bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chd7 Is a Cell Autonomous Regulator of Chromatin In Hematopoietic Stem Cells.
Authors: Huang Hsuan-Ting , Kathrein Katie , Huang Yue-Hua , Gitlin Zachary , Barton Abby , Song Anhua , Zhou Yi , Zon Leonard I. 
Published: October 2019

Abstract
Abstract 1596
Hematopoietic stem cells (HSC) are specified during embryogenesis, and the induction process involves not only transcription factors but also epigenetic factors that modulate chromatin to regulate the hematopoietic transcriptional programs. Here, we performed a reverse genetic screen to identify all the chromatin factors that are required for HSC induction in zebrafish. The zebrafish homologs of 350 human chromatin factors were identified by reciprocal BLAST and knocked down by injecting morpholinos designed against each homolog into the single cell embryo. Morphants were then analyzed for changes in blood formation by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 16 somite stage and for c-myb and runx1 in definitive stem cells at 36 hours post fertilization. From the screen, we have identified known regulators of hematopoiesis such as bmi1, in which knock down results in loss of stem cell formation. We have also identified one novel HSC regulator chd7. Chd7 is a member of the chromodomain helicase DNA-binding domain family that functions at gene enhancer elements and in ribosomal RNA synthesis. Zebrafish embryos injected with chd7 morpholino had higher levels of β-globin e3 and c-myb/runx1 expression. Additional markers such as scl, gata1, fli1, and lmo2 were also upregulated, although vascular markers flk1 and ephrinB2 were downregulated. Early mesodermal markers eve1 and ntl expression appeared normal, suggesting that the effects of chd7 knock down occurs when the mesodermal precursor cell population becomes an HSC. Transplants of chd7 deficient Tg(c-myb:GFP) blastomeres into Tg(lmo2:DsRed) blastulas resulted in more chimeric embryos compared to controls, demonstrating that the phenotype is cell autonomous. In humans, haploinsufficiency for CHD7 is the main cause of CHARGE syndrome, and it has been recognized more recently that these patients are immunodeficient, though the etiology remains unknown. Our studies indicate a new role for chd7 in hematopoiesis in which it functions to repress HSC formation during embryogenesis.

Disclosures:
Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
The Novel Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation and Migration of Lymphatic Endothelial Cells
Authors: Osada Makoto , Inoue Osamu , Ding Guo , Hirashima Masanori , Suzuki-Inoue Katsue , Ozaki Yukio 
Published: October 2019

Abstract
Abstract 160
CLEC-2 has been described recently as playing crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis. The snake venom rhodocytin is known as a strong platelet activator, and we have shown that this effect is mediated by CLEC-2. Podoplanin, which is expressed on the surface of tumor cells, is an endogenous ligand for CLEC-2 and facilitates tumor metastasis by inducing platelet aggregation. Mice deficient in podoplanin, which is also expressed on the surface of lymphatic endothelial cells, show abnormal patterns of lymphatic vessel formation. We have recently reported on the generation and phenotype of CLEC-2-deficient mice. These mice are lethal at the embryonic/neonatal stages associated with disorganized and blood-filled lymphatic vessels and severe edema, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, CLEC-2 is expressed in both platelets and neutrophils in mice and whether CLEC-2 in platelets, but not that in other cells, plays a role in the separation has not been directly proved yet. Moreover, the mechanism how CLEC-2 regulates of blood/lymphatic vessel separation has not been elucidated to date. In the present study, we found that specific deletion of CLEC-2 from platelets mediated by PF4-Cre conferred the defect of blood/lymphatic vessel separation, identifying that CLEC-2 expressed in platelets is required to regulate lymphatic vascular development. Tube formation of lymphatic endothelial cells, but not that of vascular endothelial cells, was inhibited in the presence of platelets. Further, proliferation and migration of lymphatic endothelial cells, but not those of vascular endothelial cells, were inhibited by CLEC-2+/+ platelets, but not by CLEC-2−/− platelets. We propose that CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation and migration of lymphatic endothelial cells through the interaction between CLEC-2 in platelets and podoplanin in lymphatic endothelial cells. CLEC-2 may be a novel therapeutic target protein for lymphatic metastasis of tumor if the interaction of CLEC-2 and podoplanin also regulates lymphangiogenesis by tumor cells.

