bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bmi1 Confers Stress Resistance to Self-Renewing Hematopoietic Stem Cells.
Authors: Nakamura Shunsuke , Iwama Atsushi 
Published: October 2019

Abstract
Abstract 1607
The polycomb group (PcG) proteins form chromatin-modifying complexes that implement transcriptional silencing. There are two major PcG complexes, polycomb repressive complex (PRC) 1 and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Among PcG proteins, Bmi1, a core component of PRC1, plays an essential role in the self-renewal and maintenance of various kinds of stem cells including hematopoietic stem cells (HSCs), neural stem cells, and leukemic stem cells. We previously reported that forced expression of Bmi1 using a Bmi1 retrovirus promotes symmetrical cell division of HSCs, resulting in a marked ex vivo expansion of multipotent progenitors and an enhancement of repopulating capacity of HSCs in vivo. However, the impact of overexpression of Bmi1 in HSCs remains to be precisely addressed. To this end, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the Rosa promoter in a tissue-specific fashion by the Cre-LoxP system. We crossed the mice to Tie2-Cre mice (Tie2-Cre; Rosa-Bmi1fl/+) and induced overexpression of Bmi1 in hematopoietic cells in vivo. Real-time PCR analysis demonstrated that expression of Bmi1 inpurified bone marrow (BM) c-Kit+Sca-1+Lineage-marker- (KSL) cells was 6-fold higher in Tie2-Cre; Rosa-Bmi1fl/+ mice than control Tie2-Cre mice mice. Overexpression of Bmi1 did not significantly affect steady state hematopoiesis. The number of HSCs and progenitors (multipotent progenitors, CMPs, GMP, MEPs, and CLPs) and lineage composition (myeloid cells, B cells, and T cells) in BM of Tie2-Cre; Rosa-Bmi1fl/+ mice was not significantly changed compared to those in control mice. We then performed serial transplantation assay. The repopulating capacity of Tie2-Cre; Rosa-Bmi1fl/+ BM cells was comparable to those of the control cells in primary recipients. However, Tie2-Cre; Rosa-Bmi1fl/+ cells retained higher repopulating capacity during serial transplantation compared to those of the control cells. Moreover, ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs for 10 days contained approximately 2-fold more high proliferative potential colony-forming cells (HPP-CFCs; colony diameter>1mm) and colony-forming unit-neutrophil/macrophage/erythroblast/megakaryocyte (CFU-nmEM) which retain multi-lineage differentiation potential along myeloid lineage than the control. Of note, cells in ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs exhibited significantly augmented repopulating capacity in recipient mice and we are now engaged in competitive repopulation unit (CRU) assays to determine the net HSC expansion by overexpression of Bmi1 during ex vivo culture. These results indicate that overexpression of Bmi1 confers stress resistance to HSCs during ex vivo culture and serial transplantation. Our findings also provide Bmi1 as a potential target for efficient manipulation of HSCs ex vivo.

Disclosures:
No relevant conflicts of interest to declare.
Filamins Regulate Differentiation of Megakaryocytes and Platelets From Cultured Embryonic Stem (ES) Cells.
Authors: Kanaji Taisuke , Okamura Takashi , Newman Peter J. 
Published: October 2019

Abstract
Abstract 1618
Filamins (Flns) A and B are major non-muscle actin binding proteins that play important roles in cross-linking cortical actin filaments into three-dimensional networks. In addition to their role as cytoskeletal scaffolding molecules, filamins are also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. The role of filamins in hematopoietic stem cell differentiation, however, remains unclear, in part because gene-targeted mice lacking filamins die early on in embryonic development. To investigate the role of filamins A and B in the differentiation of embryonic stem cells (ESCs) along the megakaryocyte/platelet axis, we designed shRNA-containing vectors that targeted both FlnA and B under the control of either the CMV immediate-early promoter (CMV-FlnABLow), or an endogenous Rosa26 promoter (Rosa26-FlnABLow). Compared with wild-type ESCs, FlnABLow ESCs formed small, tightly packed undifferentiated colonies that expressed high levels of the ESC transcription factor, Nanog, and low levels of ERK activity – all indicators of an undifferentiated state. Embryoid prepared from FlnABLow ESCs, were allowed to differentiate, and examined for markers of mesoderm differentiation (Flk-1) and megakaryocyte differentiation (CD41). Whereas Day 6 EB-derived FlnABnormal wild-type cells were 8% Flk-1 positive and 13% CD41 positive, Day 6 FlnABLow cells were 9% Flk-1 positive and only 4% CD41 positive, consistent with the notion that loss of Fln A and B results in a delay of mesoderm to hematopoietic differentiation. To evaluate the effect of Fln knockdown on the ability of the CD41-positive cells to further differentiate into megakaryocytes, form proplatelet extensions, and produce platelets, CD41 positive cells isolated from day 8 EBs were cultured in the presence of a thrombopoietin (TPO)-producing TERT stromal cell line. We found that FlnABLow CD41-positive cells formed far fewer and smaller megakaryocytes compared with their FlnABnormal wild-type counterparts. Proplatelets derived from FlnABLow cells exhibited an abnormal, enlarged morphology with swellings and thick shafts that released platelets prematurely, yielding platelets that were nearly twice the size of those derived from FlnABnormal cells. Taken together, we conclude that not only do filamins function prominently in hematopoietic cell differentiation, they also play an important role in platelet production, likely via their ability to by recruit and organize the necessary signaling molecules near the inner face of the plasma membrane.

