bluuebunny/crossref_metadata_2025 huggingface dataset:
p38 MAPK Inhibition Prevents TACE-Mediated Receptor Shedding and Improves the Hemostatic Function of Stored Platelets.
Authors: Canault Matthias , Duerschmied Daniel , Brill Alexander , Patten Ian Sean , Bergmeier Wolfgang , Wagner Denisa D.
Published: October 2019

Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding from platelet stored for 18 hours at 37ºC or 22ºC. Using pharmacological inhibitors, we show that TACE -dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during the storage of mouse platelets also markedly improved their posttransfusion recovery and hemostatic function in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved their adhesion to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed after platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.
Transcription Factor Integration from Pathways Regulating Adult Blood Stem Cell Production and Self-Renewal.
Authors: Zon Leonard I.
Published: October 2019

Self-renewal of hematopoietic stem cells (HSCs) involves multiple signaling and transcription factors. We recently showed that prostaglandin (PG) E2 regulates the induction and engraftment of vertebrate HSC pathways. Yet, the targets and transcriptional output of the PGE2 pathway remains to be defined. The wnt pathway similarly affects HSC formation; activation of wnt signaling in heat-shock inducible transgenic zebrafish led to enhanced HSC formation, while inhibition of wnt/β-catenin signaling at the membrane level (dickkopf), in the cytosol (axin) or in the nucleus (dominant negative TCF), reduced HSC numbers. Using wnt transcriptional reporter zebrafish with multimerized TCF-binding sites. PGE2 was found to increase activity during embryonic development, demonstrating a direct interaction between these signaling pathways. The transcriptional output of the wnt pathway is linked to the interaction of TCF with β-catenin. Based on the transcriptional increase in multimerized TCF binding sites in vivo, we had hypothesized that prostaglandin signaling directly stimulates β-catenin activation. β-catenin is known to be phosphorylated on critical residues that modulate its activity. In marrow-derived cells, we demonstrate that prostaglandin increases a cyclic AMP pathway that leads to the PKA phosphorylation of β-catenin, thereby increasing its transcriptional activity. In HSCs, the interaction of multiple signaling pathways with transcriptional output is a method for modulating self-renewal in the stem cell pool as well as tissue differentiation.
Switching Genes On and Off During Hematopoiesis.
Authors: Higgs Douglas R.
Published: October 2019

We have studied how transcriptional and epigenetic programs are played out on chromatin spanning the terminal 500kb of human chromosome 16 (16p13.3) as hematopoietic cells undergo lineage fate decisions and differentiation. This region includes the alpha globin cluster and its regulatory elements, which are silenced in early progenitors, poised for expression in later progenitors, and fully expressed during terminal erythroid differentiation. Other genes in this region are also upregulated in an erythroid specific manner. Using a variety of approaches, we have established the order in which silencing factors are removed, activating transcription factors bind and epigenetic modifications occur. In addition, we have shown how chromosomal conformation and nuclear sublocalization change during hematopoiesis. Natural cis- and trans-acting mutations that cause alpha thalassaemia provide additional insight into how the long-range regulatory elements may interact with the promoters of the globin genes and other flanking genes to activate their expression. Together, these observations establish some of the general principles by which genes within their natural chromosomal environment are switched on and off during hematopoiesis.
Platelet Glycoprotein 1b-1X in Hemostasis and Malignancy.
Authors: Ware Jerry , Jain Shashank
Published: October 2019

