bluuebunny/crossref_metadata_2025 huggingface dataset:
Sox4 Is Essential for Initiation and Progression of Acute Lymphoblastic Leukemia Induced by P190-BCR-ABL Transformation
Authors: Ma Haiqing , Mallampati Saradhi , Sun Baohua , Gong Yun , Lefebvre Véronique , Sun Xiaoping
Published: October 2019

Abstract 564

Sox4 is an Sry-related HMG box transcription factor that plays critical roles in development. Sox4-null mice die at embryonic day 14 due to cardiac malformation. Recent studies have suggested that SOX4 may contribute to tumorigenesis, based on the fact that SOX4 is upregulated in many human tumor types, including brain, bladder, prostate, colon, and lung cancers. In addition, increased expression of Sox4 induced by retroviral insertional mutagenesis in mice has been shown to be associated with leukemia and lymphoma. We also found that SOX4 is highly expressed in human acute lymphoblastic leukemia (ALL) cells. We therefore set out in this study to determine the function of Sox4 and its underlying mechanisms in ALL. To overcome embryonic lethality caused by conventional Sox4 inactivation, we used Sox4 conditional null mice (Sox4fl/fl) to establish Sox4-null ALL cell lines. First, we obtained pure B220+ pro-B cells by co-culturing Sox4fl/fl fetal liver cells and OP-9 bone marrow stromal cells in the presence of IL-7. Subsequently, pro-B cells were transformed with p190-BCR-ABL and Sox4 inactivation was induced with Cre recombinase. We found that Sox4−/− pro-B transformed cells exhibited a significantly slower proliferation rate than Sox4+/− pro-B cells. Notably, cell cycle analysis indicated a lower percentage of S phase cells in Sox4−/− pro-B transformed cells and these cells yielded much fewer colonies than transformed Sox4+/− cells in colony formation assay. We further labeled the transformed cells with a luciferase reporter and injected them intravenously into sublethally irradiated NOD/SCID mice. Bioluminescence imaging showed that mice injected with Sox4−/− cells developed leukemia significantly more slowly and survive longer than mice injected with Sox4+/− cells. In conclusion, our data show that Sox4 significantly contributes to initiation and progression of leukemia from p190-BCR-ABL-transformed ALL cells in the mouse and thereby strongly support the notion that SOX4 overexpression critically contributes to acute lymphoblastic leukemia development in humans.

Disclosures:

No relevant conflicts of interest to declare.
MYC
+ Diffuse Large B Cell Lymphomas (DLBCL) Treated in Randomized Prospective Salvage Therapy, RICE or RDHAP Followed by BEAM Plus Autologous Stem Cell Transplantation (ASCT). A BioCORAL Report
Authors: Cuccuini Wendy , Briere Josette , Mounier Nicolas , Voelker Hans-Ullrich , Rosenwald Andreas , Sundstrom Christer , Cogliatti Sergio , Bron Dominique , Hirchaud Edouard , Soulier Jean , Gaulard Philippe , Houlgatte Remi , Gisselbrecht C. , Thieblemont Catherine
Published: October 2019

Abstract 594FN2

Background

. Approximately 5% to 10% of DLBCLs harbor a MYC/8q24 oncogene rearrangement. We determined the prognostic significance of MYC+ DLBCL in a selected population of patients with relapsed/refractory DLBCL included in the CORAL and prospectively treated by RICE or RDHAP followed by BEAM and ASCT1. METHODS. Among the 396 patients included, 150 patients were analysed with break-apart FISH probes for MYC/8q24, BCL2/18q21, and BCL6/3q27. Correlations with clinical characteristics at diagnosis and at relapse, immunohistochemistry (IHC) for CD10, BCL6, MUM1, FOXP1, and BCL2 expression, cell of origin determined according to different IHC algorithms2-5 and by transcriptomic analysis, response to treatment and survivals were analysed considering presence or absence of MYC rearrangement. RESULTS. Twenty-four (16%) cases exhibited a MYC rearrangement, associated in 87.5% (n=21) of the cases with a BCL2/18q21 rearrangement (n=16), with a BCL6/3q27 rearrangement (n=4), and with both BCL2/18q21 and BCL6/3q27 rearrangements (n=4). Among the matched cases analysed (n=57), biological characteristics including tumor immunophenotype and breakpoints occurrence, were identical between primary and secondary biopsies. Presence of MYC+ rearrangement was significantly associated with CD10 expression (p= 0.0067) and a GC phenotype, based either on Hans algorithm (p<104) or on Alizadeh GC signature (p=0.0015). Patients with MYC+ DLBCL presented significantly with more advanced aaIPI (p =.03). Sixty-three percent of them presented a time to relapse < 12 months, compared to 51% in patients with MYC− DLBCL (p=0.2925). Before HDT+ASCT, CR and ORR (PR+CR) were observed in 56% and 63% in patients with MYC+ DLBCL, compared to 75% and 87% in patients with MYC− DLBCL (p=0.3442). Outcome of patients with MYC+ DLBCL was significantly worse than MYC− DLBCL, with a 4-y PFS at 22% vs 45% (p =.0105) and a 4-y OS at 33% vs 62% (p =.0192). Type of treatment, RDHAP or RICE did not have any impact on survival with a 4-y PFS at 23% vs 22% (p=NS) and a 4-y OS at 44% vs 28% (p=NS), respectively. No difference of outcome was observed between patients with simple hit and with complex hits MYC+ DLBCL. CONCLUSION. MYC rearrangement is an early event in DLBCL and is significantly associated with a GC phenotype. Patients with MYC+ DLBCL presented with a more advanced disease, and have a significant inferior prognosis than patients with MYC− DLBCLs. Their outcome seems not influenced by the proposed salvage therapy.