Disclosures:
No relevant conflicts of interest to declare.
Endoglin Identifies the First Wave of Hematopoietic Progenitors During Embryogenesis.
Authors: Borges Luciene , Iacovino Michelina , Baik June , Mayerhofer Tim , Letarte Michelle , Perlingeiro Rita 
Published: October 2019

Abstract
Abstract 1600
Endoglin (Eng), an ancillary receptor for several members of the Transforming Growth Factor (TGF)-beta superfamily, plays a critical role in early development. Eng-/- embryos dye around 10.5 dpc due primarily to vascular and cardiac abnormalities. Yolk sacs (YS) of 9.5 dpc Eng-/- embryos present abnormal vasculature and anemia. However, the mechanism by which endoglin leads to an anemic phenotype is unclear. Using in vitro differentiation of Eng-/- ES cells, we have previously demonstrated that endoglin is required for proper hemangioblast and primitive hematopoietic development. To test the hypothesis that endoglin might have a direct effect on blood formation, here we investigated the role of endoglin during hematopoiesis in vivo. We first evaluated YS from 9.5 dpc Eng-/- embryos and observed significantly reduced numbers of hematopoietic colony-forming cells (CFCs), in particular GEMMs and BFU-Es, when compared to both Eng+/− and Eng+/+ embryos. Real-time PCR analysis revealed decreased expression of embryonic globin, Gata-1, and SCL in the mutant YS. We then investigated the function of endoglin in wild-type mice by sorting the endoglin positive and negative population from E7.5 to 9.5 CD1 mouse embryos. When cultured on the stromal cell line OP9 in the presence of hematopoietic cytokines, only cell cultures derived from the Eng+ fraction gave rise to hematopoietic progenitors, as evidenced by colony assays and FACS analyses. Engneg cells did not produce any hematopoietic colonies. We then separated cells into 4 fractions using Eng and Flk-1, and found that from E7.5-9.5, the hematopoietic progenitor activity resided in the Eng+Flk-1+ double-positive fraction. FACS and microarray analyses of the 4 fractions in E7.5 embryos demonstrated that the Eng+Flk-1+ fraction highly expresses both endothelial and hematopoietic markers, including VE-Cadherin, Tie-2, PECAM1, Gata-1, SCL, and Lmo2, indicative of hemogenic endothelial potential. Taken together, our findings demonstrate that endoglin marks the first wave of hematopoietic progenitors during development, pointing to a defined role for endoglin in the specification of YS hemogenic endothelium as early as E7.5, in addition to its well defined role in vascular branching and remodeling.

Disclosures:
No relevant conflicts of interest to declare.
Ga13 and Talin Coordinate integrin Inside-out and Outside-In Signaling by “time-sharing” of b3 cytoplasmic Domain
Authors: Gong Haixia , Du Xiaoping 
Published: October 2019

Abstract
Abstract 162
The bidirectional signaling of integrins mediates cell adhesion, spreading, retraction and migration. The binding of talin and kindlins to the cytoplasmic domain of integrin b3 subunit transmits inside-out signals to induce integrin activation. Ligand-induced outside-in signaling requires the binding of a G protein subunit, Ga13, and a tyrosine kinase, c-Src, to the b3 cytoplasmic domain. It is unclear how the short cytoplasmic domain of b3 accommodates these molecules and allows coordinated bidirectional signaling. Here we show that Ga13 and talin are mutually exclusive in binding to b3 both in vivo and in vitro. Increasing expression level of talin head or full-length talin in CHO123 cell decreases Ga13-b3 association. Ga13 also competes with talin head for GST-b3 binding in purified binding system. More importantly, talin is associated with b3 only in inside-out signaling during platelet aggregation. Following integrin ligation, however, Ga13 binds to b3, replacing talin. The Ga13 binding site located between K729-T741 within the talin binding region. However, Ga13 binding and signaling require a distinct ExE733 motif (EEE in b3) conserved in most integrin b subunits that is not required for talin binding but flanked by talin binding sequences on both sides. Interference of Ga13 binding to integrin b3 cytoplasmic domain by myristorylated b3 peptide (Myr-EEERA735) or by point-mutating the EEE motif to AAA selectively inhibits outside-in signaling, thus inhibited cell spreading on fibrinogen, accelerated RhoA activation and inhibited c-Src activity. But they have no effect on talin-dependent inside-out signaling judged by fibrinogen binding assay. In conclusion, our data suggest that the timed share of binding sites in b3 between Ga13 and talin coordinates bidirectional integrin signaling.

