bluuebunny/crossref_metadata_2025_split huggingface dataset:
Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells
Authors: Fischer Stephen E , Ma Yiwei , Smith Caitlin , Sodha Anirudhasingh , Zhao Yukang 
Published: October 2019

Abstract
Abstract 4411
Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo.
In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation.
Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells.

Disclosures:
No relevant conflicts of interest to declare.
Effect of Knockdown of CD44 Expression by shRNA On Biologicalfeatures of Activated B-Cell-Like Diffuse Large B Cell Lymphoma Cell Line
Authors: Feng Ru , Wei Xiaolei , Duan Wenbing , Huang Fen , Yu J. Jessica , Ye B. Hilda 
Published: October 2019

Abstract
Abstract 5080

Objective
To study the effect of CD44 down-regulation with lentivirus-mediated small hairpin RNA (shRNA) interference on the biological features of activated B-cell-like diffuse large B-cell lymphoma (DLBCL) cell line SUDHL-2.

Methods
Lentiviral vector for RNAi of CD44, which contained green fluorescent protein(GFP) was constructed. Package cells phoenix293 was used to produce virus stocks. Then SUDHL-2 cells were infected with the recombinant lentivirus and the cells with CD44 knock-down were selected by FACS for GFP expression. CD44 expression in the cells was determined by RT-PCR and FACS. The proliferation, apoptosis and invasion were evaluated by MTT methods, FACS and transwell migration assay, respectively.

Results
DNA sequencing demonstrated that the lentivirus RNAi vector was constructed successfully. Clones of SUDHL-2 cells infected with the recombinant lentivirus were selected and exhibited substantial knock-down of CD44 mRNA and protein expression compared with the control cells. The proliferation ability of cloned SUDHL-2 cells were inhibited. The apoptosis rates at early and late phase were 6. 26%, 36. 40% respectively in cloned SUDHL-2 cells and in control cells were 2. 9%, 2. 56% at early phase and 6. 1%, 6. 58% at late phase. The migrating number of cloned SUDHL-2 cells(34. 53±8. 05)% was also significantly decreased compared with the control cells, 78. 67±2. 64% and 78. 00±6. 13% (P=0. 290).

Conclusion
The lentivirus-mediated shRNA of CD44 is efficient in down-regulating CD44 expression and inducing apoptosis, inhibiting proliferation and invasiveness of SUDHL-2 cells, suggesting that CD44 might have an oncogene role in the tumorigenesis and progression of ABC-DLBCL.

Disclosures:
No relevant conflicts of interest to declare.
Leukemogenic Transformation of Hematopoietic Cells by Constitutive Activation of Canonical Wnt Signaling in Osteoblast Precursors
Authors: Kode Aruna , Manavalan Sanil , Mossialou Ioanna , Bhagat Govind , Vundavalli Murty , DePinho Ronald , Teruya-Feldstein Julie , Berman Ellin , Kousteni Stavroula 
Published: October 2019

Abstract
Abstract 509
Osteoblasts, the bone forming cells, are implicated in the fate of healthy and malignant stem cells. They affect self-renewal and expansion of hematopoietic stem cells (HSCs) and homing of tumor cells into the bone marrow. Here we show that constitutive activation of canonical Wnt signaling in osteoblast precursors disrupts hematopoiesis in mice by shifting the differentiation potential of HSC progenitors to the myeloid lineage which results in accumulation of granulocyte/monocyte progenitors and concomitant development of acute myeloid leukemia (AML). B-lymphopoiesis is also decreased. The AML phenotype is associated with clonal evolution at the cytogenetic level since clonal abnormalities could be detected in leukemic blasts from mice with constitutive activation of the canonical Wnt target β-catenin in osteoblast precursors (βcateninosb mice). Bone marrow transplantation experiments from βcateninosb mice to wild type lethally irradiated mice resulted in development of AML within 8 weeks following transplantation, demonstrating progression towards AML. At the molecular level, cell-specific gene inactivation mouse models demonstrate that β-catenin interacts with FoxO1 in osteoblasts to induce development of AML. Downstream signaling events that confer osteoblast signaling to normal HSCs and lead to their leukemogenic transformation will be presented. Importantly, malignancy-inducing osteoblasts, detected by nuclear accumulation of β-catenin in bone marrow biopsies, were identified in > 25% of patients with myelodysplasia (MDS), acute myeloid leukemia (AML) or AML arising from a prior MDS. Specifically, 15 out of 53 patients with MDS (n=17 patients), AML (n=20 patients), or MDS that had transformed to AML (n=16) chosen at random showed nuclear localization of β-catenin in osteoblasts. Of note, 12 of the 15 (80%) patients with nuclear localization of β-catenin in osteoblasts had abnormalities of chromosome 5 and/or 7, very common cytogenetic abnormalities in patients with MDS and AML. The same signaling pathways mediating AML development in βcateninosb mice were also found to be activated in osteoblasts and hematopoietic cells from the patients with nuclear accumulation of β-catenin in osteoblasts. These findings demonstrate that genetic alterations in osteoblast precursors (1) can induce AML in mice and (2) are associated with AML development in humans. They also provide a molecular basis for the leukemogenic transformation.