Disclosures:
Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.
Sickle-Cell Disease and Malaria: Evaluation of Seasonal Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Senegalese patients—a Randomized Placebo-Controlled Trial
Authors: Diop Saliou , Seck Moussa , Gueye Youssou Bamar , Soudre Fabienne , Fall Awa Oumar Touré , Sall Abibatou , Thiam Doudou 
Published: October 2019

Abstract
Abstract 1633
Malaria is a real public health problem in Africa, more than 300 million new cases and approximately two million deaths arise every year. Sickle-cell disease (SCD) patients are at high risk of developing malaria which is a major contributor to morbidity and mortality in this disease. In Senegal, malaria transmission is high during rainy season, between July and October, and it was noted that sickle-cell crisis are frequent during this period. Then we carried out a double-blind randomized controlled trial to compare the impact of monthly sulfadoxine-pyrimethamine (SP) during the high-transmission season versus placebo on malaria incidence and morbidity of sickle-cell anemia. Sixty (60) SCD patients were randomized either to receive three intermittent preventive treatment (ITP) with SP or placebo using the random permutation table with nine elements. The drug was administrated as follows: sulfadoxine 25 mg/kg and pyrimethamine 1.25 mg/kg and this treatment was given once during the following months: September, October, and November. Overall four episodes of malaria disease were diagnosed, all these cases in the placebo arm. Thus, overall prevalence was 6.6% and there was no other case of malaria in the SP arm during the study period. Parasitological diagnosis confirmed the presence of Plasmodium falciparum in all four cases. No patient death was encountered during the study. SP treatment was well tolerated as only one patient (1.6%) in the SP arm reported pruritis. A significant reduction of patients’ complaints (p= 0.002) and blood requirements (p = 0.001) was noted in the SP group; whereas, no impact was observed on vaso-occlusive crisis and hospitalization occurrence. Malaria prophylaxis by monthly intake of SP during the transmission period of the parasite reduced the prevalence of malaria and was safe in SCD patients leaving in malaria endemic area.

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Stem Cell Transplantation Overcomes the Adverse Prognostic Impact of CD20 Expression In Both Adult and Pediatric Acute Lymphoblastic Leukemia.
Authors: Bachanova Veronika , Sandhu Karamjeet , Weisdorf Daniel J. , Verneris Michael , Burke Michael J , Cao Qing 
Published: October 2019

Abstract
Abstract 1681
The heterogeneous expression of CD20 antigen on leukemic blasts may harbor prognostic value and therapeutic potential. CD20 positivity (CD20+) has been recently associated with disease recurrence and shorter overall survival in adults with pre-B acute lymphoblastic leukemia (ALL). The influence of CD20 expression on outcomes following allogeneic donor hematopoietic stem cell transplantation (HCT) is not known.