The platelet paradigm in hemostasis and thrombosis involves an initiation step dependent upon platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. This paradigm has been established from decades of research. The platelet-specific receptor, glycoprotein (GP) Ib-IX, is critical in this process and can initiate the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib-IX have been identified, but von Willebrand factor (vWF) and the resultant GP Ib-IX/vWF axis has emerged as a major initiator of platelet thrombus formation in the arterial circulation. Newer, emerging data supports a role for platelets in pathological events beyond the prevention of blood loss. We have obtained data supporting the hypothesis that platelet GP Ib-IX contributes to malignancy. Knockout and transgenic mouse colonies have been generated in our laboratory and bred to C57BL/6J animals to generate several congenic strains (> 10 generation backcrosses) with dysfunctional platelet GP Ib-IX. These colonies have been used to characterize tumor metastasis and primary tumor growth in syngeneic models where the host animals are immunocompetent. The results demonstrate platelet GP Ib-IX contributes to tumorigenesis, as a functional absence of GP Ib-IX correlates with a significant reduction in the number of metastatic foci using models of experimental metastasis. In addition, an absence of GP Ib-IX significantly reduces primary tumor burden. A second series of experiments has determined that platelet GP VI, a key activation receptor on the surface of platelets also contributes to progression of experimental metastasis. The importance of metastasis in the prognosis for recovery from cancer can not be over-emphasized. Indeed, the spread of metastatic disease represents a fundamental change in significantly shortening the lifespan of patients with breast cancer. Thus, understanding the molecules that regulate metastasis identifies potential targets for therapeutic intervention that could significantly improve the prognosis for the breast cancer patient. Several decades of prior work in hemostasis and thrombosis has placed this project in a position to quickly provide new information and understanding on the links between hemostasis and cancer.
Mechanisms Coupling Thrombin-Mediated Proteolysis to Metastasis.
Authors: Palumbo Joseph S.
Published: October 2019

Multiple lines of evidence point to a link between coagulation factors and malignancy. Numerous clinical studies have demonstrated a strong correlation between tumor cell-associated tissue factor (TF) expression and poor outcome for many types of cancer. While TF expression by tumor cells could promote metastasis through several mechanisms, comparative analyses of TF-sufficient and TF-deficient tumor cell lines in mice with and without key circulating hemostatic factors point to thrombin as the premier downstream effector coupling TF to metastasis. Furthermore, it appears that thrombin supports metastasis through mechanism(s) that are highly dependent on the availability of fibrinogen, factor XIII, and platelet function. The metastatic potential of TF-expressing tumor cells has been shown to be strongly diminished in mice with genetic defects in either fibrin formation or stabilization (e.g., Fib−/−, fXIII−/−), or platelet disorders (e.g., NF-E2−/−, Gαq−/−, PAR-4−/−). Interestingly, one mechanism through which tumor-cell-associated TF, prothrombin, and the platelet/fibrinogen axis all appear to support metastasis is by impeding natural killer (NK) cell-mediated clearance of newly formed micrometastases. While these results demonstrate a novel link between the hemostatic and innate immune systems, limiting NK cell function is clearly not the only mechanism coupling thrombin to tumor cell metastasis. Signaling through protease activated receptors (PARs) on cells other than platelets has been suggested to support tumor stroma formation and angiogenesis. Furthermore, thrombin-mediated signaling via PARs expressed by tumor cells themselves has been shown to support metastatic potential. Finally, thrombin may support metastatic potential by acting in concert on multiple substrates (e.g., platelet and endothelial cell-associated PARs, fibrinogen, fXIII, etc.) to promote the sustained, stable adherence of circulating tumor cells in distant vascular beds. In summary, thrombinmediated proteolysis appears to influence both extracellular and intracellular events vital to tumor cell dissemination. Therapeutic strategies designed to block tumor cell-associated thrombin generation/activity could prove effective in preventing or eliminating metastatic disease.
The Pokemon/LRF-Notch Paradigm.
Authors: Pandolfi Pier Paolo
Published: October 2019

Text Not Available.
Nitric Oxide: An Introductory Primer.
Authors: Dzik Walter H.
Published: October 2019

