bluuebunny/crossref_metadata_2025_split huggingface dataset:
Active Chromatin Structure near MYB Occupancy at the Mir-155 Host Gene Promoter Coincides with Increased Mir-155 and MYB Levels In Chronic Lymphocytic Leukemia
Authors: Stopka Tomas , Vargova Karin , Curik Nikola , Burda Pavel , Basova Petra , Pospisil Vit , Kulvait Vojtech , Savvulidi Filipp , Necas Emanuel , Berkova Adela , Obrtlikova Petra , Karban Josef , Trneny Marek , Zavadil Jiri 
Published: October 2019

Abstract
Abstract 3589
Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In our study, B-CLL patients (N=71, F/M 31/40, median age 65) were subject to molecular analyses that found significant overexpression of MYB and miR-155 in B-CLL and physical association of MYB with the promoter of MIR155 host gene (MIR155HG, also known as B-cell integration cluster). In CD19+ B-cells derived from healthy control individuals (N=13), MYB and miR-155 expression was significantly lower. Next, we found that MYB positively regulates MIR155HG transcription in reporter assays. The endogenous chromatin structure of the MIR155HG promoter in B-CLL cells is characterized by spreading of active chromatin mark, histone H3K9 acetylation. Gene expression arrays on B-CLL patients (N=11) identified a set of predicted miR-155 target genes (N=94) that are downregulated. Their expression pattern displayed significant negative correlation with pri-miR-155 levels. Similarly, a number of MYB target genes were found deregulated (N=99) in B-CLL. Gene annotation of differentially expressed miR-155 and MYB targets in B-CLL revealed their biological functions as regulators of apoptosis (BCL2, API5), proliferation (CCND1, CCND2) and mediators of B-cell function (CD200, F11R). In addition, expression patterns of miR-155, MYB and their targets are currently being compared in a larger patient group with prognostic hallmarks of CLL (ZAP70, CD38, del(17)(p13.1), and IgVH). Our data collectively support novel candidate mechanism in B-CLL that includes MYB directly stimulating MIR155HG promoter coincident with its epigenetic dysregulation. (Grant # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).

Disclosures:
Trneny: ROCHE: Honoraria, Research Funding.
High IGSF4 expression In Pediatric Acute Monoblastic Leukemia with t(9;11)
Authors: Coenen Eva A , Kuipers Jenny , Balgobind Brian V , Stary Jan , Baruchel Andre , Haas Valerie de , Bont Evelina S. De , Reinhardt Dirk , Kaspers Gertjan J. , Cloos Jacqueline , Oorschot Astrid Danen-van , Boer Monique L. Den , Marschalek Rolf , Meyer Claus , Pieters Rob , Zwaan Christian M. , Heuvel-Eibrink Marry van den 
Published: October 2019

Abstract
Abstract 3614
Pediatric MLL-rearranged acute monoblastic leukemia with t(9;11)(p22;q23) has a good outcome as compared to other MLL-rearranged AML. The biological background for this difference is unknown. Therefore, we compared gene expression profiles (GEP) of -t(9;11)(p22;q23) patients with other MLL-rearranged AML patients to identify differentially expressed genes.
We performed GEP (Affymetrix HG U133 plus 2.0) in 245 pediatric AML patients (237 de novo and 8 secondary AML patients) and used RT-qPCR and Western Blot to validate expression. Methylation specific PCR was used to investigate epigenetic regulation. We tested the effect of the demethylating agent decitabine and the effect of knock-down by siRNA on both proliferation and drug sensitivity in AML cell lines.
IGSF4, a cell-cell adhesion molecule, was highly expressed in AML-t(9;11). Expression within AML-t(9;11) was 18.5 times higher in FAB-M5 versus other FAB-types (p=0.013). RT-qPCR and Western Blot confirmed this. Methylation status investigation showed that high IGSF4 expressing AML-t(9;11) patients with FAB-M5 have no promoter hypermethylation, whereas all other cases do. This was also seen in cell lines. Cell line incubation with decitabine resulted in promoter demethylation and increased expression of IGSF4. Downregulation of IGSF4 by siRNA did not affect proliferation nor drug sensitivity in suspension culture.
In conclusion, we identified IGSF4 overexpression to be discriminative for AML-t(9;11) with FAB-M5, regulated partially by promoter methylation.