Disclosures:

No relevant conflicts of interest to declare.
Development of a Novel Small Molecule CRM-1 Inhibitor for Non Hodgkin's Lymphoma
Authors: Azmi Asfar Sohail , Al-Katib Ayad , Aboukameel Amro , Kauffman Michael , McCauley Dilara , Mohammad Ramzi , Shacham Sharon
Published: October 2019

Abstract 598

Recent evidence demonstrates that Non Hodgkin's Lymphoma (NHL) tumors have elevated expression of the XPO1 gene that codes for the nuclear exporter protein CRM-1 controlling the localization of critical tumor suppressors including p53 family members. For effective apoptosis and cell cycle regulation, p53/p73 nuclear localization and DNA binding are highly critical making CRM-1 an attractive therapeutic target for NHL that carries >90% wild type/functional p53 and only rare mutations in p73. We have identified novel small molecule inhibitors of CRM-1 (KPTs) that bind irreversibly and lock target proteins (including p53 and p73) in the nucleus leading to apoptosis of tumor cells. We demonstrate for the first time that KPTs can induce apoptosis in resistant NHL cell lines and corresponding xenograft models. The most potent CRM-1 inhibitor (KPT-185) induced growth inhibition and apoptosis in a panel of NHL cell lines with a median IC50 ∼25 nM. Western blot and confocal microscopy analyses demonstrated that KPT-185 treatment resulted in nuclear localization of p53 in wt-p53 and p73 in mut-p53 cell lines. Additionally, we observed KPT-185 mediated activation of p21 and Bax, known downstream executioners of p53/p73 cell cycle control and apoptosis, respectively. Most significantly, siRNA knockdown of p53 in WSU-FSCCL and p73 in WSU-DLCL2 abrogated the apoptotic potential of KPT-185, confirming that these were indeed p53/p73 dependent apoptotic events. KPT-185 showed a substantial enhancement in apoptosis when combined with genotoxic p53/p73 re-activating regimen CHOP. The KPTs selectively kill cancer cells with minimal toxicity to normal tissues, and possess clinically acceptable pharmacokinetic parameters. Using WSU-DLCL2 SCID models, we further show that oral administration of related CRM-1 inhibitor KPT-276 (75 and 150 mg/Kg p.o) resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of KPT-251 (25 and 75 mg/Kg) resulted in 70 and 74% suppression of tumor growth with no observed toxicity to the host. Remnant tumor tissue analysis (histology and protein markers) fell in line with our in vitro results with clear activation of p73 pathway. Our study verifies CRM-1 as a potential therapeutic target in NHL irrespective of the functional status of p53. These results build a strong case for the clinical use of our novel CRM-1 inhibitors either as single agents or in combination with CHOP.

Disclosures:

Kauffman: Karyopharm: Equity Ownership. McCauley:Karyopharm: Employment. Shacham:Karyopharm: Equity Ownership.
Mini-Hepcidins Prevent Iron Overload In A Mouse Model of Hereditary Hemochromatosis
Authors: Nemeth Elizabeta , Ramos Emilio , Ruchala Peter , Preza Gloria , Ganz Tomas
Published: October 2019

Abstract 689

Mini-hepcidins are synthetic peptide analogues of the hepcidin N-terminus which is crucial for hepcidin interaction with ferroportin. Due to their small size and relative ease of synthesis, mini-hepcidins are better candidates than native hepcidin as therapeutics for the prevention and treatment of iron overload. In our previous studies, the first 9 amino acids of hepcidin (DTHFPICIF) were sufficient for in vitro activity (measured as ferroportin-GFP degradation). We modified the amino acid sequence of hepcidin-9 to improve resistance to proteolysis and to enhance the biophysical interactions with ferroportin. From >70 modified mini-hepcidins, we selected several that were more potent than native hepcidin in vitro, and tested them in mouse models. Bioactivity was assessed by measuring the hypoferremic effect of minihepcidins in C57BL/6 mice 4h after administration. Several minihepcidins administered by intraperitoneal or subcutaneous injection were more potent than the native hepcidin in reducing serum iron. Remarkably, retroinverted mini-hepcidins modified by palmitoylation or bile acid conjugation were active also by gavage. None of the tested mini-hepcidins altered endogenous hepcidin production. We also examined the ability of parenteral minihepcidins to prevent tissue iron loading in hepcidin knockout mice. Daily injections for 12 days completely prevented iron loading of the liver, decreased serum iron and increased splenic iron content. At high doses, minihepcidins were sufficiently potent that they caused iron-restricted microcytic anemia in hepcidin knockout mice. Minihepcidins may be useful for the treatment of human iron overload conditions caused by hepcidin deficiency.

