bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Nucleotide Polymorphisms (SNPs) Associated with Pulmonary Embolism (PE): A Genome-Wide Association Study (GWAS)
Authors: Heit John A , Armasu Sebastian M. , de Andrade Mariza
Published: October 2019

Abstract 1148

Background:

Factor V Leiden (F5rs6025) has been suggested as a disease-susceptibility SNP for deep vein thrombosis (DVT) but not PE. Given the significantly reduced survival after PE, identifying individuals at risk for PE is important for targeting prophylaxis and improving survival.

Objectives:

To identify PE disease-susceptibility genes.

Methods:

We performed in silicoGWAS analyses of VTE cases using genotype data imputed to ∼2.5 million SNPs from adults with objectively-diagnosed PE ± DVT (n=745), and DVT only (n=758). We tested these SNPs for an association with PE ± DVT after adjusting for age, sex, stroke and/or myocardial infarction, and USA state of residence using unconditional logistic regression.

Results:

Of all SNPs, three were most strongly associated with PE ± DVT: one at BDH1 on chromosome (chr) 3q29 (rs1309873, OR=1.56, p=4.02E-07) and two at SLC39A10 on chr 2q32.3 (rs7578216, OR=2.55, p=4.57E-07; and rs4589778, OR=2.49, p=7.52E-07). Individually adjusting for F5 rs6025 (chr 1), F2 rs1799963 (prothrombin G20210A; chr 11) and ABO rs8176719 (ABO blood type O allele; chr 9) did not materially affect these associations. None of these three SNPs associated with PE were intragenic. BDH1 encodes for 3-hydroxybutyrate dehydrogenase 1, a key mitochondrial enzyme in fatty acid catabolism, and SLC39A10encodes for solute carrier family 39, member 10, an important protein for zinc transport.

Conclusions:

Chromosomes 3q29 (BDH1) and 2q32.2 (SLC39A10) may harbor PE disease-susceptibility gene regions (genes). These findings require independent confirmation. If confirmed, additional studies addressing the biologic mechanisms through which these genes and/or gene regions operate to increase PE risk also will be required.

Disclosures:

No relevant conflicts of interest to declare.
Tolerated, Low-Dose Deferasirox: Successful Three Cases of Aplastic Anemia and 34 Cases of MDS
Authors: Hirai Manabu , Yamamura Ryousuke , Nakamae Hirohisa , Terada Yoshiki , Inoue Atsushi , Hashimura Mitsuharu , Ichihara Hiroyoshi , Sakamoto Erina , Hino Masayuki
Published: October 2019

Abstract 1187

It has been shown that serum ferritin (SF) levels are associated with mortality in transfusion-dependent patients with MDS. Optimal dosing with deferasirox has been shown to decrease SF level even in transfusion-dependent patients and to be able to decrease the adverse effects of repeated transfusion. Previous studies have however shown that about 50% of patients withdraw from treatment because of side effects in the early phase of treatment.

Low-dose deferasirox (mean 6.3 mg/kg, range 1.6∼25.6) was administered to 53 patients (MDS 34, AA 13, ALL 2, MF 2, AML 1,ML 1) with dose escalation up to the maximum dose tolerated (mean 9.8 mg/kg, range 1.6∼31.0). In 47 eligible cases, a mean SF of 1579 ng/ml (range 320∼4477) decreased to 1101 ng/ml (range 233∼1680) at 18 months with 71% of continuous rate. Just six cases had treatment stopped, solely because of renal dysfunction. In three cases with transfusion-dependent aplastic anemia, deferasirox (6.25∼10.8 mg/kg) was given with G-CSF, cyclosporin A, and metenolone with the successful abreaction from transfusion dependency and even with elevation of the platelet count (from 10 to 30∼50×109/L).

Continuous administration of low-dose deferasirox, as tolerated, was thought to be sufficient to prevent a rise in SF in transfusion-dependent patients. A prospective study that will examine OS in such MDS patients has been started.