Disclosures:
No relevant conflicts of interest to declare.
Gene Expression-Based Chemical Genomics Identifies Valproic Acid to Revert the Oncogenic Effect of GATA1s In Down Syndrome Megakaryoblastic Leukemia.
Authors: Heitmann Kirsten , Li Zhe , Klusmann Jan-Henning , Thakur Basant Kumar , Schöning Jennifer , Bourquin Jean-Pierre , Orkin Stuart H. , Reinhardt Dirk 
Published: October 2019

Abstract
Abstract 3646
Children with Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the antecedent transient leukemia (DS-TL). Acquired mutations in the hematopoietic transcription factor GATA1, leading to expression of a shorter GATA1 variant (referred to as GATA1s) truncated at its N-terminus, are consistently present in the affected cells of children with DS-AMKL and DS-TL. Mechanistically, we recently found that in fetal megakaryocytic progenitor cells, GATA1 coordinates proliferation and differentiation by repressing E2F target genes through a direct interaction with E2F activators. Failure of this GATA1-E2F interaction in mutated GATA1s likely converges with overactive IGF signaling to promote cellular transformation.
The treatment of DS-AMKL is hampered by their sensitivity against current cytostatic agents, resulting in treatment-related mortality as the main cause of death. To develop novel targeted and less toxic treatment options for DS-AMKL and DS-TL, we conducted a gene expression-based chemical genomic screen. We connected a DS-AMKL gene expression signature (compared to non-DS-AMKL, i.e. GATA1s vs. GATA1) to a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules (Connectivity Map). We discovered the histone deacetylase (HDAC) inhibitor valproic acid (VPA) reverses the DS-AMKL gene expression program.
Cell viability assays, cell cycle analyses, growths curves and colony-forming assays revealed exceptional sensitivity of DS-AMKL cell lines (CMK, CMY; IC50 1mM) and primary DS-AMKL and DS-TL blasts to VPA treatment compared to control cell lines K562 (IC50 4.75mM), M07 (IC50 6.75mM) and CD34+ hematopoietic stem and progenitor cells (IC50 4.75mM). VPA induces apoptosis (26.8% 7-AAD-)Annexin V+ and 38.8% 7-AAD+ CMK cells after 48h at 2mM VPA) and cell cycle arrest (50% reduction of CMK cells in S-phase at 2mM) via activation of the cell cycle inhibitor p21 and the proapoptotic genes BAX and BAK. Gene expression profiles indicated that VPA interferes with the oncogenic effects of GATA1s by globally repressing the deregulated E2F targets. The effects of VPA on leukemic growth in DS-AMKL could be attributed to its HDAC inhibitory function, as the global HDAC inhibitors SAHA and TSA induced a similar response.
Thus, by using a gene expression-based chemical genomic approach, we identified VPA as an efficient and well-tolerated treatment option for DS-AMKL and DS-TL by targeting GATA1s-mediated deregulation of the E2F transcription network.

Disclosures:
No relevant conflicts of interest to declare.
Congenital Factor VII Deficiency and Surgery In a Single Center Experience.
Authors: Park Young sil , Park Ji Kyoung 
Published: October 2019

Abstract
Abstract 3658
Congenital factor VII (FVII) deficiency is rare hemorrhagic disorder, and, in affected patients, surgery is likely to cause excessive bleeding. Recombinant form of activated factor VII (Novoseven® from Novo Nordisk, Bagsvaerd, Denmark), that was developed as a second-generation bypassing agent, is used in management of bleeds in patients with congenital FVII deficiency recently. Doses lower than those recommended for patients with hemophilia are usually effective in patients with FVII deficiency.
We review the results of 6 surgical procedures in 4 patients with congenital FVII deficiency in East-West Neo Medical Center. Between January 2008 and February 2010, 6 surgeries for patients with congenital FVII deficiency were performed. We administerated rFVIIa pre- and postoperatively. Plasma FVII:C levels were measured in baseline, before and after the intravenous administration of routine dose FVIIa.
Between January 2008 and February 2010, 6 surgeries for 4 patients with congenital FVII deficiency were performed successfully without complications in our center. The median level of FVII:C was 2% (range 2–7%). The median age of patients was 29.5 years (range, 7–37). Types of surgery were 4 orthopedic surgeries, 1 tonsillectomy, and 1 dental extraction. The median duration of hospitalization was 14 days (range 6–15). rFVIIa was used at all surgery except dental extraction. FVIIa was administrated every 4 hours with the first dose being administered 1 hour preoperatively at least 48 h with dose 15–30 μμ¤/kg. After 48 h, the frequency was reduced according to the patient's clinical status. Plasma FVII:C levels were measured before testing and then again after the intravenous administration of routine dose FVIIa. FVII:C levels increased to about 200 %. Dental extraction was performed with only fibrinolytic agents without any replacement. There was no bleeding and thrombogenic complication.
rFVIIa was well-tolerated, maintained effective haemostasis with good clinical outcome. Patients with congenital FVII deficiency requiring surgery can be treated by the use of rFVIIa and antifibrinolytic agents efficiently and safely.