As humans evolved in an atmosphere in which the most plentiful gases are Nitrogen and Oxygen, it is not surprising that nitric oxide (NO) plays an important role in human physiology. Originally described as an “endothelial relaxing factor,” we now recognize NO as not only a neurotransmitter, but also an important agent in blood physiology and vascular biology. In this symposium, three experts in NO research discuss emerging knowledge of NO’s role in hematology and transfusion medicine. NO is derived from arginine by three isoforms of NO synthetase, including NOS3 found in endothelial cells. NO binds avidly to free hemoglobin (Hg) through several chemical interactions, some of which depend upon the degree of oxygen saturation of Hg. Scavenging of NO by free plasma Hg accounts for the hypertensive effects of RBC substitutes and likely contributes to the renal lesion that accompanies acute hemolytic transfusion reactions. Under non-hemolytic conditions, the RBC membrane plays an important role to limit scavenging of endothelial-derived NO by Hg. The importance of disrupted NO physiology is exemplified by sickle cell anemia, in which chronic intravascular hemolysis, abnormal hemoglobin shape change during deoxygenation, and membrane abnormalities may all contribute to the disruption of NO physiology. Reduced NO in sickle cell anemia may contribute pulmonary hypertension, right ventricular strain, vasculopathy, and thrombosis. A better understanding of the role of NO in sickle cell anemia holds promise for new therapeutic approaches to this devastating blood disorder. Recent research is exploring changes in the concentration of NO that occur in stored red cells prior to and following transfusion. Whether or not these changes may account for clinical effects of blood transfusion is an area of active investigation. NO also plays a fundamental role in platelet physiology where it serves to reduce platelet activation. Thus, depletion of NO during intravascular hemolysis promotes a thrombotic signal. Examples may include TTP, transfusion reactions, PNH, DIC, sickle cell anemia, and others. The molecular mechanism of NO’s action on platelets, including its effects on vesicle and protein trafficking within the cell, is the subject of active research that will be summarized. Attendees of this symposium should gain new insights into the important role that NO plays in blood, vascular, and transfusion physiology.
Nitric Oxide and Platelet Function.
Authors: Lowenstein Charles J.
Published: October 2019

Nitric Oxide (NO) is a versatile messenger molecule of the vascular system. Three different NO synthase (NOS) isoforms found in the vasculature can synthesize NO: endothelial NOS, neuronal NOS, and inducible NOS. Once produced, NO can diffuse across cell membranes and modulate the cell biology of leukocytes, endothelial cells, and platelets. NO has several classes of molecular targets, including proteins with heme moieties, such as guanyly cyclase, cysteine residues of proteins, and radicals, such as superoxide. NO influences platelets through at least two distinct pathways. NO activation of a cGMP pathway mediates NO inhibition of eicosanoid metabolism, NO suppression of gpIIb/IIIa conformational changes, and NO deactivation of thromboxane receptors. However, NO also regulates platelets by chemically modifying cysteine residues of proteins critical to platelet activation. For example, NO modifies key cysteine residues of the enzyme N-ethylmaleimide sensitive factor, thereby blocking exocytosis of alphagranules. Recent studies have shown that metabolites of NO such as nitrite may also regulate platelet function.
Donor CD4+ T Cells That Possess Both Allo- and Autoreactivity in Transplants Mediate Chronic Graft Versus Host Disease.
Authors: Zhao Dongchang , Chen Yu-Hong , Young James , Shen Elizabeth , Yi Tangsheng , Liu Hong-Jun , Chu Peiguo G. , Zeng Defu
Published: October 2019

Abstract 3555

Poster Board III-492

Chronic graft versus host disease (GVHD) is considered an autoimmune-like disease mediated by donor CD4+ T cells, but the role and origin of the autoreactive T cells remain controversial. Here, we report that, in a chronic GVHD model of MHC-matched DBA/2 (H-2d) donor to BALB/c (H-2d) host, donor spleen cells induced autoimmune-like chronic GVHD in thymectomized allogeneic BALB/c but not in syngeneic DBA/2 recipients. The spleen cells from the former but not the latter recipients induced autoimmune-like disease in the secondary DBA/2 recipients, indicating that autoreactive donor CD4+ T cells from transplants are expanded and contribute to chronic GVHD pathogenesis. In addition, we found that both auto- and alloreactive donor CD4+ T cells generated from primary chronic GVHD recipients via serial in vivo and in vitro expansion proliferated to donor and host DC stimulation and both induced autoimmune-like disease in syngeneic and allogeneic recipients. Furthermore, the clonal expansion and TCR spreading of the autoreactive T cells in chronic GVHD recipients were following the alloreactive T cells, as revealed by TCR-spectrum typing and skewing of TCR-CDR3 length; No dual TCR was expressed by the donor-type T cells with both allo- and autoreactivity; and the autoreactive hybridoma T clones proliferated to stimulation by both syngeneic and allogeneic DCs. Taken together, these results demonstrate that donor CD4+ T cells that possess both allo- and autoreactivity in transplants are expanded in recipients and contribute to chronic GVHD pathogenesis, and the allo- and autoreactivity of the donor T cells can be mediated by a single TCR.