Disclosures:

No relevant conflicts of interest to declare.
Rescue of the HSC Maintenance Defects in Ku70-Deficient Mice by Overexpression of Bcl2 Reveals a Novel Role of Bcl2 in HSC
Authors: Qing Yulan , Wang Zhengqi , Matsuyama Shigemi , Bunting Kevin D. , Gerson Stanton L.
Published: October 2019

Abstract 1235

Ku70 is a key component of the non-homologous end joining (NHEJ) pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. We find that HSC from Ku70-deficient mice are severely impaired in maintenance, defective in self-renewal, competitive repopulation and BM hematopoietic niche occupancy. HSCs from Ku70-deficient mice are not prone to spontaneous apoptosis, however, they display more active proliferation, and fewer cells in a quiescent state than HSC obtained from WT mice. Further, gene expression profiling showed that multiple HSC quiescence- related genes such as c-MPL and p57 were significantly downregulated in Ku70-deficient HSCs. These data suggested that loss of quiescence results in the dramatic defect in the maintenance of Ku70-deficient HSC.

Bcl2 plays important roles in both anti-apoptosis and anti-proliferation, while the anti-apoptosis function of Bcl2 has been extensively studied and well established in various settings, including HSCs, the anti-proliferation function of Bcl2 in HSC has yet to be investigated. To exam whether overexpression of Bcl2 can rescue the HSC defect in Ku70-deficient mice, H2K-Bcl2 transgenic/Ku70-deficient double mutant mice were generated. Though overexpression of Bcl2 does not rescue the SCID phenotype in Ku70-deficient mice, overexpression of Bcl2 in Ku70-deficient HSCs almost completely rescued the impaired HSC quiescence, repopulation and BM hematopoietic niche occupancy capacities. At the transcriptional level, overexpression of Bcl2 restored the expression of c-MPL and p57 comparable to WT levels. However, additional deletion of Bax, a pro-apoptosis protein in the Bcl2 family, in Ku70-deficient mice could not rescue the HSC repopulation defect, suggesting that apoptosis is not the main mediator for the impaired HSC maintenance in Ku70-deficient mice.

Together, our data indicate that the HSC maintenance defect of Ku70-deficient mice is due to the loss of HSC quiescence populations, whereas overexpression of Bcl2 rescues the HSC defect in Ku70-deficient mice by restoration of quiescence. Our study uncovers a novel role of Bcl2 in hematopoietic stem cell quiescence regulation.

Disclosures:

No relevant conflicts of interest to declare.
Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells
Authors: Lenzo Rebecca , Dua-Awereh Martha , Carroll Martin , Shetzline Susan E.
Published: October 2019

Abstract 1241

Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents.

Disclosures:

No relevant conflicts of interest to declare.
Identifying an Acetylation Signature Following Vorinostat Treatment in AML
Authors: Hay Jodie F , Liberante Fabio G , McCourt Clare , O'Hagan Kathleen A , Mills Ken I
Published: October 2019

Abstract 1291

Acute myeloid leukemia (AML) remains incurable for most patients, particularly in the elderly, and novel therapeutic approaches are needed. Genes are often epigenetically silenced in AML through the recruitment of histone deacetylases (HDACs) and the development of agents that inhibit HDACs (HDACi) are a novel treatment approach to AML. However, AML represents a spectrum of mutations and chromosomal abnormalities and it would be of benefit to target these therapies based on epigenetic profiles and responses. One HDAC inhibitor, Vorinostat (Zolinza™), is currently being used in phase i/ii clinical trials as a therapeutic strategy in multiple myeloma with evidence that combinational therapies provide an extended disease free survival; this could be an attractive option for AML. In order to find a successful combination, a fuller characterisation of Vorinostat induced histone modifications is required.

In this study, we used a combination of ChIP-seq and microarray approaches to identify epigenetically silenced genes pathways in AML cells that could be restored by treatment with the HDACi Vorinostat.

OCI-AML3 cells were treated with Vorinostat 1 μM or control conditions for 24 hours. Changes in gene expression were measured using Affymetrix Human Genome U133 Plus 2.0 and/or Leukemia AmpliChip arrays. The ChIP-seq was done on DNA extracted from the same cells and time points as for the expression study. The chromatin was cross-linked and the protein-DNA interactions at H3K9Ac were elucidated by chromatin immunoprecipitation, with anti-acetylated H3K9 histone and Pol II antibodies. Pol II was used to identify regions of actively transcribed DNA, and high throughput sequencing using the Ion Torrent PGM (Life Technologies).