Disclosures:
No relevant conflicts of interest to declare.
Activated NRAS Mediates Self-Renewal Capacity in AML by Facilitating the Mll/AF9-Specified Gene Expression Signature
Authors: Sachs Zohar , Nguyen Hanh , Hassan Nurul Azyan Mohd , Rathe Susan K , Sachs Karen , Diaz-Flores Ernesto , Bendall Sean C , Diers Miechaleen D , Nolan Garry P. , Shannon Kevin M. , Largaespada David A. 
Published: October 2019

Abstract
Abstract 5116
RAS is a known oncogene in AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), however the specific effects of targeting RAS-activated pathways on AML physiology are unclear. NRASG12V transgene repression in an NRASG12V/Mll/AF9 transgenic murine AML model leads to apoptosis and disease remission (Kim et al. Blood 2009). To better understand possibilities for and implications of therapeutic targeting of RAS-activated pathways, we inactivate NRASG12V in this mouse model and characterize the subsequent signaling and transcriptional response. We profiled signaling intermediates and markers of apoptosis and cell cycle using mass cytometry, a next-generation flow cytometry technology, which simultaneously measured the levels of 32 antibody-labeled proteins in single cells. These analyses revealed specific signaling changes that varied with the surface immunophenotype of the AML cells and highlighted relevant RAS-directed signaling pathways. Parallel RNA sequencing and gene expression microarrays revealed that NRASG12V-expressing cells express hematopoietic self-renewal genes and repression of NRASG12V leads to loss of this program. Importantly, the NRASG12V-dependent gene expression program mimics the Mll/AF9-determined, Myb-mediated self-renewal program reported by Zuber et al. (Zuber et al. Genes Dev 2011). These data suggest a novel role for RAS in AML self-renewal capacity by maintaining the Mll/AF9-mediated self-renewal program. Furthermore, inhibition of the PI3K-mTOR pathway decreases viability, in vitro colony formation, and recapitulates the effects of NRASG12V inactivation on self renewal-associated genes, implicating this pathway as the mediator of RAS-directed leukemia self renewal capacity. These data provide rationale for therapeutic targeting of leukemia stem cells via PI3K-mTOR pathway inhibition and for using this mouse model as a tool to test such therapeutic approaches. Since a variety of AML-specific genetic mutations lead to perturbations in RAS signaling, these results may be generalizable to AML with a broader range of mutations.
Figure: NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature. Figure:. NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature.

Disclosures:
Largaespada: Discovery Genomics, Inc. : Consultancy, Equity Ownership; NeoClone Biotechnology, Inc. : Consultancy, Equity Ownership.
NPM-RAR Binds to Tradd and Impedes the Extrinsic Apoptotic Pathway
Authors: Redner Robert L. , Chattopadhyay Anuja , Hood Brian L. , Conrads Thomas P. 
Published: October 2019

Abstract
Abstract 5126
The t(5;17)(q35;q21) variant of Acute Promyelocytic Leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). The resultant NPM-RAR fusion protein blocks myeloid differentiation, and leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. The contribution of the N-terminal 117 amino acids of NPM contained within NPM-RAR has not been well studied. NPM is a molecular chaperone, and binds to a variety of proteins implicated in leukemogenesis. We performed a proteomic analysis to identify NPM-RAR interacting proteins. Vectors encoding NPM-RAR or RARA (as control) fused in frame to calmodulin binding peptide and Protein A were expressed in HEK293 cells, and interacting proteins subjected to tryptic digest. Peptides were analyzed by nanoflow reversed-phase liquid chromatography-mass spectrometry. Amongst other targets, we identified tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD) as a distinct binding partner for NPM-RAR. TRADD did not bind to wild-type RARA or NPM, suggesting that the interaction was unique to the fusion protein. The NPM-RAR/TRADD interaction was verified by reciprocal co-precipitation. Though NPM-RAR localizes primarily in the nucleoplasm, we also found a low level of NPM-RAR/TRADD dimer in the cytoplasm utilizing confocal microscopy. Expression of NPM-RAR in U937 cells impaired TNF activation of caspase 8 and caspase 3. TNF-induced acquisition of Annexin V, generation of sub-G0/G1 nuclear content, and cleavage of PARP were all blunted, indicating that NPM-RAR blocks TNF-induced apoptosis. Our studies identify a novel mechanism through which NPM-RAR impacts leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Prevalence and Risk Factors of Iron Deficiency Anemia in Korea: The Fifth Korean Health and Nutrition Examination Survey (KNHANES V-1)
Authors: Lee Jeong-Ok , Bang Soo-Mee , Choi Hyoung Soo , Ahn Soyeon , Lee Ju Hyun 
Published: October 2019