Disclosures:
No relevant conflicts of interest to declare.
Non-Invasive Management of Alloimmune Thrombocytopenia: Prediction of the Fetal Status.
Authors: Bertrand Gerald , Drame Moustapha , Martageix Corinne , Kaplan-Gouet Cecile 
Published: October 2019

Abstract
Abstract 3690
Fetal/neonatal alloimmune thrombocytopenia (F/NAIT) is the most common cause of severe thrombocytopenia in the fetus and the newborn in maternity wards. To counter the bleeding consequences of severe fetal thrombocytopenia, antenatal therapies have been implemented for subsequent pregnancies with incompatible fetuses. Predictive parameters for fetal severe thrombocytopenia are important for the development of non-invasive strategy and tailored intervention. We report here data concerning 67 HPA-1bb women with 81 managed pregnancies with IVIG. In 51% of the cases, the diagnosis of F/NAIT was made during the first gestation, following an intracranial haemorrhage (ICH) in 8 cases (12%). Severe thrombocytopenia was recorded for 88% of the newborn. Analysis of the index cases did not show any correlation between the severity of the disease and the maternal genetic background (ABO group and HLA-DRB3 allele). Subsequent pregnancies were managed and therapy effectiveness was evaluated. The highest mean newborn plt count was observed for a combination of intravenous immunoglobulin (IVIG) and corticoids (135.109/L; 54 newborn), in comparison with IVIG alone (89.109/L; 27 newborn). No ICH was recorded in these 2 groups. The maternal anti HPA-1a antibody concentration measured before any treatment and before 28 weeks of gestation was predictive of the fetal status: a high antibody concentration (≥28 International Units/mL) was correlated with a severe thrombocytopenia of the fetus (p=0.0016). Follow-up of the antibody concentrations during 34 pregnancies with antenatal management allowed demonstrating for the first time that the areas under curves (AUC) weighted by the weeks of gestation were a predictive parameter of therapy failure. The weighted AUC was significantly higher for women who delivered severely thrombocytopenic newborn than newborn with platelet count above 50.109/L (p=0.0107). To conclude, this large retrospective survey gives new insights on maternal predictive parameters for fetal status and therapy effectiveness allowing non-invasive strategies.
Table 1: Maternal anti HPA-1a alloantibody concentrations and fetal platelet counts: statistical analysis. Predictive parameter Platelet count (×109/L) p-value Se (%) Sp (%) PPV (%) NPV (%) <50 ≥50 Number of mothers with antibody concentration (<28wg) <28 IU/mL 3 Fetuses 10 0.0016* 81.2 91.7 92.3 79.9 ≥28 IU/mL 13 2 Number of pregnancies with weighted AUC <24 IU/mL/wg 5 Newborn 18 0.0153* 64.3 82.6 69.2 79.2 ≥24 IU/mL/wg 9 5 * P<0.05. AUC: area under curve; IU: international units; Se: sensitivity, Sp: specificity; PPV: positive-predictive value; NPV: negative-predictive value. Wg: weeks of gestation.

Disclosures:
No relevant conflicts of interest to declare.
Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.
Authors: Semple Kenrick , Nguyen Antony , Yu Yu , Anasetti Claudio , Yu Xue-Zhong 
Published: October 2019

Abstract
Abstract 3728
CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling.

Disclosures:
No relevant conflicts of interest to declare.
High Frequency of Alloantigen-Specific Regulatory T-Cells In Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy.
Authors: Veerapathran Anandharaman , Pidala Joseph , Beato Francisca , Yu Xue-Zhong , Anasetti Claudio 
Published: October 2019