Results:
We analyzed CD20 expression on pre-B ALL marrow blasts at diagnosis in 157 consecutive patients who underwent allogeneic HCT in first or second complete remission (CR) from 1999–2010. Thirty-two patients with no available flow cytometry data were excluded. Out of 125 evaluable patients, 52 (42%) were < 20 years of age; 73 (58%) were ≥20. All patients had high risk ALL and were in CR at HCT (57% in CR1, 43% in CR2). A majority of patients were Ph+ (60%) and received myeloablative conditioning (84%). Grafts were from sibling (36%), unrelated umbilical cord blood (UCB) (59%) and matched adult URD (5%). CD20+, defined as >20% expression on marrow blasts, was observed in 58 (46%) patients. 55% of children and 39% of adults were CD20+ and expression was similar in CR1 & CR2. Gender, donor source (sibling vs UCB), patient CMV serostatus, cytogenetics (Ph+ vs other), and conditioning (myeloablative vs reduced intensity) was similar in CD20+ and CD20- groups. Disease-free survival (DFS) at 3 years was 48% (95% CI 39–57%) for all patients; 42% (95% CI 30–54%) for CD20- patients, and 55% (95% CI 40–67%) for CD20+ patients (p=0.14). CD20+ expression did not significantly impact relapse rate or DFS in adults (Table) while in patients <20 years CD20+ expression was associated with a slightly favorable relapse rate. Similar overall survival (OS) and DFS was seen in both age groups, independent of CD20 expression. Treatment-related mortality was unaffected at 18% (95% CI 9–27%) and 19% (95% CI 9–29%) in CD20+ and CD20- cohorts, respectively (p= 1.00). In adjusted multivariate regression, the CD20+ group had a slightly but not significantly lower risk of relapse (RR 0.54 [0.27-1.09]; p=0.09), yet similar OS (RR 0.66 [0.38-1.14], p=0.13) and DFS (RR 0.67 [0.39-1.14], p=0.14) compared to the CD20- group.

Conclusion:
Pre-B ALL with a CD20+ immunophenotype reportedly confers higher risk of disease recurrence and unfavorable OS with conventional chemotherapy. Our data demonstrates that the poor prognosis associated with CD20+ ALL blasts is overcome by allogeneic HCT. In addition, post-HCT survival is promising in both adults and children, independent of CD20 expression. CD20 targeting monoclonal antibodies may further improve these outcomes.

Disclosures:
No relevant conflicts of interest to declare.
CD70 Expression on HTLV-1 Infected T Cells of Carriers and ATL Patients and Its Clinical Significance
Authors: Masamoto Izumi , Horai Sawako , Kozako Tomohiro , Yoshimitsu Makoto , Niimoto Junko , White Yohann , Uozumi Kimiharu , Suzuki Shinsuke , Arima Naomichi 
Published: October 2019

Abstract
Abstract 1730

Human T-lymphotropic virus type-1(HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL). HTLV-1 infected T cell growth or leukemogenesis in ATL is controlled by various host immune surveillance systems. Among them, CD70 on HTLV-1 infected T cells coupled with CD27 on virus specific cytotoxic T cells has been suggested to play an important role in ATL leukemogenesis. The CD70 molecule is the only known ligand for CD27, a member of the tumor necrosis factor (TNF) receptor superfamily 7. This negative immunoregulatory pathway downregulates cytotoxic T lymphocyte activity against CD70-expressing virus infected cells. In the present study, we examined CD70 expression on primary lymphocytes of HTLV-1 carriers and ATL patients, its relationship with HTLV-1 Tax protein expression, and the effect on CTL induction. CD70 expression was higher on peripheral blood mononuclear cells (PBMCs) of HTLV-1 infected carriers compared with healthy donors (p = 0.021, n = 21, Mann-Whitney U test), and higher in ATL patients compared to carriers (p = 0.045, n = 38, Mann-Whitney U test). CD70 expression may be observed in CD4 T cells, as well as B cells, but not in CD8 T cells or monocytes. CD70 expression in CD4 T cells is related to HTLV-1 infection, because of increased detection of HTLV-1 Tax protein during over night culture of CD70-expressing cells. Experiments using an ATL cell line, in which Tax expression is inducible by doxycycline stimulation, demonstrated enhanced CD70 expression when Tax protein was induced in HTLV-1 infected cells. Anti-CD70 antibody enhanced CD107a mobilization, a marker of recent cytotoxic degranulation, in HTLV-1 Tax specific CTLs in PBMCs from asymptomatic carriers in vitro, suggesting that the CD70/CD27 pathway plays an important role in the immune response to HTLV-1 infection in carriers, as well as ATL patients.

