heterotypic cell-cell interactions are regulated during vascular disease. Recently, we demonstrated that neutrophil AKT2 is a key regulator for neutrophil-platelet interactions during vascular inflammation. Since it is known that AKT2 is important for NADPH oxidase 2 (NOX2) activity and reactive oxygen species (ROS) generation during neutrophil activation, we investigated whether NOX2 regulates the heterotypic neutrophil-platelet interaction during vascular inflammation. Using real-time fluorescence intravital microscopy, we found that platelet-neutrophil interactions are nearly completely inhibited during TNF-alpha-induced cremaster venular inflammation in NOX2 KO mice, compared with WT mice. Studies with bone marrow chimera further demonstrated that both hematopoietic and endothelial cell NOX2 are crucial for neutrophil-platelet interactions on the inflamed venules. In vitro studies with neutrophils and platelets isolated from WT and NOX2-deficient mice indicated that both neutrophil and platelet NOX2 are required for neutrophil-platelet aggregation under shear conditions. Using ROS scavengers and NOX2 KO cells, we observed that neutrophil NOX2-generated ROS regulate the activation and ligand-binding activity of alphaMbeta2 integrin during neutrophil activation and that platelet NOX2-produced ROS are important for P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Ibalpha. Interestingly, neutrophil NOX2 was crucial for store-operated calcium entry (SOCE), but not calcium release from the endoplasmic reticulum, whereas platelet NOX2 modulated intracellular calcium release, but not SOCE. We further demonstrated that the differential regulation of platelet and neutrophil NOX2 in calcium signaling correlates with the differences in the phosphorylation of AKT, ERK, and p38MAPK, suggesting the importance of AKT and its downstream molecules for NOX2-mediated calcium signaling. Our results indicate that neutrophil and platelet NOX2-produced ROS play critical roles in regulating the function of surface receptors required for heterotypic neutrophil-platelet interactions during vascular inflammation.

Disclosures
No relevant conflicts of interest to declare.
STAT3 Mediates C6-Ceramide-Induced Cell Death in Chronic Lymphocytic Leukemia
Authors: Doshi Ushma Atul , Loughran Thomas P. , Kester Mark 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in adults in Western countries. Despite high incidence, its pathogenesis is still poorly understood and it remains incurable with current therapies. Ceramide is a tumor suppressor' lipid known to regulate differentiation, senescence and cell cycle arrest. Previous work from our laboratory has demonstrated the therapeutic efficacy of nanoliposomal C6-ceramide in several solid and non-solid tumor models. We have shown that nanoliposomal C6-ceramide selectively induces cell death in CLL cells by targeting the Warburg effect with no detrimental effects on normal peripheral blood mononuclear cells. Additionally, nanoliposomal C6-ceramide treatment results in tumor regression in an in vivo murine model of CLL. However, the mechanism and key molecular players mediating C6-ceramide-induced cell death in CLL remains unknown. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in many cancers. Several reports in the literature have shown that STAT3 is constitutively phosphorylated on serine 727 (S727 p-STAT3) in CLL and STAT3 might be a therapeutic target in this disease. We hypothesize that STAT3 is a target of C6-ceramide in CLL cells.
We first validated STAT3 as a therapeutic target in CLL. STAT3 knockdown in CLL cells induced cell death. C6-ceramide treatment also induces necrotic cell death in these cells. Examination of STAT3 phosphorylation state demonstrates constitutive phosphorylation at S727 and Y705 residue in CLL cell lines and patient cells. C6-ceramide treatment suppressed the levels of Y705 p-STAT3 and S727 p-STAT3, while having no effect on total STAT3 levels. The downregulation of phosphorylation was specific to STAT3 and did not affect the other STATs. C6-ceramide treatment also led to the downregulation of several downstream targets of STAT3 like p21, cyclin D1, Mcl-1 and survivin. Furthermore, the suppression in STAT3 phosphorylation preceded induction of C6-ceramide-mediated cell death in CLL cells, suggesting that this event might be critical for cell death induction. Analysis of the upstream kinases revealed that C6-ceramide-induced suppression of S727 p-STAT3 might be, in part, a consequence of suppression of MEK1/2 kinase and protein kinase C (PKC). Ongoing studies will focus on identifying the specific phosphorylation site(s) that mediates cell death and examine the effect of C6-ceramide on upstream regulators of p-STAT3 in an effort to delineate the molecular mechanism of C6-ceramide-induced cell death in CLL. In conclusion, our data suggests that targeting STAT3 with nanoliposomal C6-ceramide is a potential therapy for CLL. This work is the first body of evidence demonstrating an effect of C6-ceramide on STAT3 phosphorylation.
Figure 1 Figure 1.

Disclosures
Kester: Keystone Nano, Inc: Employment, Equity Ownership.
Impact of Beta Blocker on Clinical Outcomes of Multiple Myeloma (MM) Patients
Authors: Hwa Yi Lisa , Lacy Martha Q. , Gertz Morie A , Buadi Francis , Rajkumar S. Vincent , Kumar Shaji , Kapoor Prashant , Go Ronald S. , Hayman Suzanne R , Lin Yi , Lust John A. , Dingli David , Russell Stephen J. , Gonsalves Wilson I , Dispenzieri Angela 
Published: October 2019

Abstract

Background: A recent preclinical study revealed an antiproliferative and apoptotic effect of propranolol on MM cell lines. This study was to investigate if propranolol and other beta blocker intake improved survival in MM patients.
Methods: Among patients seen at Mayo Clinic, Rochester from 1993 to 2010, we identified those taking propranolol. Age, year of diagnosis, International Staging System (ISS), Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) were used as case-match variables to match propranolol users to two control populations: no beta blocker and other beta-blocker. Cox proportional hazard modeling was utilized to analyze the effect of propranolol and other beta blockers intake on overall survival (OS) and disease-specific survival (DSS).
Results: In 3 groups: propranolol (n=52), no beta blocker (n= 136), or other beta blocker (n=84), transplant eligibility was 63.5%, 66.9%, 66.7% respectively (p=0.90). Median age was 63 years. Median follow-up was 86 months. No statistically differences were seen in age, ISS, mSMART or year of diagnosis in 3 groups (p=0.46).In univariate analysis comparing propranolol vs no beta blocker or other beta blocker cohort, 5-year OS rate was 60.2% vs 47% or 64% (p=0.039), and 5-year DSS rate was 71.7% vs 50.3% or 69.1% (p=0.0029). In multivariate analysis adjusted for age, ISS, transplant eligibility, mSMART and year of diagnosis, compared to propranolol intake, no beta blocker group had poorer OS [HR= 2.081; 95% CI (1.353- 3.200); p=0.0008], and DSS [HR=2.833 (1.709-4.697); p<0.0001], but other beta blocker users had no difference in OS [HR =1.145 (0.728-1.803);p=0.56],and DSS [HR= 1.276 (0.745-2.185); p=0.38].
Conclusion: Propranolol and other beta-blocker intake among MM patients were equally associated with a more favorable OS and DSS.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
Lust: Senesco: PI Other.
HPRT As a Selectable Safe Harbor for Transgenesis
Authors: Conway Anthony , Laganière Josée , Paschon David E , Hacke Katrin , Kasahara Noriyuki , Gregory Philip D , Holmes Michael C. , Cost Gregory J 
Published: October 2019

Abstract
A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest.

Disclosures
Conway: Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.