Disclosures:
No relevant conflicts of interest to declare.
atpif1 regulates Mitochondrial Heme Synthesis In Developing Erythroid Cells
Authors: Shah Dhvanit I , Takahasi-Makise Naoko , Schultz Iman , Pierce Eric L , Li Liangtao , Vogin Guillaume , Brigham Benjamin S , Zhou Yi , Brugnara Carlo , Campanella Michelangelo , Danial Nika , Ward Diane M , Kaplan Jerry , Paw Barry H 
Published: October 2019

Abstract
Abstract 163
Iron plays a key role as a cofactor in many fundamental metabolic processes, which require heme synthesis and Fe/S cluster assembly in the mitochondria. Defects in the transport of iron into the mitochondria would lead to anemias due to a deficiency in heme and hemoglobin synthesis. Here we describe a zebrafish genetic mutant, pinotage (pnttq209), which exhibits a profound hypochromic, microcytic anemia. Erythrocytes from pnt mutants have a defect in hemoglobinization and decreased red cell indices (mean corpuscular volume and hemoglobin content, hematocrit, hemoglobin concentration). Through positional cloning, we showed that the mitochondrial ATPase Inhibitory Factor 1 (atpif1), which regulates the inner mitochondrial membrane potential, is the gene disrupted in pnt. The identity of the pnt gene was verified by: (a) decreased atpif1 steady-state mRNA in pnt mutants, (b) phenocopying the anemia with anti-sense atpif1 morpholinos, (c) functional complementation of the anemia with atpif1 cRNA, and (d) a genetic polymorphism in the 3'UTR co-segregating with the mutant phenotype that destabilizes the atpif1 mRNA. Consistent with the conserved function of atpif1 in higher vertebrates, the silencing of the murine ortholog of atpif1 in Friend mouse erythroleukemia (MEL) cells showed a defect in hemoglobinization by o-dianisidine staining and reduction of 59Fe incorporation into heme in 59Fe-metabolically labeled cells. Moreover, Atpif1 knockdown destabilizes their mitochondrial membrane potential and volume. Therefore, the identification of atpif1 in pnt functionally demonstrates the role of atpif1 in regulating the proton motive gradient across the inner mitochondrial membrane for mitochondrial iron incorporation in heme biosynthesis. These results uncover a novel hematopoiesis-related function of atpif1, which will directly contribute to our understanding and potential treatment of human congenital and acquired anemias.

Disclosures:
No relevant conflicts of interest to declare.
p90RSK2 Is Essential for FLT3-ITD-, but Dispensable for BCR-ABL-Induced Myeloid Leukemia
Authors: Elf Shannon , Chung Tae-Wook , Blevins Dean P. , Williams Ifor , Khoury H. Jean , Taunton Jack , Kang Sumin 
Published: October 2019

