bluuebunny/crossref_metadata_2025_split huggingface dataset:
DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING
Authors: Zhang Guoying , Xiang Binggang , Shaojing Ye , Chrzanowska-Wodnicka Magdalena , Morris Andrew J. , Gartner T. Kent , Whiteheart Sidney W. , White Gilbert C. , Smyth Susan S. , Li Zhenyu 
Published: October 2019

Abstract
Abstract 2200
Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling.

Disclosures:
No relevant conflicts of interest to declare.
Involvement of Lipid Rafts and Caveolae in gas6-Axl Signaling in Endothelial Cells
Authors: Laurance Sandrine , Lemarie Catherine A , Blostein Mark 
Published: October 2019

Abstract
Abstract 2211
Lipid rafts are localized regions of plasma membranes that contain primarily cholesterol and glycosphingolipids. Many receptor tyrosine kinases including the epidermal growth factor receptor, the platelet derived growth factor receptor, and the insulin receptor, localize specifically to these structures. Lipid rafts have been shown to play a role in intracellular signaling events. More specifically, diverse processes such as dimer formation, autophosphorylation, and intracellular adaptor protein recruitment/activation have been specifically involved in receptor tyrosine kinase/lipid raft interactions. Caveolae are defined as a subclass of lipid rafts and form well-shape invaginations on the cell surface. Caveolin-1 is the major constitutive protein of caveolae. Axl is a receptor tyrosine kinase whose association with lipid rafts and more specifically with caveolae has not been elucidated. Gas6, the ligand for Axl, is a vitamin K dependent protein that is post-translationally modified by an enzymatic process called g-carboxylation. We hypothesize that gas6/Axl mediated intracellular signaling is dependent on its localization to lipid rafts/caveolae in endothelial cells. First, we show that gas6-induced c-Src, Akt and ERK1/2 phosphorylation is mediated via Axl by using Axl siRNA in human umbilical vein endothelial cells (HUVECs). Then, lipid rafts were isolated from HUVECs by a detergent-free lysis method followed by a sucrose gradient centrifugation and western blot analysis. We found that Axl moved into the lipid raft-enriched fractions after 5 and 10 min of gas6 treatment as shown by its colocalization with caveolin-1. Interestingly, after gas6 treatment, c-Src, which is known to act as a transient docking platform for signaling molecules in lipid rafts, follow the same localization pattern to lipid raft-enriched fractions. The requirement of caveolae was then evaluated by transfecting HUVECs with caveolin-1 siRNA. Caveolin-1 knock down abolished completely gas6-induced phosphorylation of c-Src, Akt and ERK1/2 thus highlighting the role of caveolae in gas6-Axl signaling. Taken together these results demonstrate that activation of gas6-dependent signaling pathways involves Axl trafficking to lipid rafts/caveolae. These novel findings identify lipid rafts/caveolae as dynamic scaffolding systems for gas6/Axl interactions in endothelial cells.

Disclosures:
No relevant conflicts of interest to declare.
Age Dependent Alternations In Hematopoietic Stem Cell Niches
Authors: Li Geqiang , Arrieta Cesar Nombela , Wang Leo D. , Wagers Amy J. , Silberstein Leslie E 
Published: October 2019

Abstract
Abstract 2395
Age associated decline in hematopoietic system is a well-documented process which encompasses a substantial expansion of the hematopoietic stem cell (HSC) pool and a decline of their competitive repopulating ability on a per cell basis. Expansion occurs predominantly in the myeloid biased HSC subset, subsequently leading to a global skewing of lineage differentiation from lymphopoiesis toward myelopoiesis. These age-related effects are known to partially result from cell-intrinsic alterations of HSC, which include DNA damage accumulation, oxidative stress and activation of tumor suppressor pathways. HSCs exist in specialized microanatomical locations within the bone marrow (BM), where they receive critical regulatory cues that control HSC homeostasis. While the cell-autonomous effects of HSC aging have been extensively studied, little is known on how aging of the surrounding microenvironment may contribute to the age-associated alterations of HSC function, and thus the focus of our study.
Here, we have employed novel imaging approaches to analyze age-related changes of the BM microenvironment, and how these affect the global spatial distribution of HSPC-enriched subsets in the BM parenchyma. Our preliminary studies suggest during aging leads to a profound disruption of the BM microvascular compartment. By using imaging cytometry of two-dimensional (2D) sections, as well as confocal microscopy of 3D-BM slices, we demonstrate that the characteristic sinusoidal network found in young mice, degenerated with aging into a highly disorganized and expanded vascular bed, in which expression of the arterial marker Sca-1 was strongly upregulated. Flow cytometry analysis further revealed a 3–4 fold increase of CD45-Ter119-CD31+ Sca-1+cells in 22–24 month old mice. We next investigated how the changes in vasculature network impact HSC distribution. We employed imaging cytometry to track the presence in BM sections of a population of Lineage-Cd48-kit+ cells, 60% of which are long term HSC. Our preliminary results indicate that, in aged mice, HSPC-accumulation in endosteal zones (<100μm from endosteal surfaces) remains unchanged. Of note, consistent with the increase in vascularization, HSPCs display a significantly increased tendency to directly associate to BM microvessels in 22–24month old mice. We are currently investigating at the molecular level how altered vasculature influence HSC aging which would enable us to enhance the regenerative properties of the aged stem cells.