After treatment with Vorinostat, expression studies showed that 180 genes were up-regulated and 441 genes were down-regulated (FC 4, p<0.05). The ChIP-seq data identified DNA sequences that were associated with de-acetylated histones following Vorinostat treatment. Bioinformatic analysis identified regions that were also associated with actively transcribed regions of DNA. Pathway analysis identified that the principle pathways induced by Vorinostat were TGFβ signalling and glycosaminoglycan degradation. Gene set enrichment analysis showed that Vorinostat down-regulated the integrin mediated signalling and p53 binding, and up-regulated stem cell differentiation, blood vessel morphogenesis and response to fibroblast growth factor stimulus.

This study would indicate that Vorinostat is an effective regulator of gene expression in AML cells and that the integrated approach can identify specific epigenetic targets of Vorinostat and could lead to the development of tailored AML therapies.

Disclosures:

No relevant conflicts of interest to declare.
Notch/HES1 Signaling Stabilizes p53 Expression Via a Novel PARP1-Chfr-PLK1-Mediated Mechanism in B-ALL
Authors: Kannan Sankaranarayanan , Zweidler-McKay Patrick A
Published: October 2019

Abstract 1292

Background:

Notch signaling contributes to T cell leukemogenesis. However, we have found that activation of Notch signaling in human B-ALL promotes growth arrest and apoptosis. These contrasting effects of Notch in B versus T cell ALL, mirror effects seen in early lymphocyte development. As the Notch receptors are common between T and B cells, we hypothesized that these differences rely on the cell-type specific downstream mechanisms. We previously reported a critical role for Notch/HES1-mediated activation of Poly ADP-Ribose Polymerase 1 (PARP1) function in this B cell specific mechanism.

Approach:

To explore the cell-type specific downstream mechanisms of Notch activation in B-ALL, we used cell fractionation, westerns and immunoprecipitation to identify cell cycle regulators which were altered by Notch activation via HES1 expression in human B-ALL lines.

Results:

Notch activation in a panel of human B-ALL lines led to consistent growth arrest and apoptosis. Indeed, ligands, activated receptors and the Notch target gene HES1 all induced these leukemia lihibiting effects in B-ALL but not T-ALL lines. In this study we report a mechanism whereby HES1-mediated activation of PARP1 leads to PARylation of the E3 ligase Checkpoint with FHA and RING finger (CHFR) (Panel A) which results in targeting and ubiquitination of the cell cycle regulator Polo-Like Kinase 1 (PLK1) (Panel B). PLK1 is highly expressed in B vs. T-ALL and plays a critical role in B cell growth and survival. Following Notch activation, loss of ubiquitionated PLK1 through proteosomal degradation leads to cell cycle arrest through two mechanisms, namely cytoplasmic relocalization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at S20, which leads to decreased weakened p53-MDM2 interaction and accumulation of p53 (Panel C). siRNA to CHFR reveal that this mechanism is dependent on CHFR (Panel C). Importantly this mechanism is not seen in T-ALL cells as the activation of PARP1 by HES1 does not occur in T-ALL cells.

Conclusions:

Our findings reveal a novel molecular mechanism whereby Notch signaling induces disruption of the cell cycle in a cell type specific manner in B-ALL. Activation of PARP1, PARylation of CHFR, ubiquitination of PLK1 resulting in loss of nuclear cyclin B and accumulation of p53 demonstrates a series of events which can be initiated through activation of Notch in B-ALL. This mechanism reveals a potentially targetable approach to B-ALL.

Disclosures:

No relevant conflicts of interest to declare.
TET2, ANPM1 and FLT3-ITD Gene Mutations in Normal Karyotype Acute Myeloid Leukemia
Authors: Sun Aining , Tian Xiaopeng , Yin Jia , Li Weiyang , Chen Suning , Depei Wu
Published: October 2019

Abstract 1413

Objective:

Analyze the molecular genetics characteristics of acute myeloid leukemia with normal karyotype and explore the relationship between different genetic mutation patterns and prognosis.