Disclosures:

No relevant conflicts of interest to declare.
Thrombotic and Inflammatory Mediators in Cancer Patients Are Downregulated by Enoxaparin. Biochip and Protein Array Analysis
Authors: Hoppensteadt Debra , Litinas Evangelos , Khan Hussein , Cunanan Josephine , Thethi Indermohan , Iqbal Omer , Fareed Jawed
Published: October 2019

Abstract 713

Introduction:

The pathogenesis of cancer is known to upregulate inflammatory and thrombotic processes which contribute to the increased mortality in these patients. We hypothesized that the baseline inflammatory and thrombotic mediators are upregulated in cancer and treatment with LMWHs such as enoxaparin may downregulate these mediators. Methods: To test this hypothesis plasma samples were retrospectively analyzed from an open label multi dose active comparator parallel design study in which all patients (n=110) were initially treated with enoxaparin (1-1.5 mg/kg sc) for 5 days. These patients were subdivided into two groups. Group A continued to receive enoxaparin whereas Group B received warfarin. Baseline blood samples, 5 days and 12 weeks post treatment samples were analyzed using bio chip arrays (Randox analyzer) and protein chip array using surface enhanced laser desorption ionization (SELDI) technique.Results: In the cerebral biochip array analysis, levels of CRP, TNFRI, D DIMER, NGAL and TM were elevated at baseline which reduced after treatment with enoxaparin at three months except for NSE and TNFRI. In the cytokine biochip array, IL2, IL4, IL6, IL8, IL10, VEGF, IFNG, TNFA, IL1A, IL1B, MCP1 and EGF showed marked upregulation at baseline with enoxaparin treatment resulting in a decrease of IL6 alone. In contrast the warfarin treated group, both the cerebral and cytokine biochip array showed only marginal decreases.The baseline plasma samples from the patients recruited with multiple cancers in the Oncenox study showed a greater prevalence of the 11.6kDa biomarker (76%) with average amplitude of 23.6. The samples collected after 3 Months of enoxaparin treatment revealed markedly reduced prevalence (38%) and average amplitude of 5.4, whereas the warfarin treated group showed a modest decline in the 11-6kD a biomarker with a 52% prevalence and an average amplitude of 18.2. Conclusions: These results confirm that inflammatory and thrombotic mediators are differentially downregulated by treatment with enoxaparin in comparison to warfarin. The biochip and protein arrays provide unique tools to profile the known mediators and identify newer biomarkers. which may be useful in differentiating target sites for the newer drugs in cancer management.

Disclosures:

No relevant conflicts of interest to declare.
A “canonical” Npm1 mutation Knock-in Mouse Model Revealed Subtle but Definitive Myeloid Expansion with Poor HSC Niche Interaction
Authors: Chou Wen-Chien , Chou Shiu-Huey , Chiou Ji-Shain , Ko Bor-Sheng , Lin Shu-Wha , Hsu Yueh-Chwen , Tien Hwei-Fang
Published: October 2019

Abstract 762

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein, which has nucleocytoplasmic shuttling activity. Somatic mutations in NPM1 gene result in cytoplasmic dislocation of NPM1 (NPM1c) and are frequently associated with acute myeloid leukemia (AML). The pathogenetic effects of mutated NPM1 protein have been explored by animal models including transgenic or “humanized” knock-in mouse models. Here, we demonstrate the first “canonical” mouse Npm1 mutant knock-in model. Different from the previously report of humanized NPM1 mutant knock-in model, we inserted TCTG after nucleotide c.857 of murine Npm1 coding sequence (c.854–857dupTCTG), a pattern identical to human NPM1 mutation, without any “humanized” sequence. This mutation caused a shift of peptide sequence from WQWRKSL* (amino acid 286–292) to LCLAVEEISLRKGFKQFEIFCLHFCNS* (amino acid 285 to 311), a pattern mildly different from the change in human NPM1 mutation, but is still predicted to generate a nuclear export signal. NPM1c+ genotype and protein accumulation in cytoplasm were confirmed with PCR and immunocytochemistry, respectively. We found that the homozygous NPM mutant (NPMc+/c+) mice were embryonic lethal before E10.5 day, while hetrozygote (NPMwt/c+) mice survived and were fertile, and born with Mendelian ratio. Most NPMwt/c+ mice had normal hematologic parameters and remained disease-free, however, these mice developed a delayed-onset aberration on the distribution of granulocyte-monocye progenitors (GMP), monocytes, and B lymphocytes in blood, spleen, and bone marrow. Colony forming unit assay showed normal hematopoietic development of marrow hematopoietic stem cells (HSC), but poor cobblestone formation while HSCs contacted with stroma microenvironment in NPMwt/c+ mice, suggesting the NPMc mutant may affect the ability of HSCs on contact signal expression. Three (12.5%) of 24 NPMwt/c+ mice developed leukocytosis and splenomegaly mimicking myeloproliferative neoplasm of human. Microscopic examination showed panmyelosis of the bone marrow and existence of hematopoiesis in the spleen. In addition, the immune activities of NPMwt/c+ mice's splenocytes and thymocytes with mitogen stimulation were decreased. In summary, our “canonical” NPMwt/c+ mouse model demonstrated subtle but definitive phenotypes in hematopoietic cells and provided insight into the pathogenesis of NPM1 mutation in human acute myeloid leukemia. A second hit may be necessary for the development of AML in NPMwt/c+ mice since no AML was detected in these mice till 20 months of age.