Disclosures:
Wagers: iPierian, Inc.: Membership on an entity's Board of Directors or advisory committees.
Alternative Methods of Activation of the Stem Cell Niche Reveal a Spatial Localization of Primitive Cells in the Bone Marrow
Authors: Rashidi Narges M , Spindler Tassja J , Barsky Lora W , Adams Gregor B. 
Published: October 2019

Abstract
Abstract 2396
Osteoblasts are key constituents of the murine hematopoietic stem cell (HSC) endosteal niche, evidenced by the fact that increasing their activity leads to an increase in the number of HSCs. However, recent studies have also suggested a role of the bone resorbing osteoclast in the HSC niche. In these studies we wished to examine whether activation of the osteoblast or the osteoclast had differing effects on the primitive hematopoietic cell populations. To achieve this we pharmacologically activated osteoclasts and osteoblasts using receptor activator of nuclear factor-κB ligand (RANK-L) and parathyroid hormone (PTH), respectively. Our in vitro results demonstrated that both PTH and RANK-L treatment of bone marrow stromal cell populations activated osteoblasts and osteoclasts. Yet, RANK-L treatment, which is able to expand the osteoclastic population to a much higher degree, demonstrated enhanced support of primitive hematopoietic cells in cobblestone area forming cell assays. We next examined the effects of in vivo treatment with RANK-L and PTH by performing histological analyses to study the effect of these treatments on bone composition, and examining the effects on different HSC sub-populations. These data suggested that the effects of our treatment regimens were different according to the region of the bone, with RANK-L treatment having no effect or leading to a reduction in trabecular bone in the metaphysis, but an increase in bone remodeling activity at the endosteal surface of the cortical bone in the diaphysis, while PTH treatment increases bone formation and remodeling activity both in the diaphysis and the trabecular bone of the metaphysis. Correlating with this was our observations that LT-HSC frequency was increased following RANK-L and PTH treatment, yet an increased ST-HSC frequency was only observed following PTH treatment. Direct examination of HSCs in specific regions of the bone indicated that RANK-L treatment preferentially increased the LT-HSCs in the diaphysis region of the bone marrow, while PTH treatment specifically increased the ST-HSC population in the metaphysis. These data not only reveal a supportive role for osteoclasts in the HSC niche suggesting that osteoclast interaction with osteoblasts is an essential component for maintaining HSC population in the bone marrow niche, they also suggest a possible structural organization for localization of LT-HSC vs. ST-HSCs in the long bone with LT-HSCs being more localized in the endosteal surface of the diaphysis area of the long bones as oppose to the ST-HSCs having a preference for the metaphysis of these bones.

Disclosures:
No relevant conflicts of interest to declare.
The TLE1 Tumor Suppressor Regulates Myc Induced Leukemogenesis
Authors: Sweetser David , Ramasamy Selvi , Blackburn Jessica S , Langenau David M. 
Published: October 2019

Abstract
Abstract 2473
The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies.
Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability.
We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies.

