bluuebunny/crossref_metadata_2025_split huggingface dataset:
Akt-Mediated Phosphorylation of Bmi1 Regulates Its Chromatin Association and Growth Promoting Properties.
Authors: Liu Yan , Liu Fan , Zhao Xinyang , Sashida Goro , Deblasio Anthony , Chen Zhenbang , Lin Hui-kuan , Di Giandomenico Silvana , Elf Shannon , Yang Youyang , Miyata Yasuhiko , Huang Gang , Menendez Silvia , Mellinghoff Ingo , Pandolfi Pier Paolo , Hedvat Cyrus V. , Nimer Stephen D. 
Published: October 2019

Abstract
Abstract 3605
Poster Board III-541
The Polycomb group (PcG) protein Bmi1 maintains silencing of the Ink4a-Arf locus and plays a key role in stem cell self-renewal and oncogenesis. The phosphoinositide 3-kinase-Akt (PI3K-Akt) signaling pathway regulates cell survival, growth, metabolism, migration and angiogenesis. In response to acute Pten loss (which results in Akt activation), mouse embryonic fibroblasts (mefs) accumulate p16Ink4a and p19Arf and undergo senescence. Similarly, Bmi1 −/− mefs undergo premature senescence and accumulate p16Ink4a and p19Arf. PTEN and Bmi1 have similar effects on hematopoiesis; Pten deletion promotes hematopoietic stem cell (HSC) proliferation, resulting in HSC depletion, whereas loss of Bmi1 impairs HSC self-renewal capability, also leading to bone marrow failure. These similarities led us to examine whether the PI3K/Akt pathway functions upstream of Bmi1 to negatively regulate its function and indeed we found that PKB/Akt phosphorylates Bmi1 in vivo, which results in its dissociation from chromatin and in de-repression of the Ink4a-Arf locus. Furthermore, activation of the PI3K/Akt pathway suppresses the ability of Bmi1 to promote cell growth and tumourigenesis and decreases the global level of histone H2A ubiquitination. PI3K/Akt signaling is not active in hematopoietic stem cells, but it is active in more committed progenitor cells. Thus, phosphorylation and inactivation of Bmi1 by Akt may limit HSC self-renewal. Our study also provides a mechanism for the upregulation of p16Ink4a and p19Arf seen in cancer cells that have activation of the PI3K/Akt signaling pathway, and identifies important crosstalk between phosphorylation and chromatin structure.

Disclosures:
No relevant conflicts of interest to declare.
Continuous Single Cell Observation Proves Lineage Instruction by Hematopoietic Cytokines.
Authors: Rieger Michael A. , Hoppe Philipp S , Smejkal Benjamin M , Eitelhuber Andrea C , Schroeder Timm 
Published: October 2019

Abstract
Abstract 3607
Poster Board III-543
The significance of cytokines in hematopoiesis is irrevocable, but their contribution in lineage choice of multipotent progenitors is still controversial. Can late acting cytokines actively induce lineage decisions (instructive model), or do they only allow the survival and proliferation of already unilineage-restricted precursors, which have been differentiated by cell-autonomous mechanisms (selective/stochastic model)?
A major reason for contradictory results is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. Both models can only be distinguished by constantly observing the fate of each cell and its progeny, but not by endpoint analyses of whole populations. Therefore, real-time tracking of individual cells in culture, tissues or whole organisms is an extremely powerful approach to fully understand the exact effect of cytokines on fate decisions.
A computer aided culture and imaging system was developed to follow the fate of individual cells over long periods of time with high temporal and spatial resolution. New software modules were written, which help to record, display and analyze the divisional history, position, properties etc. of individual cells.
Purified progenitor cells from mouse bone marrow of lineage-reporter mice were cultivated in defined media with the addition of selected cytokines. Video-time-lapse experiments enabled the tracking of individual cells and their progeny over many days. Pedigrees of hundreds of progenitor cells under each condition were generated and statistically analyzed. This novel single cell tracking technology revealed an unexpected instructive action of M-CSF and G-CSF directing lineage decisions of granulocyte-macrophage progenitors (GMPs) to differentiate into the monocytic or granulocytic lineage, respectively. Additionally, both late acting cytokines accelerated the differentiation process of GMPs. First results indicate an instructive in vivo action of these cytokines on progenitor lineage choice.

