bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heme-Regulated eIF2α Kinase in Erythropoiesis and Oxidative Stress
Authors: Chen Jane-Jane 
Published: October 2019

Abstract SCI-23
Besides serving as a prosthetic group for hemoglobin, heme also regulates translation by inhibiting the kinase activity of heme-regulated translational inhibitor (HRI). HRI is the only known protein kinase that senses the intracellular heme concentration and does so through its two heme-binding domains. HRI is activated in heme deficiency by autophosphorylation, and phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), which impairs the recycling of eIF2 for further translational initiation and results in cessation of protein synthesis. In this manner, HRI serves as a feedback inhibitor and coordinates the translation of globin mRNAs with the intracellular heme concentration to ensure that globin proteins are not made in excess of the heme available for the formation of hemoglobin. Excess of globin chains or heme is cytotoxic. In the absence of HRI, uncontrolled protein synthesis in heme deficiency results in globin aggregation and precipitation in red blood cells and their precursors. HRI is responsible for the physiological adaptation that produces hypochromic, microcytic erythrocytes in iron deficiency. The expression of HRI was increased during late stages of erythropoiesis with higher expression in Ter119high erythroblasts and reticulocytes, correlating with the active synthesis and regulation of globins at these stages of erythroid differentiation. In addition to inhibiting global protein synthesis, the second important function of eIF2α phosphorylation is to reprogram translation and the subsequent transcription of genes required for stress response. In mammalian cells, translation of the transcription factor ATF4 mRNA is upregulated specifically by eIF2α phosphorylation via upstream open reading frames in the 5’UTR. Activation of the HRI-ATF4 stress response pathway in nucleated erythroid precursors is required for adaptation to acute and chronic oxidative stress. Furthermore, this HRI-dependent ATF4 pathway is also operative and necessary for erythroid differentiation, especially under stress conditions. In chronic iron deficiency, HRI is necessary for adaptive gene expression for erythroid differentiation as well as for iron heme and redox homeostasis. Beyond heme deficiency and oxidative stress, HRI is also activated by osmotic shock and heat shock. HRI deficiency in mice exacerbates erythropoietic protoporphyria and renders β-thalassemia intermedia embryonically lethal. Hri−/− mice also develop ineffective erythropoiesis during iron/heme deficiency and are severely compromised upon phenylhydrazine-induced acute hemolytic anemia. Thus, translational regulation by HRI plays a critical role in the manifestation of red cell diseases in mice and may be a significant modifier of such diseases in humans.

Disclosures:
No relevant conflicts of interest to declare.
HSC Transplantation for Hemoglobinopathies: Allogeneic or Autologous Gene-Modified HSC?
Authors: Cavazzana-Calvo Marina , Payen Emmanuel , Negre Olivier , Hacein-Bey-Abina Salima , Leboulch Philippe 
Published: October 2019

Abstract
Abstract SCI-46
The hematopoietic stem cell (HSC) transplantation pioneered by the Pesaro group is now applied wildly worldwide. The European Group for Blood and Marrow Transplantation (EBMT) has established the hemoglobinopathy registry, which now contains detailed epidemiological data on over 3000 patients. Today, allogeneic transplantation remains the only definitive curative therapy for thalassemia and other hemoglobinopathies. The development of iron chelation did not change this position. However, this has not settled the debate on how this curative but potentially lethal treatment stands vis-à-vis a medical noncurative therapy for adult and advanced disease patients. Nevertheless, despite the substantial improvements, a number of problems remain unsolved, such as the absence of an HLA genoidentical donor for all the diseased patients especially when they come from Outreach countries and the long-term morbidity due to HSCT (i.e., infectious complications, acute and chronic graft-versus-host disease, sterility). In this context and despite the unsolved debate, the development of gene therapy is highly justified. Sadelain and Leboulch pioneered the use of lentiviruses in human globin gene therapy, showing that anemia in mice affected with diseases mimicking the human pathological conditions can be abbreviated significantly by using lentiviruses containing a normal human β-globin gene. These pre-clinical studies set the basis for the current human clinical trial in Paris and for the upcoming trials. In our gene therapy trial, the human β-globin gene sequence has been modified by mutating a single amino acid at position 87 of the β-globin sequence. The major reason for using the b87 globin gene in lentiglobin in patients with β-thalassemia is that Hbb87 expression can easily be distinguished from that of normal Hb (HbA). In this trial, the patients received a full dose of Busulfex in the aim to obtain a myeloablation allowing the successful intake of gene modified autologous HSC. Two patients with thalassemia have been treated to date. One out of the two patients obtained a clinical benefit and is stably transfusion independent more than 3 years after the last transfusion. He currently has approximately 9 to 10 g percent of Hb in his blood, approximately one third being Hbb87, one third human HbF, and one third HbE. The patient is healthy up to now, with a full-time job. Integration site profile data will be discussed in detail during the meeting. Additional patients affected with thalassemia are currently being recruited in the Paris trial.