Disclosures:
No relevant conflicts of interest to declare.
Studying Evolution of Multiple Myeloma with CanEvolve
Authors: Shah Parantu K , Samur Mehmet Kemal , Li Cheng , Munshi Nikhil C. 
Published: October 2019

Abstract
Abstract 2886
Multiple Myeloma (MM) evolves from monoclonal gammopathy of undetermined significance (MGUS), its pre-malignant stage at the rate of 1% per year. Moreover all MM patients eventually develop drug resistance. Currently, very little is known about this process of MM evolution, specifically about the changes in regulatory networks and signaling pathways responsible for it. One major difficulty in studying this is the lack of suitable functional genomics profiles covering all the stages of MM evolution from the same patients.
We have now modeled this process using the knowledge stored in the canEvolve portal for cancer integrative genomics and evolution (available at http://www.canevolve.org). The current version of the portal stores gene and miRNA expression and copy number alterations information derived from microarray and next generation sequencing platforms for more than 10 cancer types. It also stores hundreds of thousands of instances of protein-protein interactions, and metabolic and signaling pathway information for the Human proteome. The primary analysis capabilities include identification of differential gene expression, survival analysis and reconstructed gene regulatory networks and their visualization. Finally, canEvolve stores integrative analysis carried out using Gene set enrichment (GSEA) and GemiNI.
We carried out meta analysis of gene expression profiles of MGUS, smoldering Myeloma, MM and relapsed patients publicly available from multiple Gene expression omnibus (GEO) datasets. We carried out geneset enrichment analysis of normal-MGUS, normal-MM, normal-relapsed, MGUS-SMM, MGUS-MM and MM-relapsed pairs with different gene sets available from MSigDB. We identified transcription factors, miRNA, metabolic and signaling pathways whose targets/members significantly change expression. For example, targets of mir-17 and NF-Kappa B significantly change as MGUS turns to MM. Similarly, targets of miR-484 and CREL significantly changes as MM patients relapse. The CanEvolve portal thus provides us the ability to serially monitor and analyze various genomic datasets to provide targets that may play a crucial role in myelomagenesis as well as propgression of the disease. We are now validating some of the identified targets.

Disclosures:
No relevant conflicts of interest to declare.
Successful Treatment with Bortezomib in Combination with Bendamustine and Prednisone of Patients with Newly Diagnosed/Untreated Multiple Myeloma and Light Chain Induced Renal Failure
Authors: Pönisch Wolfram , Andrea Marc , Wagner Ina , Hammerschmidt Doreen , Kreibich Ute , Schwarzer Andreas , Zehrfeld Thomas , Schwarz Maik , Winkelmann Cornelia , Petros Sirak , Bachmann Anette , Lindner Tom , Niederwieser Dietger 
Published: October 2019

Abstract
Abstract 2938

Introduction:
Renal impairment is one of the most severe complications of Multiple Myeloma (MM) at diagnosis. These patients are at increased risk for infections and have a significantly worse prognosis. Small phase I/II studies suggest that treatment with chemotherapy and/or new substances results in recovery of renal function in up to 25%. The window of opportunity to reverse renal impairment is rather small, making an immediate and highly active treatment strategy mandatory. Bortezomib as well as Bendamustine have turned out to be effective, rapid action drugs in the treatment of MM. Bendamustine is a bifunctional alkylating agent with low toxicity that produces both single- and double-strand breaks of DNA, and shows only partial cross resistance with other alkylating drugs.

Methods:
Between June 2006 and May 2011, 18 patients (median age 69; range 43 – 86 years) with newly diagnosed/untreated MM and renal insufficiency (creatinine clearance < 35 ml/min) were treated with Bendamustine 60 mg/qm day 1 and 2, Prednisone 100 mg on day 1, 2, 4, 8 and 11, and Bortezomib 1.3 mg/qm on day 1, 4, 8 and 11 (BPV). Cycles were repeated every 21 days up to the stage of maximum response or disease progression. MM response was assessed using IMWG criteria modified to include near complete response (nCR) and minimal response (MR). Eight patients were on dialysis at the time of diagnosis.

Results:
Fifteen patients (83%) responded after at least one cycle of chemotherapy with three sCR, five nCR, five VGPR, and two PR. With a median follow up of 17 months, PFS at 12 months was 57 % and OS was 61 %. The median number of the BPV-treatment cycles was 2 (1–5) cycles. The myeloma protein decreased rapidly, reaching the best response after the first cycle in 4 patients and after the second cycle in a further 7. Six patients showed a complete remission of the kidney function (creatinine clearance > 60 ml/min) and in seven patients a partial remission (creatinine clearance > 30 ml/min) was attained. Transient grade 3 – 4 neutropenia was reported in one patient, and grade 3 – 4 thrombocytopenia occurred in 6 patients. One patient experienced a new grade 3 polyneuropathy.