Disclosures:
No relevant conflicts of interest to declare.
Gata4 Regulates Expression of the Erythropoietin Receptor in Cardiomyocytes.
Authors: von Salisch Sandy , Klar Martin , Thurisch Boris , Bungert Jorg , Dame Christof 
Published: October 2019

Abstract
Abstract 3610
Poster Board III-546
Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. Using reporter gene assays, we identified in murine HL-1 cardiomyocytes a 774 bp regulatory domain in the 5′-flanking region of the EpoR gene. Fragments that include an E-box, CACCC motifs, GC-rich elements, and binding sites for members of the Gata and Sp transcription factor families contribute to EpoR promoter activity. EMSA experiments indicated the specific binding of Gata4 and Sp1 (a known interaction factor of Gata4) to the minimal EpoR promoter. Forced expression of Gata4 resulted in a significant induction of EpoR mRNA expression, while Sp1 overexpression did not affect EpoR expression. Depletion of Gata4 by treating mice with high-dose doxorubicin led to the downregulation of EpoR expression, followed by the upregulation of EpoR when Gata4 levels recovered after 5 days. Transgenic mice with an inducible short hairpin RNA against Gata4 confirmed the suppression of EpoR expression if Gata4 levels were reduced. Under hypoxia, however, a 2-fold induction of EpoR mRNA expression in cardiomyocytes was independent from Gata4, since Gata4 levels decreased. Our data indicate that Gata4 activates EpoR mRNA expression in cardiomyocytes. This is a novel mechanism that differs from EpoR regulation in neurons and hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.
Impaired Bone Marrow Microenvironment in Fanconi Anemia Murine Model Is Responsible for the Defective Hematopoietic Stem/ Progenitor Cell Mobilization.
Authors: Li Yan , Chen Shi , He Yongzheng , Li Xiaohong , Yang Fengchun 
Published: October 2019

Abstract
Abstract 3629
Poster Board III-565
Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients.

Disclosures:
No relevant conflicts of interest to declare.
Expression of CD1c, CD1d and Presence of Invariant NKT Cells in Hodgkin Lymphoma.
Authors: Xu Chuanhui , de Vries Riemer , Visser Lydia , Diepstra Arjan , Gadola Stephan D , Poppema Sibrand , Van Den Berg Anke 
Published: October 2019

Abstract
Abstract 3659
Poster Board III-595

Introduction
Hodgkin lymphoma (HL) is a B-cell neoplasm characterized by a minority of neoplastic cells, the so-called Hodgkin and Reed-Sternberg (HRS) cells. The HRS cells are located within an extensive infiltrate of reactive cells, including T cells, B cells, plasma cells, stromal cells, eosinophils and macrophages. CD1 molecules are non-classical MHC I-like molecules that present lipid antigens to T cells, triggering a specific immune response. Of the five CD1 isoforms (CD1a, CD1b, CD1c, CD1d and CD1e) expressed in human tissue, only CD1c and CD1d are expressed in B cells. The T cell receptors (TCRs) of T cells that recognize CD1c are indistinguishable from those that recognize MHC class I or II complexes. In contrast, CD1d presents lipid antigens to invariant Natural Killer T cells (iNKT cells), which are characterized by the expression of a semi-invariant Vα24Jα18 chain plus a limited set of β chains. CD4− and CD4+ iNKT cells were demonstrated as two distinct functional subsets in terms of cytokine production and cytotoxic activation. Immunoregulatory CD1 restricted T cells have been implicated in the pathogenesis of many cancers, but its role in HL is still unknown. In this study, we analyzed the expression of CD1c, CD1d and presence of iNKT cells in HL.

Methods
Expression of CD1c and CD1d was determined by immunocytochemistry in four HL cell lines KMH2, L1236, L428 and U-HO1. CD1c and CD1d expression in both HRS cells and reactive cells was studied in 46 HL patient tissues by immunohistochemistry. Cell suspensions of ten HL cases were studied for the presence of iNKT cells using monoclonal antibodies against human iNKT cell (clone 6B11) and TCR Vβ11 (clone C21) by flow cytometry. The percentage of CD4+ iNKT cells was determined in the gated iNKT cell population.

Results
Cytoplasmic CD1d expression was found in four HL cell lines and membranous CD1d expression was found in KMH2 and L1236. CD1c expression was negative in all four HL cell lines. CD1d expression was detected in HRS cells in 46% (21/46) of the HL cases. No correlation was observed between CD1d expression and presence of EBV in HRS cells. In contrast, CD1c expression was negative in HRS cells in all HL cases. Both CD1c and CD1d were detected in reactive cells in all HL cases albeit at varying frequencies. The mean percentage of CD1d restricted iNKT cells was 4% (range 0.8-8%) in HL cell suspensions irrespective of CD1d expression status in HRS cells. In reactive lymph node (RLN) the mean percentage of iNKT cells was also 4% (range 0.4-7%). Approximately half of the iNKT cells were CD4+ in both HL and RLN cell suspensions.