Disclosures:
No relevant conflicts of interest to declare.
Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.
Authors: Yasunaga Jun-ichirou , Ma Guangyong , Fan Jun , Matsuoka Masao 
Published: October 2019

Abstract
Abstract 2405
Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Disclosures:
No relevant conflicts of interest to declare.
Expression of CD56 Is an Independent Prognostic Factor to Predict Relapse in Acute Myeloid Leukemia with t(8;21): Results of Japan Adult Leukemia Study Group (JALSG) AML97 Protocol.
Authors: Iriyama Noriyoshi , Hatta Yoshihiro , Takeuchi Jin , Ogawa Yoshiaki , Ohtake Shigeki , Sakura Toru , Mitani Kinuko , Ishida Fumihiro , Takahashi Masatomo , Maeda Tomoya , Izumi Tohru , Sakamaki Hisashi , Miyawaki Shuichi , Honda Sumihisa , Miyazaki Yasushi , Taki Tomohiko , Taniwaki Masafumi , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 2511

Background:
Although prognosis of acute myeloid leukemia (AML) with t(8;21) is better than other types of AML, outcome of the patients has not been satisfied. Previously, aberrant antigen expression has been reported as risk factor for AML with t(8;21). However, in the reported series, number of cases was not large enough and chemotherapy regimens were variable. We investigated the association of prognosis and several biomarkers including immunophenotype, WBC count, age, and performance status for large number of AML patients with t(8;21) uniformly treated in JALSG AML97 regimen.

Patients and Methods:
Seven hundred eighty-nine eligible AML patients were evaluated for the multicenter JALSG AML97 study. Adult patients with de novo AML except for APL, ages 15–64 years, were registered consecutively from 103 institutions that participated in JALSG from December 1997 to July 2001. One hundred forty-four patients with AML with t(8;21) were analyzed in this study with a median 1205 days of observation term from diagnosis. Complete remission (CR), relapse-free survival (RFS), and overall survival (OS) rates were analyzed by Fisher's exact test and log-rank test. Factors that would affect clinical outcome were analyzed by multivariate Cox proportional hazard regression model.

Results:
AML with t(8;21) frequently expressed CD19, CD34, and CD56 compared to other subtypes of AML. CD11b was rarely expressed. Expression of CD19 favorably affected on CR rate (96% in CD19 positive and 87% in negative patients, p<0.05). Univariate analysis showed WBC>20×109/L, CD19 negativity, and CD56 positivity were adverse factors for RFS. CD56 expression was the only independent adverse factor for RFS by multivariate analysis (73.7% in CD56 negative and 48.2% in CD56 positive patients at 3 yrs) although its expression did not affect on OS. There was no difference of age, sex, WBC count, presence or absence of Auer rod, performance status, or CD15 expression between CD56 positive and negative cases. Expression of CD19 was more common in CD56 negative patients (50% in CD56 negative and 30.6% in CD56 positive patients, p<0.05).

Conclusions:
We demonstrated that the expression of CD56 was a distinctive adverse factor in a large number of AML patients with t(8;21) treated with JALSG AML97 regimen. CD56 positive AML patients with t(8;21) are possible candidates for hematopoietic stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Preimplantation Genetic Diagnosis for Benign Hematological Disorders Combined with HLA Matching and Stem Cells Development.
Authors: Altarescu Gheona , Elstein Deborah , Zimran Ari , Geva Talia Eldar , Lahad Ephrat Levy , Renbaum Paul 
Published: October 2019