Disclosures:

No relevant conflicts of interest to declare.
CCL3 and CCL4 Plasma Levels Correlate with Established Prognostic Markers in Chronic Lymphocytic Leukemia: Towards a Simple, ELISA-Based Assay for Risk Assessment.
Authors: Sivina Mariela , Hartmann Elena , Krupnik Diana , LaPushin Ruth , Keating Michael , Kipps Thomas J. , Rosenwald Andreas , Wierda William G , Burger Jan A.
Published: October 2019

Abstract 358

During the induction of normal immune responses, activated B cells secrete the chemokines CCL3 and CCL4 for recruitment of regulatory T cells (Nat Immunol. 2:1126-32, 2001). This may represent a mechanism enabling cognate interactions between rare antigen-specific T and B cells. We previously reported that CCL3 and CCL4 RNA and protein are induced in CLL cells by co-culture with nurselike cells and after B cell antigen receptor (BCR) cross-linking (Blood 113:3050-8, 2009). We found higher levels of CCL3 and CCL4 in ZAP-70 positive cases, and CCL3 and CCL4 secretion was abrogated by the Syk inhibitor R406. These findings suggest that CCL3/CCL4 secretion by CLL cells correlates with the signaling capacity of the BCR. Also, we previously noticed higher plasma levels of CCL3 and CCL4 in CLL patients, when compared to healthy controls.

In this study, we measured CCL3 and CCL4 plasma levels by ELISA in 351 CLL patients, and correlated CCL3 and CCL4 levels with various prognostic markers, including RAI stage, immunoglobulin heavy chain variable region gene (IgVH) mutational status, ZAP-70, β2 microglobulin, CD38, white blood count (WBC), and cytogenetic subgroups. We found that the concentrations of CCL3 and CCL4 were significantly higher in plasma samples from CLL patients who had CLL cells that used unmutated IgVH genes or that expressed ZAP-70 or CD38, or who had an advanced stage of their disease (see Table 1). For example, the mean CCL3 plasma level was 56.4 ± 8.8 pg/ml in patients with CLL cells that used unmutated IgVH (mean ± SEM, n=123) versus 14.5 ± 2.4 in patients with CLL cells that used mutated IgVH (mean ± SEM, n=139, p<0.001). The mean CCL4 level was 171.4 ± 26.3 in patients that had CLL cells that used unmutated IgVH and 92.5 ± 17.5 in patients that had CLL that expressed mutated IgVH. On the other hand, the relative absolute white cell count or presence of chromosomal abnormalities did not correlate with high plasma levels of CCL3 or CCL4. Immunohistochemistry revealed that CCL4 was predominantly expressed in proliferation centers in approximately 50% of the cases. Currently, we are evaluating whether high plasma levels of CCL3 and/or CCL4 are associated with a relatively short time from diagnosis to requiring initial treatment by the IWCLL-working group criteria. Also, we are exploring whether high plasma levels of CCL3 or CCL4 are associated with high proportions of infiltrating T cells in proliferation centers.

These studies suggest that patients can be stratified by their relative plasma levels of CCL3 and/or CCL4 and that high plasma-levels of theses chemokines define a characteristic that may be associated with aggressive disease.