Conclusion
Expression of CD1d was found in four HL cell lines and in HRS cells of ∼50% of the HL cases. In HL cell suspensions, about 4% (range 0.8-8%) of the reactive background cells were iNKT cells.

Disclosures:
No relevant conflicts of interest to declare.
Ten Years of Experience with Invasive Aspergillosis: Has Anything Changed?.
Authors: Mattiuzzi Gloria , Cortes Jorge , Ho Jennifer , Kantarjian Hagop M. 
Published: October 2019

Abstract
Abstract 3687
Poster Board III-623
Invasive Aspergillosis (IA) has represented a threat for patients and physicians for many years. This is especially true for patients with hematologic malignancies. Several new antifungal agents with broad antifungal activity became available in 2000 ushering in a new millennium with hopes that with this new armamentarium of antifungal drugs will decrease mortality due to IA and perhaps even prevent them better. Mortality rates due to IA have been described in recent reports as acceptable and even in some cancer centers significantly lower than before. The objective of the present study was to evaluate IA in patients with hematologic malignancies in the leukemia department at MDACC during the past 10 years (1998 to 2007) and compare the incidence and mortality rates between these two times periods.

Methods
retrospective review of all patients that presented at the leukemia department at MDACC from January 1998 to December 2007. Proven or probable IA was defined as per EORTC criteria.

Results
From 1998 to 2007, 12,845 patients were presented to our department, including 4,243 pts with acute myelogenous leukemia (AML); 940 with acute lymphocytic leukemia (ALL); 3,642 with chronic lymphocytic leukemia (CLL); and 4,276 with other type of leukemia (chronic myelocytic leukemia, low risk-myelodysplastic syndrome and miscellaneous). Overall median age was 58 (range 17-83); 60% were male, 83% did not have history of diabetes mellitus; 71% were neutropenic (ANC < 500) at the moment of the infection; and 11% had a previous bone marrow transplantation.
The overall incidence of IA has significantly decreased from 4 % to 2% over the 10 study years (p= 0.005, IRR=0.45), however, the overall mortality rate (60%) has not decreased significantly. Comparison between the incidence of IA and mortality in the different subsets of patients between 1998-1999 and 2000-2007 is shown in Table 1
The incidence of IA decreased significantly between the two time periods only in patients with AML and in patients with ALL (p<0.001 and 0.004, respectively). However, although there has been a reduction in the mortality rate in all the subsets of patients in the two periods, this difference is not significant.
Overall, A. fumigatus was the pathogen more frequently isolated (30%), followed by A. flavus (21%), A. terreus (18%) and A. niger (11%). In 10% of the episodes more than one Aspergillus species was isolated.
In conclusion, our data suggests that the incidence of IA in patients with leukemia traditionally at high risk for IA has significantly decreased since 2000. Despite the availability of new antifungal drugs for the treatment of IA, the mortality rate in patients with hematological malignancies has not significantly decreased.