Abstract
Abstract 3167
Preimplantation genetic diagnosis (PGD) for molecular disorders involves development of disease- and family-specific protocols that allow simultaneous amplification of the mutation with multiple polymorphic markers in single cells. We present the development and results of PGD for 6 non-lethal hematological disorders: dyskeratosis congenita, Gaucher type 1, Fanconi C and A, hemophilia A, and beta thalassemia. For beta thalassemia and Fanconi A we performed combined PGD for the disease and HLA matching since each family had an affected child requiring bone marrow transplant. For each of the 6 diseases we developed a fluorescent PCR protocol that included the familial mutation and at least 6 informative markers surrounding the mutation. Mechanical biopsy technique was performed and a single cell of a 6-cell embryo was analyzed. For 17 families, 43 PGD cycles were performed by blastomere analysis. The efficiency of single cell amplification was 96%. 12 healthy children were born (and 3 on-going pregnancies). PGD for dyskeratosis congenita was performed in 2 families: one carrying the Arg 631Gln mutation and the other the Arg901Trp in the TERT gene. A total of 3 cycles were performed and 1 healthy child was born. Analysis for Gaucher type 1 disease was performed in 4 families (1 couple was carriers of both Tay-Sachs and Gaucher mutations) in which the offspring were predicted to have severe type 1 Gaucher involvement based on genotypes of the following mutations in the beta-glucocerebrosidase gene: IVS2+1 and IVS12+1, 84GG and R359Q, 84GG and R496H, N370S homozygous patient and 84GG carrier with 1 affected child requiring treatment. 13 PGD cycles were performed by blastomere biopsy and 6 healthy children were born (3 children to the same couple from different cycles). Mutant embryos were used for development of stem cell lines and 1 line (N370S/84GG) was achieved. PGD for Fanconi C and A was performed in 4 couples. For Fanconi A combined PGD for the disease and HLA matching was performed in 9 cycles; 2 healthy children were born and 1 on-going pregnancy. For Hemophilia A, 3 PGD cycles were performed for 2 families resulting in birth of 1 healthy boy. PGD for thalassemia beta was performed in 5 families with a total of 10 cycles, 5 of them combined with HLA matching; 1 healthy child was born. The overall pregnancy rate/embryo transfer was 34% (highest for Gaucher, 53%, lowest for beta thalassemia, 10%). We suggest that PGD is a safe and effective method for hematological disorders to prevent birth of an affected child. Combining analysis for the specific disease with HLA matching is useful for hematological diseases requiring bone marrow transplant. Mutant embryos can be developed in stem cell lines that are a valuable source for research.

Disclosures:
No relevant conflicts of interest to declare.
Whole Genome Sequencing Reveals Novel Recurring Somatic Mutations Affecting HUWE1 and DIAPH2 Genes in Multiple Myeloma
Authors: Tomasson Michael H. , Shen Dong , Hucthagowder Vishwanathan , Schierding William , Mullins Chelsea D. , Fiala Mark , Hall Ira M , Wallis John , Fulton Robert S. , Fulton Lucinda A. , Kulkarni Shashikant , Mardis Elaine R. , Wilson Richard K , Ley Timothy J. , DiPersio John F. , Maher Christopher A. , Vij Ravi , Ding Li 
Published: October 2019

Abstract
Abstract 320
Multiple myeloma (MM) is an incurable malignancy of antibody secreting plasma B-cells whose etiology is still poorly understood. We used whole genome sequencing (WGS) and deep-read capture validation to thoroughly characterize the mutation landscape in four newly diagnosed MM patients and further examined mutation recurrence in 89 additional MM patients. WGS cases were selected to be racially diverse and represent both hyperdiploid and non-hyperdiploid MM with otherwise “simple” karyotypes. All studies to date have used peripheral blood as controls, however, abnormal B cells and circulating tumor cells frequently contaminate the peripheral blood of MM patients and these studies may have missed the early genetic events potentially important in disease pathogenesis. We therefore chose to use matched skin samples as normal controls. The use of skin DNA controls and deep read count capture validation of single nucleotide variants (SNV) allowed us, for the first time, to demonstrate clear separation between normal and malignant cell populations. Our analysis pipeline detected both somatic SNVs and structural variants (SV, ie. translocations, deletions, insertions). In all four patients, we observed chromosomal translocations at the Ig heavy chain locus, VDJ recombination, and Ig locus somatic hypermuation. We found somatic mutations affecting the E3 ubiquitin ligase HUWE1 in 4% (4/94) of cases, as well as recurring deletions affecting the Rho pathway regulator DIAPH2 (recurrence data will be presented). RNA sequencing was preformed for 3 of the WGS patients to explore transcriptional effects of mutations observed. Statistical analysis identified significantly mutated genes, i.e. mutation hotspots, including ROBO2, BCL6 and cadherin/catenin genes. Genes involved in cell polarity, cell adhesion and axon guidance were affected in all four WGS patients. Chromosomal translocations present in all four WGS patients involved both known oncogenes (CCND1, MYC and MAFB) and putative oncogenes. A novel translocation implicated KCNT2, encoding a sodium-activated potassium channel, as a potential oncogene in MM. Recurrent point mutations were relatively rare in MM, suggesting that SVs are more common driver mutations and/or that SNVs in MM disrupt a diverse array of genes to affect key pathways. For genomic studies in MM moving forward, our results emphasize the importance of matched normal controls uncontaminated by tumor cells, and suggest that careful analysis of SVs be included to find novel oncogenes and important clinical correlations.