Disclosures:

No relevant conflicts of interest to declare.
Role of Microparticles in Hemostasis and Thrombosis
Authors: Mackman Nigel
Published: October 2019

Abstract SCI-10

Microparticles (MPs) (also known as or microvesicles) are submicron plasma membrane vesicles released from activated or apoptotic cells. Current estimates indicate that MPs are ∼200 nm in diameter. They are formed as membrane blebs and have phosphatidylserine exposed on the surface due to a loss of membrane asymmetry. MPs containing tissue factor (TF) are highly procoagulant. We recently developed an assay to measure TF activity of MPs isolated from plasma. We found that healthy individuals contain low levels of TF+ MPs, but levels are increased in many pathological conditions associated with thrombosis, such as sepsis, cardiovascular disease, sickle cell disease, diabetes mellitus, and cancer. We have analyzed the role of TF+ MPs in various murine models of thrombosis and in different patient populations. Work in collaboration with Bruce and Barbara Furie demonstrated that leukocyte-derived TF+ MPs contributed to microvascular thrombosis in a model of laser injured cremaster arterioles using healthy mice. Further studies have been performed using different murine models of disease. Arterial thrombosis is mostly caused by rupture of atherosclerotic plaques and leads to myocardial infarction and stroke. In hypercholesterolemic LDLR−/− mice we observed increased levels of hematopoietic cell-derived TF+ MPs that were associated with a prothrombotic state. These TF+ MPs may enhance thrombosis after plaque rupture. Indeed, we observed more fibrin deposition in the laser injury model of thrombosis in hypercholesterolemic mice than controls. Importantly, we found that simvastatin reduced TF expression in mononuclear cells and levels of TF+ MPs in the plasma in these hypercholesterolemic mice. Venous thrombosis occurs without significant damage to the vessel wall and is associated with embolization of clot fragments that can block pulmonary arteries. Venous clots appear to form due to changes in the vessel wall, stasis, and/or increased coagulability of the blood. We have analyzed levels of TF+ MPs in murine cancer models and cancer patient samples. We and other have found that tumors release TF+ MPs into the circulation. In one study, we found that pancreatic patients with the highest levels of MP TF activity develop thrombosis. These results suggest that TF+ MPs may enhance venous thrombosis in cancer patients by binding to activated endothelium. Further studies are needed to determine if levels of TF+ MPs in the circulation can be used to identify patients at risk for thrombosis.

Disclosures:

Mackman: Merck: Consultancy; Daiichi: Consultancy; Daiichi: Research Funding.
State-of-the-Art and Current Controversies in the Detection and Quantitation of Tissue Factor-Bearing Microparticles
Authors: Zwicker Jeffrey
Published: October 2019

Abstract SCI-11

Microparticles are submicron vesicles that circulate in blood and are derived from a variety of vascular cells. Microparticles expressing tissue factor have been shown in animal thrombosis models to contribute to thrombus formation in vivo, which has led to the evaluation of tissue factor bearing microparticle populations in a variety of thrombotic disorders including malignancy, sepsis, and coronary thrombosis. Central to the evaluation of tissue factor-bearing microparticle populations in health and disease is the establishment of accurate means for qualitative or quantitative assessment of circulating microparticles. Whilst there is considerable interest in evaluating whether pathologic disturbances in tissue factor-bearing microparticles contribute to thrombosis, analytic techniques for microparticle measurement remain in the validation stage. The method most commonly employed for the detection of tissue factor-bearing microparticles is light-scatter flow cytometry, which relies on the sizing of particles based on light-scatter profile. Light scatter, however, is also influenced by variables independent of particle size, including the wavelength of incident light, particle shape, presence of surface absorptive material, and relative refractive indices of particles and suspension medium. A number of potentially more sensitive methodologies are currently under investigation such as impedance-based flow cytometry, quasi-elastic light scattering based on Brownian movement, and atomic force microscopy. Alternatively, assays have been developed that measure tissue factor antigen or activity following isolation of microparticles from plasma or whole blood but without determination of particle size. Absent a gold standard for the measurement of tissue factor-bearing microparticle populations by which these approaches can be validated, the development of accurate methodology will require inter-assay and intra-assay precision as well as validation of clinical relevance in human studies.