Abstract
Abstract 5170
Iron deficiency (ID) is the most important cause of anemia in the world. The contribution of ID to anemia and causes of iron deficiency anemia (IDA) is heterogeneous between different populations. We analyzed the prevalence and risk factors of iron deficiency anemia among participants of the fifth Korean Health and Nutrition Examination Survey in 2010 (KNHANES V-1). In this survey, stratified, multistaged, probability-sampling designs and weighting adjustments were conducted to represent the entire Korean population. Anemia was defined in accordance to WHO criteria: Hemoglobin (Hb) less than 11. 5 g/dL in aged 10 to 11 years, less than 12. 0 in aged 12 to 14 years, less than 13 g/dL in men >15 years, and less than 12 g/dL in non-pregnant women. ID was defined as a transferrin saturation lower than 10% or serum ferritin levels lower than 15 μg/L. IDA was confirmed with concomittent anemia with ID. Persons with active malignancy were excluded. In addition to age and sex, we analyzed the correlation between IDA and individual socioeconomic status including income, education and marriage, nutritional status based on Korean dietary reference intakes, body mass index, eating habits, body weight changes and so on. In a total of 6689 (3010 men and 3679 women) over 10 years of age, the prevalence of IDA was 0. 82% (95% CI 0. 25–1. 39) for men and 8. 01% (6. 84–9. 18) for women. (p <0. 001) Among 470 anemic women, 56% was associated with iron deficiency. In women of reproductive age (15 to 49 years), the prevalence of ID and IDA was 31. 4% and 11. 5%, respectively. IDA accounted for 13. 5% of all anemia in the elderly (>65 years). IDA prevalence in the elderly had no significant sexual difference. (0. 78 % in men and 1. 21% in women aged 65 to 74 years, 4. 11% in men and 4. 92 % in women aged > 75 years). IDA was more prevalent in persons with low income (the bottom 25% of participants vs others, 5. 6% vs 4. 2%, p=0. 024), high education level (at least a high school graduate vs others, 5. 3% vs 2. 4%, p=0. 0002), low BMI (mass(kg)/((height(m))2) (<18. 5 vs ≥18. 5 and <25 vs ≥25, 11. 1% vs 4. 8% vs 2. 9%, p< 0. 0001), and deficient daily iron intake (less than the estimated average requirement (EAR) vs EAR and more; 8. 7% vs 3. 4%, p<0. 0001). In conclusion, iron deficiency is major cause of anemia especially in women of reproductive ages, and elderly regardless of gender. A variety of socioeconomic and nutritional factors can affect the development of IDA. Screening for IDA in high risk population, modification of correctable risk factor and proper early treatment with nutritional support are mandatory.

Disclosures:
No relevant conflicts of interest to declare.
Gas6 Regulates Thrombin-Induced of VCAM-1 Expression by a FoxO1 Dependent Mechanism
Authors: Robins Richard , Lemarie Catherine A. , Blostein Mark D. 
Published: October 2019

Abstract
Abstract 5192
Forkhead proteins play a broad role in endothelial cell biology. These factors mediate cell adhesion to extracellular matrix, regulate the expression of pro-inflammatory and pro-thrombotic genes, and participate in cell repair, proliferation and apoptosis. FoxOs are known downstream targets of the PI3K/Akt signaling pathway. Phosphorylation of FoxO transcription factors results in their translocation from the nucleus to the cytoplasm, thereby inhibiting their transcriptional activity. It has recently been shown that the deletion of the three FoxO isoforms in endothelial cells protects mice from vascular dysfunction. Gas6, a member of the vitamin K-dependent family of proteins, has been shown to protect endothelial cells from apoptosis and promote endothelial cell activation in vivo. It has been shown that the expression of ICAM-1 and VCAM-1 were blunted in the absence of gas6. Interestingly, a role for VCAM-1 in the pathogenesis of venous thrombosis has been proposed. Elevated levels of the soluble form of VCAM-1 have been detected in the serum of patients with venous thrombosis. We previously demonstrated that the anti-apoptotic effect of gas6 was mediated partially through FoxO1, but overall, the signalling mechanisms occurring downstream of gas6 remain largely unknown. We hypothesize that gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO1 in endothelial cells. Western blot analysis demonstrated that thrombin induced time dependent phosphorylation of FoxO1 with a maximum at 30 minutes in WT (p<0. 05) but not in gas6 deficient (−/−) cells. In addition, thrombin reduced the nuclear content of FoxO in WT (p<0. 05) but not in gas6−/− endothelial cells. Using qPCR, we found that mRNA expression of VCAM-1 was increased after 30 minutes of stimulation with thrombin in WT cells (p<0. 05). More importantly, thrombin-mediated induction of VCAM-1 was blunted in gas6−/− endothelial cells. We found that FoxO1 siRNA increased basal VCAM-1 expression in WT endothelial cells. Taken together, our data demonstrate that gas6 is a crucial mediator of FoxO1 that regulates thrombin-induced VCAM-1 expression. This pathway may explain the pro-thrombotic and pro-inflammatory role of gas6.