Disclosures:
No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Regulates Macrophage Migration Through Control Recycling of alpha5beta1 Integrin in Endosome
Authors: Aerbajinai Wulin , Liu Lunhua , Chin Kyung , Rodgers Griffin P 
Published: October 2019

Abstract
Abstract 3273
Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. Macrophage migration is a critical step in the regulation of immune response and is also associated with many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling macrophage migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of macrophages to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate macrophage motility is therefore important for gaining a better understanding of macrophage function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on macrophage and the cellular mechanism for these effects by using small-interfering RNA technic to knockdown GMFG in human macrophage like THP-1cells. Knockdown of endogenous GMFG results in significantly reduced chemotactic migration toward fMLP and enhanced adhesion on fibronectin compared with control siRNA transfected cells. Flow cytometry analysis shows that GMFG-knockdown cells expresses significant amount of α5β1 integrin on the surface compared with control cells. Confocal microscopy analysis exhibited that GMFG was distributed throughout the cytosol and colocalized with the multiple endocytic compartment such as early endosome marker EEA1, Rab5, Rab4 and Rab11. Importantly, we found that most of the α5β1 integrins accumulated in clusters at the rear of the GMFG-knockdown cells, along with colocalization with endosome marker EEA1, rather than localization toward the leading edge in response to fMLP stimulation in control cells. These results suggest that impaired efficient α5β1 integrin recycling cause to increased adhesion, which is responsible for reduced macrophage migration on fibronectin in GMFG depletion macrophages. Accordingly, GMFG is required for proper trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-mediated macrophage motility.

Disclosures:
No relevant conflicts of interest to declare.
Prophylactic Program in fxiii Deficient Patients From Iran
Authors: Naderi Majid , Eshghi Peyman , Karimi Mehran , Alizadeh Shaban , Dorgalaleh Akbar 
Published: October 2019

Abstract
Abstract 3376

Background:
Prophylaxis is the management strategy in FXIII deficient patients. Life-long prophylactic therapy by 10–20 U/kg FXIII (every 4–6 weeks) is recommended in patients with severe FXIII deficiency to prevent life-threatening spontaneous bleeding. In this study we evaluated of the efficacy and safety of prophylaxis program in a large study group consisting of 182 FXIII deficient Iranian patients. Material and methods: A total of 182 FXIII deficient patients (98 male and 84 female, mean age 26 years old) from south and south-east Iran were selected as a study group. Continual prophylaxis was done in all patients for periods of 6–168 months (mean 66.6 months). The patients received Fibrogamin-P (Dade Behring, Marburg, Germany) 10–20 U/kg every 4–6 weeks and before availability of this concentrate in Iran the prophylaxis was done by FFP 15–20 ml/kg every 20–30 days. All of patients were followed up for any bleeding diathesis and adverse effects during the prophylaxis period. Results: A total of 189 bleeding episodes including epistaxis, gum bleeding, bleeding after dental extraction, hemarthrosis, hematoma, hematuria, wound bleeding, menorrhagia and abortion were detected in 182 patients after prophylaxis program. Life-threatening intracranial bleeding was found in 34 patients before starting prophylaxis; however, there were no reports after prophylaxis. The bleeding episodes and bleeding score were significantly decreased after prophylaxis in compare to before starting prophylaxis (P<0.05) Discussion: The prophylaxis of FXIII deficiency by FXIII concentrate is seems clinical significance in prevention of spontaneous and life-threatening bleeding and also abortion in affected women. Also it is recommended to start prophylaxis in patients as soon as possible for prevention of life-threatening bleeding episodes.

Disclosures:
No relevant conflicts of interest to declare.