Disclosures:
No relevant conflicts of interest to declare.
NK-Cell Acquisition of Chemokine Receptors From Engineered Antigen Presenting Cells
Authors: Somanchi Srinivas S. , Gururajan Anitha , Cooper Laurence J.N. , Lee Dean A. 
Published: October 2019

Abstract
Abstract 1744
Natural Killer (NK) cells are able to extract fragments of cell membrane from antigen presenting cells through the immunological synapse, functionally incorporating the membrane receptors contained therein by a process called trogocytosis. Recently it was demonstrated that NK cells can acquire functional CCR7 from dendritic cells through this process and migrate in response to chemokines (CCL19 and CCL21). We investigated whether this process could be used to transiently modify NK cells ex vivo without genetic intervention.
In previous work, we developed a K562-based artificial antigen presenting cell (aAPC) expressing membrane-bound IL-21 (K562-cl9-mIL21) which enables robust NK-cell expansion, and determined that NK cells did not express detectable CCR7 during this process. To investigate trogocytosis in this system, we genetically modified K562-cl9-mIL21 to express membrane-bound CCR7 (K562 cl9 mIL21CCR7) using the Sleeping Beauty transposon/transposase system. After 24 hours of co-culture the NK cells cultured with K562-cl9-mIL21CCR7 demonstrated marked surface expression of CCR7 compared to NK cells cultured on K562-cl9-mIL21 (Figure 1a). In kinetic experiments using three independent donors, CCR7 peak uptake occured at 24 hours (Figure 1b), followed by a decline that corresponded with the loss of aAPCs in the cultures due to lysis by NK cells. This demonstrates that NK cells can be transiently modified during in vitro expansion to bear receptors that are otherwise not a normal part of their transcriptional repertoire. We are currently establishing the functionality of trogocytosed receptors in vitro and in vivo, and investigating the kinetics of CCR7 persistence after removal of the aAPCs. However, even transient expression as demonstrated might be sufficient for bestowing novel NK-cell migration ability in vivo in response to chemokine signaling, giving the engineered NK cells ability to reach desired tissue targets.

Disclosures:
Lee: Altor BioScience Corp: Research Funding; Celgene: Research Funding.
Phosphatase RNA-Interference Lethality Screening with Suberoylanilide Hyrdoxamic Acid In Myeloid Cell Lines
Authors: Bogenberger James , Kassner Michelle , Chow Donald , Yin Holly , Tibes Raoul 
Published: October 2019

Abstract
Abstract 1851

Background:
In previous work we performed RNAi sensitizer screening of epigenetic modulation with 5-Azacytidine and the human kinome. Few kinase hits were identified. Therefore we asked if phosphatases might be involved in modulating epigenetic therapies. Thus in order to identify potentiators of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) we performed high-throughput RNA-interference (RNAi) lethality screening using the myeloid cell lines HEL, THP-1 and TF-1 in combination with SAHA.

Methods:
A small-interfering RNA (siRNA) library targeting 206 phosphatases with 4 sequences per target gene was used for this study. siRNA were transfected in a single-siRNA-per-well format in 384-well plates using commercial lipid-based transfection reagents. Non-silencing siRNA were included as negative controls to access non-specific siRNA toxicity and universal lethal siRNA were included as positive controls to indirectly assess functional transfection efficiency. After a 96 hour incubation period, cell viability/proliferation was measured with Cell Titer Glo. For analysis of screen data, log2 values of (SAHA + siRNA treatment/siRNA treatment only) were calculated. Potentiation was defined as two standard deviations from the median log2 ratio of all siRNA (excluding positive controls) on a plate-by-plate basis. Hits were defined as ≥ 2/4 siRNA sequences meeting potentiation criteria.

Results:
Transfection efficiency was 95, 90 and 70% for HEL, THP-1 and TF-1, respectively. All three cell lines were similarly sensitive to SAHA with IC50 values of 0.3, 0.4 and 0.6 μM for HEL, THP-1 and TF-1, respectively. Resulting SAHA concentrations for screens were between the IC5 to IC30. For THP-1, 5 phosphatases qualified according to the hit selection criteria applied indicating strong potentiation to SAHA. Of these 5 hits, 3 did not exhibit strong reductions in viability with siRNA alone, suggesting a SAHA-dependent relationship. For HEL and TF-1, although no phosphatases met the strict selection cutoff, there were several phosphatases exhibiting potentiation, suggesting biological relevance. The primary screening data and preliminary analysis will be validated with rigorous secondary experimentation and top hits for all cell lines will be investigated and advanced.

Conclusions:
While there are numerous kinase inhibitors in the clinic and in preclinical development, phosphatases are much less explored as therapeutic targets. Validated potentiating targets from this RNAi screen can facilitate the development of rational SAHA combination therapies.