Disclosures:

No relevant conflicts of interest to declare.
Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD+ / SIRT1 Mediated Deacetylation of p53 in Myeloid Cells.
Authors: Thakur Basant Kumar , Lippka Yannick , Skokowa Julia , Welte Karl
Published: October 2019

Abstract 3589

Poster Board III-526

Recently we found that NAMPT, a protein involved in biosynthesis of NAD+ was significantly increased in Severe Congenital Neutropenia (CN) patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD+ levels correlated with the elevated levels of SIRT1, a NAD+-dependent deacetylase involved in the deacetylation of histone and non-histone proteins. CN is considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. SIRT1 mediated deacetylation of p53 at the lysine 382 has been shown to attenuate the transcriptional activity of p53. We asked if inactivation of p53 by NAMPT/ NAD+ / SIRT1 dependent deacetylation plays a role in the leukemic transformation in patients suffering from CN. Here we can demonstrate that presence of NAMPT or NAD+ enhances the SIRT1 mediated deacetylation of acetylated lysine peptide in vitro. Further in 293T cells overexpression of NAMPT induces the SIRT1 mediated deacetylation of p53. Using the promyelocytic cell line NB4 we show that endogenous p53 interacts with SIRT1 and the presence of NAMPT leads to decreased acetylation of p53 at the lysine 382. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. Acetylation of p53 is considered to be important for its tumor suppressor function and we asked if inhibition of NAMPT using FK866 increases the acetylation of p53. Indeed we demonstrate that the acetylation level of p53 is enhanced when NB4 cells were treated with FK866 and increased acetylation of p53 correlates with the decreased interaction between p53 and SIRT1. Treatment of NB4 with NAMPT further leads to the decreased expression of p53 target gene cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein. p21 is a well known target of p53 and is involved in cell cycle arrest after the cell undergoes stress. Using p21 promoter-luciferase construct we show that SIRT1 inhibits the p53 mediated activation of p21 promoter and this inhibition was further enhanced in the presence of NAMPT or NAD+. Taken together our working hypothesis is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of p53 protein thereby leading to p53 inactivation and downregulation of downstream target genes with tumor suppression functions (e.g. p21) which might possibly resulting in the leukemic transformation in CN patients.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
Low-Dose Alemtuzumab Is Uniquely Effective in Refractory Leukemic Cutaneous T Cell Lymphoma (L-CTCL).
Authors: Fisher David C. , Tawa Marianne , Walsh Michele , Clark Rachael A , Kupper Thomas S.
Published: October 2019

Abstract 3748

Poster Board III-684

Sezary Syndrome and other Cutaneous T Cell Lymphomas with peripheral blood involvement (collectively termed leukemic CTCL, or L-CTCL) are often refractory to multiple therapies. Alemtuzumab, an antibody directed against the pan-lymphocyte antigen CD52, has been used to treat multiple hematologic malignancies, including L-CTCL. However, conventional treatment regimens are associated with an increased risk of infections, and this is of particular concern in CTCL patients with compromised skin integrity. We have treated six L-CTCL patients with a modified regimen of alemtuzumab: 10mg/kg, subcutaneously, three times weekly for six weeks (1/3 of the conventional dose). All patients had had disease for >3 years duration, skin biopsies consistent with CTCL, elevated absolute CD4 counts, CD4/CD8 ratios of >10 and were refractory to at least two prior therapies, including extracorporeal photochemotherapy, interferon, and single and combination agent chemotherapy. In five of six patients, the malignant clone was identifiable using a Vb family specific monoclonal antibody; all cells of the malignant clonotype strongly expressed CD52. Within three weeks, all six patients achieved clearance of detectable T and B cells in peripheral blood, including loss of the malignant clone. In parallel, all patients developed clearing of erythroderma, relief of pruritus, and by the end of the six week treatment period, all patients had complete to near complete resolution of all clinical signs of disease. Isolation of T cells from a skin biopsy of one patient in complete remission revealed that <6% of T cells resident in skin expressed the malignant Vb subunit. At this reduced dose, no infectious complications were encountered. We conclude from that low-dose Aletuzumab is a well tolerated, safe, and highly effective therapy in a carefully selected population of patients with refractory L-CTCL.

Disclosures:

No relevant conflicts of interest to declare.