Disclosures:

No relevant conflicts of interest to declare.
Autophagy and Metabolism in Lymphoid Malignancies
Authors: Distelhorst Clark W.
Published: October 2019

Abstract SCI-26

Cell stress, if left unchecked, leads to apoptosis and autophagy. Both are highly conserved, genetically encoded processes. Apoptosis is a programmed death mechanism, designed to eliminate cells that are defective or no longer needed as part of developmental programs. Autophagy, on the other hand, is a programmed survival mechanism designed to provide cells a temporary source of energy when nutritionally deficient. Since in autophagy (“self eating”) the cell generates energy at the expense of its own organelles, temporary survival leads to death if nutrition is not restored. Both apoptosis and autophagy are induced by glucocorticosteroid hormone (GH) treatment of lymphoid malignancies. Since the main effects of GH on lymphocytes are metabolic, it is likely that apoptosis and autophagy are responses to metabolic stress. Metabolic stress occurs because GH inhibits both glucose uptake and metabolism, as well as calcium signals required for optimal mitochondrial function. Gene expression profiling has provided insight into the complex pathways that mediate apoptosis and autophagy in GH-treated leukemia and lymphoma cells. Ultimately, the balance of pro-apoptotic and anti-apoptotic factors determines cell fate. Elevation of the pro-apoptotic protein Bim is a critical step in apoptosis induction by GH and is due to GH-mediated repression of a microRNA cluster (miR17-95) that normally represses Bim expression. In metabolically stressed cells, Bim elevation is counterbalanced by a proton-sensing G protein-coupled receptor, TDAG8, that signals an elevation of the anti-apoptotic protein Bcl-2, counterbalancing Bim elevation and inhibiting apoptosis. Gene expression profiling also provided insight into the mechanism of autophagy by detecting GH-mediated repression of the Src kinases Fyn and Lck and GH-mediated elevation of Dig2 (REDD1, RTP801). Fyn and Lck interact with the inositol 1,4,5-trisphosphate calcium channel and promote physiological calcium signals. By decreasing Fyn and Lck, GH inhibits calcium signals necessary for optimal mitochondrial metabolism, contributing to autophagy induction. Dig2 is a stress protein that inhibits mTOR signaling. The absence of the Dig2 stress response in Dig2 knockout mice significantly impairs autophagy induction, decreasing lymphocyte survival in GH-treated mice. In summary, both apoptosis and autophagy occur in lymphoid malignancies in response to metabolic stress, as illustrated by the effects of GH treatment. A goal for future studies is to tie these signaling components together into a logical signaling network and to target components of the signaling network for therapeutic purposes.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Effects of IDH1/IDH2 Mutations
Authors: Melnick Ari M. , Levine Ross L , Figueroa Maria E , Thompson Craig B. , Abdel-Wahab Omar
Published: October 2019

Abstract SCI-33

Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ∼20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss-of-function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCpG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using α-ketoglutarate (αKG) as a substrate. 2HG can disrupt the activity of enzymes that use αKG as a cofactor, including TET2 and the jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibits TET2 hydroxymethylation and jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards increased histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is in abundance in hematopoietic stem cells and displays specific distribution patterns, yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning.

Disclosures:

Thompson: Agios Pharmaceuticals: Consultancy.
Platelets, Obesity, and the Metabolic Syndrome
Authors: Freedman Jane
Published: October 2019

Abstract SCI-34

Cardiovascular disease continues to be the major cause of morbidity and mortality in North America and a growing problem in many other parts of the world. The risk of premature atherosclerosis and cardiovascular events rises with increasing obesity and excess adiposity has been linked to increased all-cause mortality. Current estimates show that one in four ischemic heart disease events are attributable to excess weight. Inflammation of adipose tissue orchestrated by monocyte/macrophage infiltration and overproduction of proinflammatory and proatherogenic cytokines may mediate metabolic dysfunction and vascular disease in human obesity. Importantly, obesity has been associated with increased cardiovascular disease as well as enhanced platelet function and thrombosis. We recently measured expression of 48 genes by high-throughput quantitative reverse transcription PCR (qRT-PCR) in over 2250 participants of the Framingham Offspring and Omni minority cohorts (FHS) using RNA from isolated platelets and found that specific inflammatory transcripts including ICAM1, IL1R, IFNG, IL6, COX2, TNF, TLR2, and TLR4 were significantly and positively associated with higher body mass index (BMI) by regression analysis. With the additional measurement of 196 genes, the data has remained extraordinarily consistent. Other cardiovascular risk factors rarely associate with platelet gene expression and BMI remains specifically associated with inflammatory transcripts related to the NFkB-innate immune and inflammatory pathways. These surprising data led to the question: Why would the anucleate platelet have gene expression changes specifically in the setting of increased BMI and can this powerful clinical association be explored using both pre-clinical and clinical studies to find new targets for disease and define new mechanisms for platelet function beyond thrombosis?