Methods:

A total of 373 acute myeloid leukemia (AML) with normal karyotype diagnosed and treated in the First Affiliated Hospital of Soochow University from 2005 to 2010 were recruited in this research to assess the genetic mutation patterns. The target genes which was extracted from bone marrow cell were amplified by PCR and analyzed by massively DNA sequencing. All of the TET2, DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1, RUNX1, c-KIT, FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations were detected in our study.

Results:

(1). A total of 16.1% of patients had TET2 mutations, 31.6% had FLT3 internal tandem duplications (ITDs), 6.2% had FLT3 tyrosine kinase domain mutations, 2.4% had c-KIT mutations, 37.8% had NPM1 mutations, 11.3% had WT1 mutations, 5.9% had RUNX1 mutations, 11.5% had ASXL1 mutations, 3.8% had MLL partial tandem duplications (PTDs), 7.8% had IDH1 mutations, 7.8% had NRAS mutations, 12.3% had IDH2 mutations, 1.6% had EZH2 mutations, 14.7% had DNMT3A mutations and no mutations were found of CBL and JAK2V617F. In conclusion, there are 77% (287/373) gene mutations hide in normal karyotype AML patients.(2). We found that the TET2 gene mutations were associated with DNMT3A (P = 0.041) and RUNX1 (P <0.001) mutations, but mutually exclusive with IDH2 (P = 0.021), or IDH1/2 (P = 0.006) gene mutations. NPM1 gene mutations were highly correlated with DNMT3A mutations (P <0.0001), IDH1 mutations (P <0.0001) and IDH2 mutations (P = 0.001), but mutually exclusive with RUNX1 mutations (P=0.003). IDH2 mutations and WT1 mutations were mutually exclusive (P = 0.01); DNMT3A mutations were associated with NRAS mutations (P = 0.01). In addition, study have shown that the number of gene mutations was closely associated with older age, high white blood cell and high bone marrow blast cell percentage, but wasn't correlated with gender, hemoglobin and platelet levels.(3). In the NPM1m+ patients, TET2 mutations were associated with shorter median OS in contrast to TET2 wild type (9.9 vs. 27.0 months, P= 0.023). Surprisingly, in NPM1m+/FLT3-ITDm- group, TET2 mutations was also an unfavorable prognostic factor, which was closely associated with shorter median OS compared to TET2 wild type (9.5 vs. 32.2 months, P=0.013).

Conclusion:

Gene mutations incidence was high in normal karyotype AML patients. TET2 mutations was an unfavorable prognostic factor which was closely associated with shorter median OS in contrast to TET2 wild type in NPM1m+/FLT3-ITDm-group. In addition, The number of gene mutation was closely associated with older age, high white blood cell levels and high bone marrow blast cell percentage.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide Consolidation Followed by Maintenance After Autologous Transplantation for Multiple Myeloma Decreases Thymic Output and Terminally Differentiated CD4 and CD8 T Cells but Increases Treg
Authors: Clave Emmanuel , Douay Corinne , Coman Tereza , Busson Marc , Bompoint Caroline , Moins Helene , Carmagnat Maryvonnick , Gorin Norbert Claude , Charron Dominique , Toubert Antoine , Garderet Laurent
Published: October 2019

Abstract 4046

Treatment with lenalidomide, an immunomodulatory drug, increases the time to progression in relapsed/refractory multiple myeloma. However, due to its pleiotropic effect, it is not known whether the efficacy of this drug is due only to direct tumor toxicity or also to immunomodulatory effects. We assessed in vivo the changes in T-cell reconstitution induced by lenalidomide consolidation and maintenance treatment following autologous peripheral blood stem cell transplantation (ASCT) in a cohort of multiple myeloma patients.

Twenty-nine newly diagnosed myeloma patients were treated with the induction combination bortezomib plus dex followed by high dose melphalan (140–200 mg/m2) and ASCT. A first group of 11 patients were treated with lenalidomide consolidation initiated 3 to 6 months post transplantation: 25 mg/day, days 1–21 of a 28 day cycle for 2 months, followed by maintenance (10 mg/day) until disease progression. This group was compared with the 18 patients who did not receive any treatment after ASCT. Blood samples were collected at diagnosis, before the transplant and 1, 3, 6, 9, 12 and 18 months after ASCT. Thymic function was assessed by real-time PCR quantification of T cell receptor excision circles (sjTREC) and percentages and absolute counts of T lymphocyte subpopulations were determined by multicolor flow cytometry. Statistics were performed using the Log-Rank or Mann-Whitney test.