Abstract
Abstract 1716
p90 ribosomal S6 kinase 2 (p90RSK2) is a serine/threonine kinase that plays an active role in diverse cellular processes, including gene expression, cell proliferation, and survival. We previously demonstrated that RSK2 signaling plays a key role in the pathogenesis and disease progression of leukemogenic FGFR3 associated hematopoietic malignancies, including FGFR3-expressing t(4;14) multiple myeloma and TEL-FGFR3-expressing t(4;12)(p16;p13) peripheral T cell lymphoma. In this study, we found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including K562 (BCR-ABL), Molm14 and Mv4;11 (FLT3-ITD), HEL (JAK2 V617F), and EOL-1(FIP1L1-PDGFR alpha). We next examined the role of RSK2 in myeloid transformation induced by BCR-ABL and FLT3-ITD due to their high frequency of occurrence in CML and AML, respectively. Interestingly, although RSK2 is activated by BCR-ABL in both stably transduced Ba/F3 cells and K562 human leukemia cells, we found that genetic deficiency of RSK2 does not affect the pathogenesis or disease progression of myeloproliferative disease induced by BCR-ABL in a murine bone marrow transplant (BMT) model using wild type or RSK2-/- donor bone marrow cells. This finding suggests that RSK2 is dispensable for BCR-ABL induced myeloproliferative disease. Moreover, targeting RSK2 by treatment with potent and highly specific RSK inhibitor fmk did not effectively induce apoptosis in K562 human leukemia cells expressing BCR-ABL, or in primary leukemia cells from BCR-ABL positive CML patients. In contrast, we found that targeting RSK2 may represent an effective therapy to treat patients with FLT3-ITD positive AML. Treatment with fmk induced significant apoptotic cell death in Molm14 and Mv4;11 human leukemia cells expressing FLT3-ITD, as well as in primary leukemia cells from FLT3-ITD positive AML patients. In consonance with these results, FLT3-ITD induced T-cell lymphoma in a BMT assay using RSK2-/- donor bone marrow cells, phenotypically distinct from the myeloproliferative disease induced by FLT3-ITD using wild type donor bone marrow cells. These results suggest that RSK2 is required for FLT3-ITD induced hematopoietic transformation, likely playing a role in pathogenesis and lineage determination. Together these findings suggest that the role of RSK2 in hematopoietic transformation may depend on different upstream oncogenic signals mediated by different leukemogenic tyrosine kinases. Our data also demonstrate that RSK2 may represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress
Authors: O'Farrell Courtney , Shamay Meir , Kalu Nene , DuFresne Andrew , Biswas Sunetra , Ambinder Richard F. 
Published: October 2019

Abstract
Abstract 1736
EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta.

Disclosures:
No relevant conflicts of interest to declare.
Herpesviridae Viral Infections Following Rituximab Combined Chemotherapy In Patients with Diffuse Large B Cell Lymphoma
Authors: Lee Ho Sup , Park Lee Chun , Shin Seong Hoon , Ye Byeong Jin , Song Moo-Kon , Oh Sung Yong , Lee Sang Min , Lee Won Sik , Kim Yang Soo 
Published: October 2019

Abstract
Abstract 1748

Backgroud:
Herpesviridae family includes herpes simplex virus, varicella zoster virus, Epstein-Barr virus, and cytomegalovirus, etc. Herpesviridae viral infections(HVIs) can lead to serious complications including dissemination, secondary infection, bacterial superinfection in patients with diffuse large B cell lymphoma (DLBL) undergoing rituximab combined chemotherapy. But there was no consensus on the dose and duration of antiviral agents prophylaxis in DLBL undergoing chemotherapy.

Method:
Three hundred seven patients were newly diagnosed with DLBL and received chemotherapy between June 2004 and August 2009. HVIs was confirmed based on clinical diagnosis, serologic test or pathologic diagnosis. The characteristics of the patients were as follows: the median age was 58years (range 15–89 years) with a female-to-male ratio of 171:136. All patients received chemotherapy with rituximab combined cyclophosphamide, adriamycin, vincristine, and prednisolone.

Results:
Forty three patients (14.0%) developed HVIs at a median of 6.63 months (range 0.37 – 51.33 months) after initial chemotherapy. The estimated cumulative incidence rates (CIR) of HVIs were 10.36%, 16.18% and 18.44% at 1years, 3years, and 5years in all patients. In univariate analysis, 5years CIR of HVIs were 14.64% vs 28.05% in low and low-intermediate IPI risk vs in high-intermediate and high IPI risk (p=0.0003), 34.07% vs 8.29% in cumulative dose of steroid ≥ 3000 mg vs < 3000mg (p < 0.001), 29.58% vs 13.56% in duration of chemotherapy ≥ 6 months vs < 6 months (p=0.0250), 38.32% vs 13.41% in relapse vs none-relapse (p=0.0056), 12.83% vs 43.80% in receiving first line chemotherapy vs second line and more than second line chemotherapy (p=0.0008), and 24.90% vs 15.49% in prolonged neutropenic fever vs none-neutropenic fever (p=0.0239). In multivariate analysis, the results confirmed 5 variables as independent predictive factors for the high IPI risk (P < 0.001, hazard ratio (HR): 3.236, 95% confidence interval (CI) 1.636–6.400), prolonged neutropenic fever (P = 0.029, HR: 0.484, 95% CI 0.253–0.927), longer duration of chemotherapy (P = 0.006, HR: 0.212, 95% CI 0.069–0.648), high cumulative dose of steroid (P = 0.008, HR: 2.889, 95% CI 1.319–6.328), and receiving autologous stem cell transplantation (P < 0.001, HR: 0.146, 95% CI 0.057–0.371).