Disclosures:

No relevant conflicts of interest to declare.
Oxygen Availability and Stem Cells: Impact On Development and Disease
Authors: Simon M. Celeste
Published: October 2019

Abstract SCI-37

Stem and progenitor cells reside in specialized microenvironments that regulate their function. While some stem/progenitor cells are perivascular, others clearly occupy hypoxic niches and may be regulated by O2 gradients. We are currently evaluating underlying mechanisms for the impact of O2 levels on stem and progenitor cells within distinct microenvironments. We have previously shown that neural stem cells within the adult hippocampus are closely associated with low O2 regions and that hypoxia-inducible factor 1α (HIF-1α), a principle mediator of hypoxic adaptations, modulates Wnt-β catenin signaling to maintain stem cell proliferation, differentiation, and neuronal maturation. We have extended these findings to other stem cell populations, such as those of adult muscle and bone marrow. Our findings will be presented at this meeting.

Disclosures:

No relevant conflicts of interest to declare.
Small Molecule Screens for Stem Cell Expansion
Authors: Cooke Michael P. , Boitano Anthony E.
Published: October 2019

Abstract SCI-42

The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening >100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a >600-fold increase in the number of CD34+ cells, and a >2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use.

Disclosures:

Cooke: Novartis: Employment. Boitano:Novartis: Employment.
Hemophilia Gene Therapy
Authors: Arruda Valder R.
Published: October 2019

Abstract SCI-48

Over the last decade, five clinical studies on gene therapy for hemophilia A and B using viral and nonviral vectors for liver-restricted or ectopic expression of clotting factor demonstrated safety with no inhibitor formation. Although efficacy in these early phase trials was not achieved because of sustained levels of clotting factor below 1%, these studies form the basis for the second generation of clinical trials. To date, two studies on adeno-associated viral (AAV) vector encoding a FIX transgene for liver-restricted expression are ongoing. In one study using a modified AAV genome pseudotyped with serotype 8, emerging data are encouraging and sustained therapeutic levels of FIX have been obtained in a dose-dependent manner. Novel approaches for hemophilia have been explored targeting hematopoietic stem cells (HSC) using lentiviral vectors for expressing FVIII or FIX genes. Transgene expression under the control of either a non-lineage specific promoter or a platelet-specific promoter showed biological activity and improvement of the disease phenotype. The platelet-restricted approaches did not increase circulating plasma clotting factor levels, but they resulted in enrichment of the factor at the injury site upon platelet activation. An advantage of targeting FVIII expression and storage in platelets is the protection of FVIII from neutralizing antibodies (inhibitors). In this model, platelet-FVIII provided superior hemostasis than elevated plasma FVIII levels upon hemostatic challenges in the microcirculation and macrocirculation. Recently, platelet-restricted expression of human FVIII gene using a lentiviral vector for ex vivo transduction of canine HSC resulted in improvement of the disease phenotype in severe hemophilia A dogs without unwanted immune responses to the transgene. Additional strategies to optimize gene- and/or cell-based approaches have also focused on use of clotting factors with enhanced biological activities will be discussed. Together with the generation of novel vectors, further enhancement of both the efficacy the safety of these approaches is envisioned.

Disclosures:

Arruda: Pfizer: Research Funding.
Ataxia Telangiectasia
Authors: Gatti Richard A.
Published: October 2019

Abstract SCI-7

Ataxia-telangiectasia (A-T) is the prototype for an expanded group of inherited radiation sensitive disorders that together define the XCIND syndrome: x-ray hypersensitivity, cancer, immunodeficiency, neurological dysfunction, and DNA repair deficiency. Although the clinical radiosensitivity of these disorders can be tested in the clinical laboratory, diagnostic methods remain limited and in need of further validation. Without exception, to date, sensitivity to ionizing radiation appears to be integrally associated with double strand break (DSB) repair defects and lymphoid cancer susceptibility, setting these disorders apart from single strand break repair disorders such as xeroderma pigmentosum. Responding within seconds to DSB damage are ATM kinase, the protein lacking in A-T, and the NMR complex (nibrin, Mre11, and Rad50). The latter three proteins are associated with three additional XCIND disorders (nibrin deficiency [aka nijmegen breakage syndrome], Mre11 deficiency [ATLD], and Rad50 deficiency). ATM kinase activates a myriad of other proteins that 1) halt DNA synthesis, replication, and the progression of the cell cycle; 2) form a complex protein “mesh” to physically stabilize the broken DNA strands; and 3) restore the integrity of the breaks before they unravel to create even larger chromosomal lesions and resulting malignancies. Another ATM-dependent cancer link involves the downregulation of ATM by microRNA-421. MicroRNA-421 is upregulated by the transcription factor N-myc. Despite this, neuroblastomas are not commonly observed in A-T or XCIND patients. Another subset of XCIND-associated disorders lacks proteins the drive the nonhomologous end joining pathway of DNA repair. Several of these diseases present in infancy as B−/T− severe combined immunodeficiency, or SCID, and are frequent candidates for stem cell transplantation. Attempts to ablate existing bone marrow prior to transplantation may further compromise such patients if they are inherently radiosensitive. Thus, attempts to preselect such patients and reduce radiation dosages may improve general post-transplantation survival. While most protein deficiencies can be diagnosed by immunoblots of appropriate cellular fractions, nonfunctional proteins are not detected by this platform. Colony survival assays (CSA) measure the ability of replicating cells (e.g., lymphoblasts or fibroblasts) to survive after exposure to radiation. Although causal proof that CSA can predict clinical radiosensitivity is lacking, the reduced percent survival fraction (i.e., radiosensitivity) of A-T, N-Bromosuccinimide, or Fanconi cell lines can be abrogated by introducing the mutated cognate gene. Other surrogate assays for radiosensitivity include kinetic studies, pre-irradiation and post-irradiation of γ-H2AX or SMC1 phosphorylation. Ultimately, DNA sequencing of a candidate gene can pinpoint the underlying pathogenesis of radiosensitivity in an XCIND disorder.