The two cohorts had similar baseline characteristics and all 29 patients were in remission after ASCT. With a median follow-up of 4 years, progression-free survival (PFS) was superior with lenalidomide treatment (69% vs 36%, p=0.05) while overall survival (OS) was similar (82% vs 75%, p=0.5). Lenalidomide treatment induced a progressive decrease in sjTREC (median at 18 months, 0.25/μL vs 1.61/μL, p<0.05) and a decrease in the percentages and absolute counts of CD4+ and CD8+ CD45RA+CCR7- effector terminal T cell subpopulations (median at 18 months, 3.2/μL vs 17.6/μL, p<0.05 for CD4+CD45RA+CCR7- and 109/μL vs 345/μL, p<0.05 for CD8+CD45RA+CCR7-). Conversely, lenalidomide treated patients displayed an increase in CD4+CD25+CD127-/low Treg populations, in both percentage and absolute count (median 13% vs 8 %, p<0.05 and 48.9/μL vs 29.3/μL, p<0.05, respectively). No correlation was found with documented infections, relapse or survival.

We confirm an increase in PFS with lenalidomide consolidation/maintenance following ASCT. However, our data also suggest that in myeloma patients, the effect of lenalidomide on the myeloma tumor may not be T cell mediated and this treatment may have a negative impact on the T cell immune surveillance.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
Imatinib Has a Negative Impact On Growth In Children With Previously Untreated Chronic Myeloid Leukaemia (CML) In Early Chronic Phase (CP): Results Of The French National Study
Authors: Millot Frédéric , Guilhot Joelle , Baruchel Andre , Petit Arnaud , Leblanc Thierry , Bertrand Yves , Mazingue Françoise , Lutz Patrick , Vérité Cécile , Berthou Christian , Galambrun Claire , Sirvent Nicolas , Yacouben Karima , Chastagner Pascal , Gandemer Virginie , Reguerre Yves , Couillaud Gérard , Cayuela Jean-Michel
Published: October 2019

Aims

The French pediatric CML working group conducted a retrospective study analysing the growth of patients under 18 years with CML treated with imatinib front-line.

Patients and Methods

Data from 78 patients receiving imatinib (260 mg/m2) for newly diagnosed CML in chronic phase between July 2001 and August 2012 were collected. Height was expressed as standard deviation score (SDS). Height SDS was calculated using French growth standard, as patient height minus mean height for age and sex, divided by standard deviation of height for age and sex. Pre-pubertal age was defined as being younger than 9 years for girls or 11 years for boys.

Results

A gradual decrease in the median of the height SDS score is observed over time since the start of imatinib. Sixty three patients were assessable for paired analysis at onset of imatinib and 12 months later. The median height-SDS in this group of patients was significantly lower (p < 10-4) 12 months after the start of imatinib. Variance analysis was performed in 36 children at onset of imatinib, 12 months and 24 months later. The median height-Z score in this group of patients was significantly lower 12 months and 24 months after the start of imatinib (p < 10-4) compared to initial diagnosis. The 63 patients assessable for paired data analysis at onset of imatinib and 12 months later were classified by sex and pubertal status. The median height Z score in boys and girls was significantly (p < 10-4) lower 12 months after the start of imatinib. When the patients were analysed according to the pubertal status at onset of imatinib, the median height SDS score was significantly (p < 10-4) lower 12 months after the start of imatinib in the prepurbertal group as well as in the postpubertal group. A similar finding was observed in the subgroups of boys and girls starting imatinib at a prebubertal or postpubertal age. No significant difference was observed when the median M12 height SDS score in patients who started imatinib at a prepubertal stage or a pospubertal stage were compared. Moreover, no significant difference was observed when the median M12 height-Z score in boys and girls were compared.

Conclusion

This retrospective analysis revealed a statistically significant deceleration in growth during the first 2 years of imatinib treatment in children and adolescents with CML and an absence of influence of sex and pubertal status on the depth of growth deceleration.

Disclosures:

No relevant conflicts of interest to declare.