Conclusion:
High IPI risk, prolonged neutropenic fever, longer duration of chemotherapy, high cumulative dose of steroid and receiving autologous stem cell transplantation were seemed to be high risk for HVIs in patients with DLBL undergoing rituximab combined chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
An Effective Salvage Treatment Using Rituximab, Ifosfamide, Etoposide, Cytarabine, and Dexamethasone (R-IVAD) for Patients with Relapsed or Refractory Aggressive B-Cell Lymphoma
Authors: Miura Katsuhiro , Takei Kazuhiro , Kobayashi Sumiko , Kobayashi Yujin , Tanaka Toshitake , Kurita Daisuke , Iriyama Noriyoshi , Inoue Yasuyuki , Hatta Yoshihiro , Kura Yoshimasa , Sawada Umihiko , Takeuchi Jin 
Published: October 2019

Abstract
Abstract 1759

Background
The prognosis of patients with relapsed or refractory aggressive B-cell lymphoma after the conventional first line chemotherapy is still disappointing. Although several rituximab combined salvage regimens were reported, the optimal treatment has not yet been established. We therefore evaluated the efficacy and toxicity of a non-anthracycline based salvage regimen, consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone (R-IVAD) in patients with relapsed or refractory aggressive B-cell lymphoma.

Patients and methods
Patients with relapsed or refractory CD20-positive diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma grade 3 (FL G3) were consecutively enrolled in this treatment. R-IVAD consisted of rituximab (375 mg/m2 on day -2), ifosfamide (1500 mg/m2 on day 1–5), etoposide (150 mg/m2 on day 1–3), cytarabine (100 mg/m2 on day 1–3) and dexamethasone (40 mg/body on day 1–3). Treatment was given every 3 weeks up to a total of 3 courses with support of granulocyte colony stimulating factor. For responders under 65 years old with good performance status (PS), we planed peripheral stem cell collection after the third cycle of R-IVAD and consolidating high-dose chemotherapy (HDC) with cycrophosphamide (60 mg/kg on day -7,-6), etoposide (500 mg/m2 on day -6, -5, -4) and ranimustine (250 mg/m2 on day -3, -2) followed by autologous stem cell transplantation (ASCT).

Results
Thirty-two patients (25 DLBCL and 7 FL G3) with a median age of 64 years old (range 38–79) participated in this study and received an average of 2.6 cycles of R-IVAD. There were 21 relapsed and 11 primary refractory patients. The overall response (OR) and the complete response (CR) rate were 72% and 56% respectively. The OR rate of relapsed patients was significantly higher than that of primary refractory patients (86% vs. 45%, p=0.035). With a median 16 months (range 2–99) of follow up, the 2-year overall survival (OS) and the event-free survival (EFS) for all patients were 55% and 36% respectively. Of the 11 eligible patients, 10 successfully proceeded to HDC/ASCT with an average of 5.5 × 106/kg of harvested CD34-positive cells, resulting in the 2-year OS of 90%. No treatment related death was observed during the investigation. Multivariate analysis revealed that the age > 60 years, PS ≥ 2, extranodal sites ≥ 2, and primary refractory disease were independently associated with worse survival.

Conclusion
R-IVAD regimen, which efficiently mobilizes peripheral stem cells, is a safe and efficacious alternative for patients with relapsed or refractory aggressive B-cell lymphoma.

Disclosures:
No relevant conflicts of interest to declare.