Disclosures:
No relevant conflicts of interest to declare.
The Impact of Rituximab and Radiotherapy On Treatment Outcome of Patients with DLBCL and Skeletal Involvement
Authors: Held Gerhard , Zeynalova Samira , Murawski Niels , Ziepert Marita , Kempf Barbara , Viardot Andreas , Dreyling Martin , Hallek Michael , Witzens-Harig Mathias , Ruebe Christian , Berdel Christian , Zwick Carsten , Schmitz Norbert , Pfreundschuh Michael 
Published: October 2019

Abstract
Abstract 690

Background:
There is limited information on the role of skeletal involvement in DLBCL patients treated with rituximab.

Methods:
In a retrospective subgroup analysis patients with aggressive B-cell lymphomas with and without skeletal involvement were compared with respect to clinical presentation, event-free and overall survival.

Results:
Of 3840 patients 292 (7.6%) had skeletal involvement. In a multivariable analysis of patients treated within the randomized MInT and RICOVER-60 trials, the two largest randomized trials addressing the role of rituximab in DLBCL to date, skeletal involvement was associated with a reduced hazard ratio (HR) of 0.8 (p=0.181) for event-free survival and 0.7 (p=0.083) for overall survival for patients treated without, but with an increased HR (1.5; p=0.048) for event-free and (1.1; p=0.828) for overall survival in patients treated with rituximab. This was due to the failure of rituximab to improve the outcome of patients with skeletal involvement. In the MInT trial, the 3-year EFS rates were 64% without and 63% with rituximab (p=0.680) and the 3-year OS rates were 83% without and 90% with rituximab (p=0.542). similarly, in the RICOVER-60 trial, the 3-year EFS rates were 45% without and 50% with rituximab (p=0.593) and the 3-year OS rates were 68% without and 68% with rituximab. In a Cox regression model for event-free survival adjusted for the IPI risk factors a relevant interaction (HR 1.5; p=0.056) term between rituximab and skeletal involvement was observed. In contrast to rituximab, additive radiotherapy to sites of skeletal involvement was associated with a better outcome: 3-year EFS rates were 40% without and 75% with radiotherapy (p<0.001), 3-year OS rates were 70% without and 86% with radiotherapy to sites of skeletal involvement (p=0.064). In a multivariable analysis radiotherapy reduced the risk for an event in EFS by 70% (HR=0.3; p=0.001) and by 50% in OS (HR=0.5; p=0.111).

Conclusion:
Addition of rituximab failed, but radiotherapy to sites of skeletal involvement did improve the outcome of DLBCL patients with skeletal involvement. Radiotherapy to sites of skeletal involvement, though abandoned by many cooperative groups world-wide, is recommended in the rituximab era, unless prospective trials demonstrate that it might be omitted in cases with a negative PET after immunochemotherapy.

Disclosures:
Dreyling: Roche: Membership on an entity's Board of Directors or advisory committees. Hallek:Roche: Membership on an entity's Board of Directors or advisory committees. Schmitz:Chugai: Membership on an entity's Board of Directors or advisory committees. Pfreundschuh:Roche: Membership on an entity's Board of Directors or advisory committees; Chugai: Consultancy.
The Origin and Function of Langerin (CD207) Expressing Cells in Mice and Humans
Authors: Merad Miriam 
Published: October 2019

Abstract
Abstract SCI-7
The current paradigm suggests that Langerhans cell histiocytosis (LCH) results from an accumulation of epidermal dendritic cells also called Langerhans cells. This concept is based on phenotypic and ultrastructural observations showing that LCH lesions are infiltrated by CD1a+langerin+ cells, two features thought to be restricted to epidermal Langerhans cells. It has been difficult, however, to understand how Langerhans cells, which are normally restricted to stratified epithelia, could give rise to such a multifocal disorder. LCH research has been handicapped by the inability to develop reliable animal models and by the fact that for a long time very few markers were available to determine the origin and stage of differentiation of histiocytes, now renamed macrophage/dendritic cell lineages. This presentation will discuss recent progress in our understanding of the regulation of the Langerhans cell, macrophage, and dendritic cell lineages and discuss the relationship of these lineages with the LCH cell.

Disclosures:
No relevant conflicts of interest to declare.