Disclosures:

No relevant conflicts of interest to declare.
Leukocyte Immunoglobulin-Like Receptor B Regulates Erythroblastic Island Size in Human in-Vitro Erythroblastic Islands
Authors: Perumbeti Ajay , Girnita Alin , Cancelas Jose A.
Published: October 2019

Abstract 3190

Erythropoiesis occurs in a unique bone marrow (BM) microenvironment known as erythroblastic islands. Erythroblastic islands are composed of erythroid precursors in close contact with specialized stromal macrophages, also referred to as central or island macrophages. Human erythroblastic island macrophages have been difficult to discern from other macrophage subpopulations by cell surface markers, although they have reduced expression of C3b receptor (CD35) (Lee et al., 1988). The unique non-antigen presenting nature of erythroblastic island macrophages led us to hypothesize that it may share surface receptors of other tolerogenic macrophages subpopulations, including leukocyte immunoglobulin-like transcript B receptors (LILRB).

We first identified a distinct subset of human bone marrow macrophages with flow cytometry, which express high amounts of LILRB1 and LILRB2 surface receptors, have low expression of C3b surface receptor (p<0.05), and have increased expression of VCAM-1 surface receptor. This macrophage subpopulation consisted of 1 in 500 (0.02%) of bone marrow mononuclear cells. Functionally, when we FACS sorted human BM enriched erythroid precursors (CD45−/CD71+) and combined them with FACS sorted LILRB1+/LILRB2+ (LILRB+) or LILRB1−/LILRB2- (LILRB−) BM macrophages, there were no significant differences in erythroblastic island reconstitution, suggesting that the adhesion of primary BM erythroblasts to island macrophages is not related to macrophage LILRB expression. Since our ex-vivo erythroid population was enriched for late precursors, we tested island reconstitution with CD34+ cell-derived cultured erythroblasts and FACS sorted LILRB+ or LILRB- BM macrophages, which resulted in a significantly higher number of large proliferative erythroblastic islands (>25 erythroblasts/island macrophage) in LILRB+ compared with LILRB- macrophages (p<0.05) (Figure 1). Incubation with neutralizing anti-LILRB1/LILRB2 antibodies did not prevent primary erythroblastic island reconstitution independent of expression of LILRB by macrophages. However, anti-LILRB1/LILRB2 did prevent the formation of large erythroblastic islands indicating a specific role of LILRB in optimal island reconstitution and erythropoiesis (p<0.05) (Figure 1). In conclusion, we demonstrate that LILRB+ BM macrophages increase the formation of large in-vitro erythroblastic islands and LILRB receptors are important candidate receptors for functional regulation of erythropoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Immunosuppressive Drugs up-Regulate the Immunoinhibitory Receptor Osteoactivin On Human Monocyte-Derived Dendritic Cells
Authors: Braun Sabine , Gutknecht Michael , Schwarzbich Mark-Alexander , Kanz Lothar , Salih Helmut R , Grünebach Frank , Rittig Susanne M
Published: October 2019

Abstract 3284

Introduction:

Dendritic cells (DC) abundantly express the type I transmembrane glycoprotein Osteoactivin (OA) - also known as transmembrane glycoprotein NMB and DC-HIL - compared to low expression levels on monocytes. Antigen-presenting cells interact via OA with the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells which inhibits T cell activation. We previously reported on increased expression of OA upon exposure of monocyte-derived DC (moDC) to immunosuppressive drugs (e.g., Gutknecht et al ASH annual meeting 2011). Here we extended these analyses and comparatively analyzed the impact of various immunsuppressive drugs (ID) on moDC phenotype and function.

Methods:

The moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Clinically relevant concentrations of ID were added to the culture medium every second day starting with the first day of culture (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml, mycophenolat-mofetil 0.1μM, methotrexat 230ng/ml). Cells were harvested for immunophenotyping by flow cytometry, western-blotting and real-time PCR. Cytokine release by moDC was determined on day 7 by ELISA. Functional properties were determined by mixed lymphocyte reactions (MLR) on day 7 of culture.