Summary:
These results indicate that the combination of Bortezomib, Bendamustine and Prednisone is effective and tolerated in patients with newly diagnosed/untreated MM and renal failure.

Disclosures:
Pönisch: Mundipharma: Honoraria, Research Funding. Niederwieser:Mundipharma: Research Funding.
Rapid and Efficient Mobilization of Murine Hematopoietic Stem and Progenitor Cells with Nox-A12, a New Spiegelmers®-Based CXCR4/SDF-1(CXCL12) Antagonist
Authors: Scheller Marina , Schwoebel Frank , Vossmeyer Doerte , Leutz Achim 
Published: October 2019

Abstract
Abstract 2995
Mobilization of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) is important in many hematological therapies. However, up to 30% of the patients respond poorly to standard granulocyte colony-stimulating factor (G-CSF) treatment, highlighting the need for more effective mobilizing strategies. The CXCR4/stromalcell-derived factor 1 (SDF-1) axis plays a crucial role in the interaction between HSCs and the marrow niche and is involved in HSC mobilization. NOX-A12 is a structured mirror-image RNA oligonucleotide, a so-called Spiegelmer®, that was identified to bind SDF-1 thereby inhibiting its activity with subnanomolar IC50. HSC/HPC mobilization by NOX-A12 was examined in the mouse. Single NOX-A12 administration induced reversible mobilization of HSC/HPC populations within a few hours. NOX-A12 synergized with G-CSF to strongly enhance HSC/HPC mobilization. In particular, the progenitor compartment mobilized by single NOX-A12 administration contained more differentiated short-term HSCs (ST-HSCs), and combined administration of NOX-A12 and G-CSF mobilized a significantly higher proportion of primitive and more potent murine long-term repopulating cells that successfully engrafted primary and secondary lethally-irradiated recipients. These results characterize NOX-A12 as a potent HSCs/HPCs mobilizing therapeutic in mammals and suggest its clinical potential.

Disclosures:
No relevant conflicts of interest to declare.
Relapsed Multiple Myeloma: Who Benefits From Salvage Autografts?
Authors: Chow Annie W.S. , Lee Cindy H.S. , Hiwase Devendra K , To Luen Bik , Horvath Noemi 
Published: October 2019

Abstract
Abstract 3059

Aim
Despite novel agents, multiple myeloma (MM) remains incurable. The majority of patients with MM will relapse at some stage after induction therapy and high dose chemotherapy with autologous stem cell transplant (ASCT). Published studies have recommended salvage ASCT as an effective option in patients with relapsed MM. However, the patient cohort who will derive maximum benefit from salvage ASCT is still undefined and is likely to evolve with increasing therapeutic options.
We aimed to evaluate the role of salvage ASCT in relapsed MM patients.

Methods
We performed a retrospective analysis of patients who underwent salvage ASCT for relapsed MM in South Australia between 1992 and 2011.

Results
During this period, autologous stem cell transplants were performed for 457 patients with newly diagnosed MM. Thirty-nine patients subsequently underwent salvage ASCT for relapsed MM. The median age of patients at salvage ASCT was 59 (34–73) years, and 85% were ISS I and II at diagnosis. The majority of patients (90%, n=35) had a progression free interval (PFI) of at least 12 months after initial ASCT, consistent with recommendations by current literature. Salvage ASCT was performed for four patients with PFI shorter than 12 months, due either to suboptimal response to novel agents (thalidomide, lenalidomide, bortezomib), or the lack of novel agent availability in the earlier years.
The median progression-free survival (PFS) and median overall survival (OS) following salvage ASCT were 22 months (95% CI: 11–32) and 52 months (95% CI: 30–74) respectively. Non-relapsed mortality at 2 years was 7%. Following salvage ASCT, the median PFS (28 vs. 12 months, p = 0.006) and OS (83 vs. 26 months, p = 0.02) were significantly longer in patients whose progression free interval after the initial ASCT was > 24 months (Figures 1 and 2). Use of novel agents in salvage induction and maintenance therapy post salvage ASCT did not appear to influence outcome.

Conclusion
Our results suggest that there remains a role for salvage ASCT, even in the era of novel therapies. Salvage ASCT may result in durable responses, particularly in patients with longer progression free interval (> 24 months) following initial ASCT. This interval could be included as a selection criterion for patients with relapsed myeloma who remain transplant eligible.

Disclosures:
No relevant conflicts of interest to declare.