Abstract
Abstract 3747
Evidence from animal studies has demonstrated that allogeneic regulatory T (Treg) cells can be used as effective therapeutic tools in the prevention of allograft rejection and graft-vs-host disease (GVHD). However, translating Treg-based therapies from animal models to human clinical trials requires methods for the isolation and expansion of functional Treg. Loss of function has been a concern for Treg expanded broadly with anti-CD3 and CD28 antibody-coated beads. Based on preclinical data in rodents, allospecific Tregs are anticipated to be more potent and exert more selective immunoregulation than polyclonal Treg in the prevention or treatment of GVHD. Currently, no effective approach has been established for selective expansion of human allospecific Treg ex vivo. In this study, we show the selective direct and indirect recognition of alloantigen by specific human CD4+CD25+CD127− Tregs. CD4+CD25+CD127− Treg were freshly isolated from normal human blood, labeled with CFSE and stimulated by MHC (HLA)-mismatched APC's, in the presence of IL-2 and IL-15 to amplify the proliferative responses, and sirolimus to suppress other T cells. The precursor frequency of the antigen specific Tregs was calculated by Limiting Dilution Analysis and found to be 1/49 -1/138 among all Tregs. The expanded antigen specific Tregs with low CFSE content were in general greater than 90% by days 12–15 in culture. CFSE-low Tregs were sorted by flow cytometry and exhibited complete suppression against CD25-negative CD4 T cell responses up to ratios of 1:100, in contrast CFSE-high Tregs were not suppressive. Ex vivo expanded antigen specific Tregs maintained high Foxp3, retained lymphoid homing receptor CD62L and chemokine receptor CCR7 expression, suggesting that they are functional and able to migrate to lymphoid tissue in vivo. Among the APC's tested, DC was found to be better stimulus with potent suppressive potential when compared to T-cell stimulated PBMC's. Specific indirect allorecognition was elicited when Tregs were stimulated with autologous APC's pulsed with allogeneic cell lysate in the presence of sirolimus, IL-15 and IL-2. Precursor frequency of antigen specific Tregs with indirect allorecognition was 100 fold lower than the precursor frequency of Tregs with direct allospecificity. Highly suppressive antigen specific Tregs show a duplication time of approximately 24 hours in the presence alloantigen, cytokines and sirolimus, and can be expanded in vitro by 400 fold in a 12 day period. This magnitude of expansion predicts the feasibility of conducting translational clinical trials. Our data may provide a platform for the selective expansion of Tregs against minor histocompatibility antigens to prevent GVHD while sparing graft-vs.-leukemia effects.

Disclosures:
No relevant conflicts of interest to declare.
Tregs In Graft-Versus-Host Disease: A Task Force In Trouble?.
Authors: Ukena Sya N. , Grosse Jens , Buchholz Stefanie , Stadler Michael , Ganser Arnold , Franzke Anke 
Published: October 2019

Abstract
Abstract 3748
Graft-versus-host disease (GvHD) remains the major clinical complication in hematopoietic stem cell transplantation (SCT) resulting in severe morbidity and significant mortality. This alloreactive immune response is mainly induced by donor T cells transplanted with the graft. Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance. In addition, data from murine models have shown that Tregs can prevent GvHD while preserving the graft-versus-leukemia effect. In order to functionally and dynamically characterize human Tregs after allogeneic SCT, we analyzed CD4+CD25highCD127dim T cells isolated from the peripheral blood of more than 80 patients with hematological malignancies every 30 days over half a year following SCT.
Patients were divided into the following clinical groups: (A) no signs of acute or chronic GvHD, (B) acute GvHD, (C) chronic GvHD and (D) acute GvHD passed into chronic GvHD. Human peripheral blood lymphocytes were separated by Ficoll gradient and CD4+CD14−CD25highCD127dim T cells were isolated by MoFlow cell sorting. Isolated RNA was pooled and microarray analysis was performed by using Affymetrix HG_U133_Plus2.0 Arrays. Results were verified by using quantitative realtime RT-PCR. Additionally, Tregs were phenotypically analyzed by FACS.
We monitored a continous but slower recovery of Tregs in GvHD within the first 6 months following PBSCT. Manifestation of acute and chronic GvHD correlated with significantly reduced frequencies of peripheral Tregs in the first month after PBSCT compared to patients without GvHD. Microarray data revealed a high stability of the Treg transcriptome in the first half year representing the most sensitive time window for tolerance induction. Moreover, comparison of the Treg gene expression profiles from patients with and without GvHD point to a reduced suppressive function of Tregs with diminished migration capacity to the target organs likely contributing to the development of GvHD.
Our findings corroborate the impact of human Tregs in the pathophysiology of GvHD and identify novel targets for the manipulation of Tregs to optimize strategies for prophylaxis and treatment of life-threatening GvHD.

Disclosures:
No relevant conflicts of interest to declare.
Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
Authors: Marin Virna , Pizzitola Irene , Biondi Andrea , Biagi Ettore , Pule Martin 
Published: October 2019

Abstract
Abstract 3753
Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.

Disclosures:
No relevant conflicts of interest to declare.