Results:

Exposure of moDC to therapeutic concentrations of prednisolone resulted in significantly reduced expression of the costimulatory molecules CD83 and CD86 and increased levels of the monocyte marker CD14, indicative of impaired differentiation. Tacrolimus significantly increased CD14 expression and reduced CD83 expression, while the other ID did not cause significant alterations. All ID altered the release of the immunomodulatory cytokines IL-10, IL-6 and TGF-ß. Notably, all ID except cyclosporine A caused a substantial upregulation of the immunoinhibitory receptor OA in moDC. The extent of OA expression increased over time of exposure to ID during differentiation and resulted in reduced capacity of the moDC to stimulate allogenic T cells which could be restored by disruption of OA/SD-4 interaction using a blocking OA antibody.

Conclusion:

Increased expression of OA on moDC upon exposure to ID contributes to inhibition of T-cell activation. The mechanisms underlying the differential effect of cyclosporine A are presently under study. Our results indicate that targeting OA/SD-4 interaction may hold promise for modulation of T cell responses in various pathophysiological conditions and immunotherapeutic strategies.

Disclosures:

No relevant conflicts of interest to declare.
C5a Depletion As a Novel Strategy to Inhibit T Regulatory Cells and Enhance Antigen-Specific Immune Responses
Authors: Veeramani Suresh , Weiner George J.
Published: October 2019

Abstract 3285

Background:

The complement system has complex activity that impacts on the immune response in a broad variety of ways. The current study was designed to assess the effect of complement components, specifically C5a, on the immune regulatory cells and on the development of an antigen-specific active immune response.

Methods:

Myeloid dendritic cells (mDCs), enriched from healthy human peripheral blood mononuclear cells, were pulsed with antigen (tetanus toxoid) and co-cultured with autologous, enriched human CD4+ T cells in the presence of various purified complement components. The percent of CD4+ T-cells that were CD25highFoxp3+ (henceforth referred to as Tregs) was determined. The presence of cytokines in supernatant of mDCs cultured with purified complement proteins was also evaluated. In murine models, the effect of C5a on in vivo induction of Tregs and on the development of immune response to ovalbumin was determined by analyzing anti-ovalbumin antibody. This was done in C5-sufficient (B10-D2-HC1) and C5-deficient (B10-D2-HC0) mice immunized with 100 μg of ovalbumin, and in wild type C57Bl/6 mice immunized with 100 μg of ovalbumin along with either irrelevant rat IgG2a (Ova+Isotype control) or rat anti-mouse C5a antibody (Ova+anti-C5a Ab).

Results:

In Vitro:

In Vivo:

Conclusions:

Presence of C5a in the immune microenvironment results in increased generation of Treg cells and leads to dampening of antigen-specific immune responses. Absence or depletion of C5a results in a drop in the Tregs and a higher antigen-specific immune response. Ongoing studies are exploring the use of C5a depletion as a novel strategy to overcome the low immunogenicity of vaccines, such as cancer vaccines.

Disclosures:

No relevant conflicts of interest to declare.
TLR3 Activation Is Involved in Venous Thrombosis Development
Authors: Lemarie Catherine A , Le Angela , Aghourian Meghedi , Wu Jianqiu , Blostein Mark D
Published: October 2019

Abstract 3309

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. The pathophysiology of VTE is now better understood from experimental studies. Leukocytes, chemokines, and proinflammatory cytokines are involved in VTE resolution and vein wall healing. This process resembles sterile wound healing with distinct phases of polymorphonucleic neutrophil and monocyte influx followed by fibrosis. Inflammation is emerging as a central mechanism in both the genesis and resolution of VTE. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3 senses dsRNA, a by-product of viral replication, in the endosome. In addition, TLR3 has been increasingly linked to tissue damage. Endogenous RNA released by damaged tissue or necrotic cells is able to induce TLR3 expression and signaling. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of VTE. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog, increases the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle control (p<0.05). In TLR3 deficient (TLR3−/−) mice, polyI:C did not induce a further increase in thrombus size compared to vehicle control. Recently, neutrophils have been shown to initiate and propagate venous thrombosis. PolyI:C injection was associated with an increased of neutrophil infiltration in the thrombus in WT but not in TLR3−/− mice (p<0.05). We found that polyI:C injection was associated with increased neutrophil activation. In the thrombus of WT mice, immunofluorescence staining for myeloperoxidase and citrulinated H3, markers for neutrophils activation, were increased by polyI:C. Interestingly, in TLR3−/− mice, no increase of these markers was found after polyI:C injection as compared to vehicle control. In vitro incubation of endothelial cells with polyI:C induces production of pro-inflammatory cytokines IL-8 and CCL5 involved in the recruitment of neutrophils as demonstrated by a cytokine array. We found that mRNA expression of TLR3, IL-8 and CCL5 were dramatically increased with polyI:C. When endothelial cells were transfected with siRNA for TLR3, mRNA expression of TLR3, IL-8 and CCL5 was blunted in response to polyI:C. These results suggest that release of IL-8 and CCL5 from endothelial cells after TLR3 activation are involved in neutrophil recruitment. Taken together, these results strongly suggest that TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment and activation of neutrophils.

Disclosures:

No relevant conflicts of interest to declare.