Disclosures:
Mattiuzzi: Merck: Research Funding.
The Role of the PI3K-AKT-mTOR Pathway in NK Cell Anti-Tumor Reactivity.
Authors: Krusch Matthias , Salih Julia , Kumbier Ingrid , Fenner Carolin , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 3690
Poster Board III-626
The phosphatidylinositol 3-kinase – protein kinase B – mammalian target of rapamycin (PI3K – AKT – mTOR) pathway was found to be abnormally activated in many malignancies. Thus, protein kinase (PK) inhibitors (PKI) targeting different signaling molecules of this pathway are presently under clinical evaluation e.g. in sarcoma, multiple myeloma, or renal cell cancer. However, PK are also responsible for most of the signal transduction in immune effector cells and control various effector mechanisms including proliferation, cellular cytotoxicity, and cytokine release. Among those immunoregulatory signaling pathways, the PI3K – AKT – mTOR pathway was found to play a central role in TLR-mediated release of cytokines in macrophages and DC as well as in the regulation of T cell functions. Little is known about the role of this pathway in NK cell-mediated anti-tumor reactivity. Here we analyzed the tumor cell-induced activation of PI3K, AKT, and mTOR in NK cells and the consequences of an inhibition of these molecules by therapeutic PKI for NK cell anti-tumor reactivity. We found that, in response to tumor target cells, PI3K, AKT, and mTOR are consecutively activated in NK cells as revealed by western blot analyses using phospho-specific antibodies. Presence of the specific PI3K-inhbitor BKM-120 concentration-dependently inhibited cytotoxicity and IFN-g production of NK cells, which is in line with available data defining PI3K as a central regulator of NK cell target recognition. The mTOR inhibitors Sirolimus, Temsirolimus, and Everolimus did not alter cytotoxicity but significantly impaired NK cell IFN-γ production. In contrast, Triciribine, a compound which inhibits the phosphorylation and thus activation of AKT, did not influence cytotoxicity and, tantalizingly, even enhanced NK cell IFN-γ production. Thus, after target cell recognition and the activation of proximal PK like PI3K, different and at least partially independent signaling events govern NK cell cytokine production and cellular cytotoxicity. While the activity of PI3K followed by the activation of mitogen-activated PK is known to be crucial for NK cell cytotoxicity, we here identified the AKT – mTOR pathway as a yet unknown central component in the regulation of NK cell IFN-γ production. Moreover, in light of the important role of NK cells in tumor immune surveillance our data indicate that the choise and dosing of the most suitable PKI for a given cancer patient requires careful consideration. In the future it will be critical to define potential differences in immunosuppressive and immunostimulatory side effects of different compounds among the rapidly growing assortment of multi-targeted PKI to enable therapeutic approaches combining targeting of crucial signaling pathways in tumor cells with immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way.
Authors: Niederfellner Gerhard J , Lammens Alfred , Schwaiger Manfred , Georges Guy , Wiechmann Kornelius , Franke Andreas , Schaefer Wolfgang , Jenewein Stefan , Slootstra Jerry , Moessner Ekkehard , Umana Pablo , Hopfner Karl-Peter , Klein Christian 
Published: October 2019

Abstract
Abstract 3726
Poster Board III-662
CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies.

Disclosures:
Niederfellner: Roche: Employment. Schwaiger:Roche: Employment. Georges:Roche: Employment. Wiechmann:Roche: Research Funding. Franke:Roche: Research Funding. Schaefer:Roche: Employment. Jenewein:Roche: Employment. Slootstra:Pepscan: Employment, Patents & Royalties. Moessner:Glycart: Employment, Equity Ownership, Patents & Royalties. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner:Roche: Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.
T Cell Responses in Patients Vaccinated with Telomerase (hTERT)-mRNA Transfected Dendritic Cells.
Authors: Suso Else Marit Inderberg , Rasmussen Anne-Marie , Aamdal Steinar , Dueland Svein , Gaudernack Gustav , Kvalheim Gunnar 
Published: October 2019

Abstract
Abstract 373
Two cancer patients were vaccinated with dendritic cells (DC) loaded with telomerase (hTERT) mRNA to investigate the safety, tolerability and immunological response to vaccination prior to the start of a new phase I/II clinical trial. Following written informed consent one primary lung adenocarcinoma with metastasis and one patient with a relapsed pancreatic ductal type of adenocarcinoma, were treated with autologus monocyte-derived DC transfected with mRNA encoding hTERT. The patients first received four weekly injections administered intradermally followed by monthly booster injections. Peripheral blood mononuclear cells (PBMC) at each vaccination time point were tested in vitro with transfected DC and a panel of 24 overlapping hTERT peptides. In addition, hTERT-specific CD8+ T cells were monitored by pentamer staining. The treatment was well tolerated with minor side effects. Immune responses against telomerase-transfected DC and some of the overlapping hTERT peptides were detected in both patients. We also detected hTERT-specific CD8+ T cells in both patients by pentamer staining in post-vaccination samples. The lung cancer patients obtained a stable disease that lasted 18 months while the patient with pancreas cancer who started the DC vaccination in July 2007 following palliative chemotherapy, still is in stable disease by continuously boost vaccination. T-cell responses against telomerase epitopes have also been identified in both non-vaccinated cancer patients and cancer patients previously vaccinated with telomerase peptide. Since patients with these findings often show extraordinary clinical courses of their disease we hypothesize that it exists a high degree of immunogenicity and HLA promiscuity for some telomerase epitopes. In this study we have shown that vaccination with hTERT-mRNA transfected DC is safe and able to induce robust immune responses to several telomerase T-cell epitopes both in CD4+ and CD8+ T cells. This opens up the possibility for a broad clinical application of mRNA hTERT DC vaccines. Furthermore, responding T cells identified in these patients are strong candidates for T-cell receptor cloning and the receptors identified can thereafter be transferred into T cells creating the next generation of immuno-gene therapy with retargeted T cells.

Disclosures:
No relevant conflicts of interest to declare.