Disclosures:
No relevant conflicts of interest to declare.
CD81-Dependent Trafficking of CD19: Restoration of CD19 Surface Expression in Human B Cells Harboring A CD81 Mutation
Authors: Levy Shoshana , Kuo Chiung-Chi , Sagi Yael , Chen Homer , Kela-Madar Neta , van Zelm Menno , van Dongen Jacques J.M. 
Published: October 2019

Abstract
Abstract 1049

Introduction:
A 6-year-old girl, who was diagnosed with a primary antibody deficiency, had B cells lacking surface CD19. However, both her CD19 alleles were normal and the impairment was actually caused by a homozygous exon splice site mutation in CD81 (1). The patient's B cells also lacked surface CD81 and produced an immature glycosylated CD19 protein that was retained intracellularly. Interestingly, this human deficiency differed from that of CD81 knockout mice as the latter still express a low level of CD19 on their B cells.

Methods:
We used an EBV-transformed B cell line from this patient to better understand i) the difference between the human and mouse CD81 deficiencies and ii) how CD81 controls the trafficking of CD19 to the cell surface. We reasoned that the truncated human CD81 mutant (CD81mut) protein might be expressed intracellularly. Indeed, whereas most anti-CD81 mAbs did not recognize CD81mut, we identified one that bound the mutated form and used it in this study. We also expressed the human CD81mut in a CD81-deficient mouse B cell line to determine if it could negatively regulate CD19 surface expression.

Results:
We show that the CD81mut protein is indeed expressed intracellularly in the patient's EBV-transformed B cells. We then used a proximity ligation assay to demonstrate that the truncated CD81mut protein interacts intracellularly with CD19. However, this interaction with the CD81mut protein abrogated carbohydrate maturation and the trafficking of CD19 to cell surface. We therefore expressed the CD81mut in CD81KO mouse B cells, which still express low levels of surface CD19, and found that it did not exert a dominant negative effect on CD19 surface expression. Finally, we used this reconstitution system to identify specific CD81 domains that restored carbohydrate maturation and cell surface expression of the CD19 molecule in the patient's B cells.

Conclusion:
This specific case of antibody deficiency was manifested because of lack of surface expression of CD19, an important B cell signaling molecule. However, the maturation of CD19 and its trafficking to the cell surface require the presence of specific domains of the tetraspanin CD81 molecule.

Disclosures:
No relevant conflicts of interest to declare.
Differential Regulation of Myeloid Leukemias by the Bone Marrow Microenvironment
Authors: Krause Daniela S. , Fulzele Keertik , Catic Andre , Hurley Michael , Lezeau Sanon , Attar Eyal C. , Wu Joy Y. , Lin Herbert Yih-Fuu , Divieti-Pajevic Paola , Hasserjian Robert , Schipani Ernestina , Van Etten Richard A. , Scadden David T. 
Published: October 2019

Abstract
Abstract 1245
Normal hematopoiesis is influenced by constituents of the bone marrow microenvironment (BMM) and is regulated by parathyroid hormone (PTH) via its actions on osteoblasts, osteoclasts and bone remodeling. The role of the niche in the biology of leukemic stem cells (LSC), frequently not eliminated by current therapies, however, is unclear. Targeting of the LSC niche is a novel concept aimed at eradication of LSC, the origin of relapse and progression of myeloid leukemias.
Using genetic and pharmacological murine models of BCR-ABL1+ chronic myelogenous leukemia (CML) and MLL-AF9+ acute myeloid leukemia (AML), we show that PTH treatment increased transforming growth factor β1 (TGFβ1) in the BMM and attenuated CML-like myeloproliferative neoplasia (MPN) without affecting engraftment, while leukemic cell-specific knockdown of the receptor for TGFβ1 (TGFβRI) exacerbated the disease. CML-like MPN is also exacerbated by treatment of mice, which have constitutively active receptor for PTH and PTH related protein, by osteoprotegerin, which inhibits osteoclasts and bone remodeling. In contrast, AML was accelerated by the PTH-modified BMM, which was reversed by overexpressing TGFbetaRI. PTH treatment reduced CML LSC frequency and cycling and, combined with the tyrosine kinase inhibitor imatinib, significantly prolonged survival of mice with CML-like MPN versus imatinib alone. PTH-treatment also reduced BCR-ABL1 transcript levels in a xenotransplantation model of human CML.
In summary, our results suggest that LSC niches in acute and chronic myeloid neoplasms are distinct and differentially modulated by PTH via TGFβ1. Combinations of TKIs with BMM-modulating agents may be a therapeutic strategy for LSC reduction and cure of CML.

Disclosures:
Lin: Ferrumax Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties.