bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells
Authors: Teramachi Jumpei , Myint Kyaw Ze Yar , Feng Rentian , Xie Xiangqun , Windle Jolene J. , Roodman David , Kurihara Noriyoshi
Published: October 2019

Abstract 888

The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα.

When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM.

Disclosures:

Roodman: Amgen: Consultancy; Millennium: Consultancy.
Cyclin A2 Plays a Critical Role in Proliferation of Lymphoid Progenitors
Authors: Kohno Kentaro , Iwasaki Hiromi , Iino Tadafumi , Mizuno Shin-ichi , Sicinski Peter , Akashi Koichi
Published: October 2019

Abstract 914

Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Activation of Vascular Cells by Microparticles and Other Danger Signals Via the CD36 Scavenger Receptor
Authors: Silverstein Roy L.
Published: October 2019

Abstract SCI-12

Clinically significant thrombotic events are common complications of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes mellitus, cancer, and chronic autoimmune rheumatologic disorders. Many of these potentially life-threatening thrombi occur in the arterial circulation and likely relate to inappropriate platelet activation and/or vessel wall injury. CD36 is a type B scavenger receptor expressed on platelets, monocytes, microvascular endothelial cells (MVEC), and vascular smooth muscle cells (VSMC) that recognizes and responds to endogenous and exogenous “danger signals” including advanced glycated proteins (AGE), microbial cell wall components, and oxidized phospholipids that are found on the surface of oxidized LDL (oxLDL) and cell-derived microparticles (MP). Mouse FeCl3 thrombosis model systems revealed that genetic deletion of cd36 rescued the prothrombotic phenotypes associated with hyperlipidemia, oxidant stress, or diabetes/hyperglycemia. Interestingly, compared to wild type mice, fewer MP were incorporated into thrombi formed in cd36 null mice after FeCl3-induced vascular injury. Cd36 deficiency also provides partial protection from atherosclerosis in experimental mouse models. On a cellular level, the prothrombotic, pro-atherogenic, and anti-angiogenic activities of CD36 relate to ligand-dependent alterations of platelet, macrophage, and vessel wall cellular functions. For example, CD36 binding to MP, AGE, or oxLDL “sensitizes” platelets to activation by low doses of agonists, such as ADP. In macrophages, CD36 interactions with oxLDL leads to lipid internalization, foam cell formation, and inhibition of migration. In the vessel wall CD36 promotes VSMC oxidant stress and MVEC apoptosis. Mechanistically, CD36 transmits intracellular signals in response to ligand engagement that involve recruitment of specific Src family kinases to its short cytoplasmic tail, with subsequent activation of specific MAP kinase pathways and Vav family guanine nucleotide exchange factors. These signaling pathways remain incompletely understood, but impact many important cellular processes, including cytoskeletal dynamics, cell polarity, transcription of antioxidant genes controlled by the transcription factor Nrf2, and activation of NADPH oxidases. Specific components of CD36 signaling pathways depend on the cellular context and in some cases involve engagement of co-receptors, including integrins, TLRs, and tetraspanins. Recent targeted genome-wide association studies suggest that cd36 polymorphisms associate with CD36 expression levels on platelets and monocytes as well as with risk of thrombotic, metabolic, and atherosclerotic disorders. Targeting CD36 may therefore provide novel approaches to many important human vascular diseases.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Activated Protein C in Cancer
Authors: Spek C. Arnold , Arruda Valder
Published: October 2019

Abstract SCI-18

Activated protein C (APC) is a natural anticoagulant that blocks the amplification of the coagulation cascade via inactivation of factors Va and VIIIa. The APC/PC pathway is initiated by complex formation of thrombin, thrombomodulin (TM), and the endothelial protein C receptor (EPCR) allowing the conversion of zymogen protein C into its activated form. Based on the well-accepted view that coagulation contributes to cancer progression and that anticoagulant treatment may benefit some cancer patient groups, it has been hypothesized that the natural anticoagulant protein C pathway may also play a role in cancer progression. Interestingly, it has recently been shown that endogenous APC limits experimental metastasis of B16 melanoma cells in mouse lungs. Notably, an APC-blocking antibody dramatically increased the number of experimental lung metastasis although not due to diminished anticoagulant activity of APC but largely due to reduced APC-driven S1P1-mediated VE-cadherin-dependent vascular barrier enhancement. In line with these findings repeated administration of recombinant human APC as well as transgenic overexpression of EPCR also diminished experimental metastasis of B16 melanoma cells. It may thus be tempting to speculate that recombinant APC could be a novel treatment strategy to limit cancer progression. However, APC has however a short half-life, needs intravenous administration, and is associated with severe bleeding complications, complicating the potential clinical application of these findings. In contrast to APC, zymogen PC has a longer half-life and is associated with significantly less bleeding complications. To prove or refute the hypothesis that zymogen PC may be an attractive alternative treatment option for APC in cancer patients, we recently compared the effect of continuous overexpression of murine APC or zymogen PC in the liver by viral-mediated gene transfer in experimental metastasis. Interestingly, both APC and zymogen PC overexpression was highly effective in limiting experimental metastasis. An APC variant (APC-5A) with reduced anticoagulant but normal signaling properties did not limit experimental metastasis, whereas the protective effect of zymogen PC remained even in the absence of protease activated receptor-1 (PAR-1), which is the main mediator of APCs cytoprotective effect. Zymogen PC may thus be a novel therapeutic target to limit cancer progression. In conclusion, the natural anticoagulant APC pathway may play an important role in limiting cancer cell extravasation and interventions seeking to modulate the PC system may ultimately benefit the cancer patient. The challenge is however to confirm these findings in alternative preclinical cancer models and eventually to translate our findings into a clinical setting.

Disclosures:

No relevant conflicts of interest to declare.
Genetics of Graft-Versus-Host Disease
Authors: Petersdorf Effie W.
Published: October 2019

Abstract SCI-49

The HLA barrier remains the primary roadblock to hematopoietic cell transplantation from alternative donors for the treatment of blood disorders. Currently over 18 million unrelated donors are represented by registries worldwide and they serve as a critically important resource for patients in need of a transplant. The basis for the selection of unrelated donors has evolved with advances in HLA typing technology. The demonstration that serologically identical HLA phenotypes have DNA-defined allelic variants that can provoke graft-versus-host reactions has served as the basis for the current criteria for the selection of donors. Although donor HLA matching lowers morbidity and mortality from graft-versus-host disease (GVHD), matching does not guarantee that the patient will not experience life-threatening GVHD and require prolonged immunosuppression after transplantation. Furthermore, the risks of acute and chronic GVHD associated with transplantation from HLA mismatched donors has lead to a reluctance to use mismatched donors for some patients. In 2011, the unmet need is two-fold. First, the vast majority of patients in need of a transplant have no HLA matched unrelated donor. To permit these patients the opportunity for a life-saving transplant, more information on the rules that govern permissible donor-recipient HLA mismatches is needed. Second, information is needed on the extent of non-HLA genetic variation that resides within the major histocompatibility complex (MHC) region and the manner in which such variation contributes to the transplantation barrier. Several research strategies have been applied to identify HLA mismatch combinations that can be used safely, including but not limited to analysis of individual amino acid residues that define the peptide binding repertoire of HLA class I and II alleles and antigens, and computational approaches that relate the sequence to structure of HLA molecules. The availability of a dense map of over 36,000 single nucleotide polymorphisms and complete sequence information for common HLA haplotypes has recently provided new information on the extent of human genetic variation and its organization on haplotypes. These data serve as a rich resource for mapping novel MHC resident variation associated with GVHD risk and transplant outcome. New information is emerging on the diversity of the MHC among transplant patient-donor pairs, the organization of simple and complex genetic variation relative to the classical HLA loci, and the putative regions within the MHC that are amenable to fine mapping. Future investigation of the genetic basis of GVHD will be enhanced with more complete information on MHC region variation in diverse human populations, haplotype content, and robust tools for both querying and analyzing complex variation.

Disclosures:

No relevant conflicts of interest to declare.
Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis
Authors: Keerthivasan Ganesan , Yang Jing , Wong Piu , Doench John , Root David E. , Ji Peng
Published: October 2019

Abstract 3199

Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development.

Disclosures:

No relevant conflicts of interest to declare.
Improved Anemia, Organomegaly and Sustained Fetal Hemoglobin Induction within Aging TR2/TR4 Overexpressing UAB Sickle Cell Mice
Authors: Campbell Andrew , Bradley Kori , Amachong Ama , Addo Jennifer , Urbonya Rebekah , Bonsu Kwaku Osei , Harro David , Giacherio David , Shi Lihong , Cui Shuaiying , Tanabe Osamu
Published: October 2019

Abstract 3248

Background:

Fetal Hemoglobin (Hgb F)has been shown to improve morbidity and mortality with SCD patients. We recently published that forced expression of TR2/TR4 resulted in 2.5 fold induction of Fetal Hemoglobin and improved phenotypes within adult 2–3 month humanized sickle cell mice (PNAS Campbell and Cui et al 2011). We wanted to determine the if the Hgb F expression remained durable by HPLC within > 6 m/o mice within SCD:TgTR2/TR4 UAB Mice when compared to 2–3 m/o SCD:TgTR2/TR4 UAB Mice. Further, we wanted to determine if the increased Hgb F expression and improved phenotype observed previously persisted within the 6–8m/o SCD:TgTR2/TR4 mice when compared to the 6–8 m/o SCD UAB Mice.

Methods:

Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate SCD-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We compared the hemoglobin F levels(HPLC), CBC, and % body weight (liver, spleen, kidney) of 6–8 m/o SCD:TgTR2/TR4(n=5) mice to 2–3 m/o SCD:TgTR2/TR4(n=5) mice. We also analyzed 6–8m/o SCD mice to the TR2/TR4 SCD Mice. Tail PCR genotyping of all SCD Mice were confirmed by HPLC Hemoglobin electrophoresis.

Results:

Fetal Hemoglobin Expression remained elevated in the 6–8 m/o SCD:TR2/TR4 Mice at 16.7% (+/− 3.94) compared to 18.5% (+/−2.91) in 2–3 month old SCD:TR2/TR4 mice. %BW of spleen (3.7% vs 3.4%) liver (7.29% v 7.8%), kidney (0.98% vs 1.1%), remained similarly lower in the 6–8 m/o compared to the 2–3 m/o mice of the SCD:TR2/TR4 Mice. Table 1 shows increased Hgb F and overall improved anemia and %BW of organs within the older SCD:TR2/TR4 SCD Mice.

Conclusions:

TR2/TR4 overexpression within >6 m/o humanized SS mouse model resulted in continued >3 fold induction of fetal hemoglobin based on HPLC compared control 6–8 m/o SCD Mice. Further, increased TR2/TR4 overexpression improved anemia, organomegaly, within sickle cell disease mice. Expression of Fetal Hemoglobin by TR2/TR4 remains sustained within older sickle cell mice with improved organomegaly with reversion phenotypically to the wild type phenotype.

Disclosures:

No relevant conflicts of interest to declare.
Initial Genomic Analysis of a Pure Erythroid Leukemia Developing in Association with Hydroyurea Treatment for Sickle Cell Anemia
Authors: Wang Zhengyuan , Darbari Deepika S. , Melenhorst J. Joseph , McIver Zachariah A. , Maric Irina , Diaw Lena , Song Young , Johansson Peter , He Jianbin , Weiyin Zhou , Jacobs Kevin B. , Wei Jun , Barrett A. John , Khan Javed , Taylor James G.
Published: October 2019

Abstract 3254

The antimetabolite hydroxyurea (HU) is the only widely available therapy for reducing complications of sickle cell anemia. It is not widely prescribed due to concerns about adverse effects, including cancer. Insufficient data currently exist regarding its leukemogenic potential. HU associated leukemia in sickle cell anemia is exceptionally rare, with only 6 literature reports. Whether such cases are coincidental or related to HU remains unanswered. We identified a 33 year old male with pure erythroid leukemia (formerly M6 AML) and a unique combination of sickle cell anemia, HU treatment, and short treatment latency. Cytogenetics showed a complex karyotype with 5q rearrangement, 7q deletion, losses of chromosomes 15–22 and Y, suggesting a therapy related etiology. To date, no sickle cell anemia AML cases have undergone in-depth genomic analysis to determine if HU has an etiologic role in susceptibility to therapy related leukemogenesis. High density SNP genotyping was performed with 2.5 million markers to understand progressive chromosomal disease changes. Comparison of germline DNA prior to HU and tumor DNA identified 10 mosaic aneuploidy events present at AML diagnosis including losses at 5q, 7q and 17p and gains at 19p. Whole exome sequencing was also performed on the patient's germline, tumor and relapsed tumor DNA. We identified 33 putative somatic missense mutations including a validated somatic TP53 mutation, however somatic mutations were not present in commonly mutated AML genes like NPM1, RUNX1 or IDH1. Genome-wide cytogenetic band enrichment analysis of exome sequence data using the Gene Set Analysis Toolkit suggests the presence of a significant enrichment of mutations along chromosome 19p, and the same analysis of peripheral blood gene expression from an unrelated cohort of 10 sickle cell anemia subjects with brisk hematopoietic activity also suggested association of chromosome 19p with stress erythropoiesis. Together, the mosaic aneuploidy and exome sequence analysis converged on a region of 19p potentially important to the pathogenesis of pure erythroid leukemia and possibly stress erythropoiesis. This region extends at least 1.9 Mb and includes 58 genes. We are validating mutations in this region and exploring the role of 19p during stress erythropoiesis to further determine the significance of this finding. Specifically, several 19p mutations have been introduced into the BaF3 cell line to investigate their role in myeloid cell growth. Investigation of this rare leukemia case offers a unique opportunity to elucidate the pathogenesis of erythroleukemia and possibly to determine if HU therapy is associated with leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin-Associated Increase in the Proportion of Kupffer Cells
Authors: Ohana Yasmin Haim , Deshet Naamit , Souroujon Miriam , Gassmann Max , Mittelman Moshe , Neumann Drorit
Published: October 2019

Abstract 3266

Erythropoietin (EPO), the hormone produced mainly by the kidney, promotes the survival, proliferation and differentiation of erythroid precursor cells. Hence, recombinant human EPO (rHuEPO) is one of the key therapies for the treatment of anemia in patients with end-stage renal disease and in cancer patients, suffering from chemotherapy-induced anemia. The past decade has revealed extra-hematopoietic sites of EPO production along with abundance of EPO receptors (EPO-Rs) in various tissues and cell lines, suggesting that this hormone may have pleiotropic activities. Indeed, previous studies in our laboratory have shown that murine bone marrow-derived macrophages express EPO-R and EPO affects their cellular phenotype.

Kupffer cells are liver macrophages that play a key role in the body's defense system against invading pathogens. These cells affect normal and pathological conditions of the liver either directly or through their influence on other cells, such as stellate cells and are important components in iron metabolism. In this study, we found that transgenic mice, overexpressing human EPO (tg6 mice), have a larger population of F4/80 positive liver macrophages compared to wild type (wt) mice. In addition, C57bl/6 wt mice, injected 3 times a week with 180 U/ml of rHuEPO, displayed a 2 fold increase in the percentage of F4/80 expressing cells compared to their untreated littermates. This increase seems to stem from EPO's effect on a subpopulation of cells expressing both F4/80 and CD11b. As for pathological conditions, we demonstrate the same effect of EPO in an experimental murine model of 5T33 multiple myeloma (MM). 5T33 MM mice that were treated with rHuEPO (10 consecutive days of 30 U/ml followed by 3 weekly injections of 30 U/ml for 2 weeks), presented a 3 fold increase in the percentage of cells double positive for F4/80 and CD11b. In addition, these mice displayed a 20% increase in the mean fluorescence intensity of CD11b, suggesting that EPO causes not only an increase in the proportion of Kupffer cells but also induces the activation of CD11b positive cells, as reflected by the elevated expression of this marker per cell.

Our findings thus contribute to the understanding of EPO's effects on the immune system in general, and on Kupffer cells in particular. These findings may have clinical significance in those cases where activation of liver macrophages is desired, and EPO treatment for anemia is recommended.

Disclosures:

No relevant conflicts of interest to declare.
Gas6-Induced Tissue Factor Expression in Endothelial Cells Is Mediated Through Caveolin-1-Enriched Domains
Authors: Laurance Sandrine , Lemarie Catherine A , Blostein Mark D
Published: October 2019

Abstract 3308

Gas6 is the ligand for the TAM family of receptors, which are composed of three members namely Tyro3, Axl and Mer. These receptors belong to the large family of type I transmembrane receptor tyrosine kinases. Our laboratory has identified important intracellular signaling pathways important in gas6-Axl mediated protection of endothelial cells from apoptosis. However, as both gas6 and Axl null mice are protected from lethal thromboembolism, we sought to explore novel gas6-Axl intracellular signaling pathways regulating thrombosis. Caveolae have been shown to play a crucial role in the activation of signaling cascades following ligand binding to receptor tyrosine kinases. Caveolae are formed from lipid rafts by polymerization of caveolins. Caveolin-1 is the most abundant protein found in caveolae. Caveolin-1-enriched microdomains are well known to play a role as a docking platform for receptor tyrosine kinases and intracellular adaptor signaling proteins. Axl association with these highly specialized domains of the plasma membrane has not been previously elucidated. In the present study, we investigated the role of caveolin-1-enriched microdomains in gas6/Axl signaling in endothelial cells. First, we demonstrated that gas6-induced Akt and Erk1/2 phosphorylation required the presence of a functional Axl receptor as shown by Axl siRNA knockdown experiments in human umbilical vein endothelial cells. Then, caveolin-1 fractions, enriched by a detergent-free cell lysis followed by sucrose gradient ultra centrifugation, were studied by western blot analysis. After 5 and 10 min of gas6 treatment, Axl colocalized with caveolin-1 suggesting Axl recruitment into caveolin-1-enriched cell fractions. We found that c-Src, a signaling molecule known to behave as a transient docking platform in lipid rafts, also moved in caveolin-1-enriched cell fractions after gas6 stimulation. Caveolin-1 siRNA abolished gas6-induced Akt, Erk1/2 and c-Src phosphorylation suggesting that caveolin-1 enriched fractions are required for gas6-Axl signaling. Interestingly, we have shown that gas6-induced Akt phosphorylation required c-Src activation using c-Src siRNA and the pharmacological inhibitor (PP2). However, gas6-induced Erk1/2 phosphorylation was independent of c-Src. Finally, we found that gas6 increased tissue factor expression through the Axl-c-Src-Akt signaling cascade. Taken together, our results demonstrate that caveolin-1-enriched domains are required for gas6-Axl signaling and lead to the upregulation of tissue factor expression by gas6 in endothelial cells. These results highlight new insights of gas6-Axl signaling and function in endothelial cells.

Disclosures:

No relevant conflicts of interest to declare.
Human and Murine Immune Thrombocytopenia (ITP) Is Associated with a Peripheral Deficiency of CD4- T Regulatory Cells (Tc-regs)
Authors: Semple John W. , Ma Daoxin , Aslam Rukhsana , Hou Ming , Ni Heyu , Hu Yu
Published: October 2019

Abstract 3333

CD8+ T suppressor cells were originally described by Gershon on Kondo more than 40 years ago, however, because of difficulties in their characterization, they fell out of favor for decades. T suppression was resurrected by the discovery of CD4+ CD25+ FoxP3+ T regulatory cells as being the critical cell in the maintenance of self tolerance. Recently, a non-conventional CD4-CD25+FoxP3+ T cell subset termed Tc regulatory cells (Tc-regs) has been described that are also important in inducing tolerance against self. A peripheral CD4+ Treg deficiency has been suggested to be the potential cause of ITP, however, the role of this newly described CD4- T cell population has not been determined. To address the role of these cells, we studied lymphocyte responses in 36 patients with ITP (18 newly-diagnosed and 18 in remission) and a murine model of ITP. Peripheral blood mononuclear cells from patients or spleens and thymi from mice were isolated and FoxP3 expressing Tc-regs were evaluated by flow cytometry. Results show that compared with healthy control subjects, the percentages of peripheral Tc-regs in newly diagnosed patients with ITP were significantly decreased. The Tc-reg deficiency was rescued in those patients in remission due to therapy. Similar results were observed in an active model of murine ITP. Additionally, the murine model of ITP revealed that the CD4- deficient population expressed CD8. These results suggest that, like CD4+ T regs, there is a significant reduction in peripheral Tc-regs in both human and murine ITP and disease remission correlates with rescuing the deficiency.

Disclosures:

No relevant conflicts of interest to declare.
Factor V Inhibits PAR2-Mediated Lethal Inflammation
Authors: Kerschen Edward J , Liang Hai-Po Helena , Fernandez Jose A. , Toso Raffaella , Ruf Wolfram , Camire Rodney M. , Griffin John H. , Weiler Hartmut
Published: October 2019

Abstract 3360

The Leiden polymorphism (Arg506Gln) in human blood coagulation factor V (fV) is the most prevalent genetic risk factor for venous thrombosis. We have now shown that heterozygous carriers, but not homozygous carriers, exhibit a robust survival advantage in murine models of lethal infection with gram-positive and gram-negative bacterial pathogens. FV Leiden augments the thrombin-mediated formation of activated protein C (aPC) and thereby enables the aPC-mediated inhibition of a specific component of the overall inflammatory response of myeloid immune cells. This specific, aPC-inhibited inflammatory response was mediated by the induction of tissue factor (TF) expression, assembly of the ternary TF-VIIa-Xa complex, and the EPCR-dependent activation of Protease Activated Receptor 2 (PAR2) by the ternary TF complex. The inhibition of inflammation-induced, PAR2-dependent gene expression by APC required factor V, protein S, and Protease Activated Receptor 3. This anti-inflammatory bioactivity of purified, plasma-derived or recombinant fV was not expressed by the fV Leiden variant. Thus, in heterozygous fV Leiden individuals, aPC levels are increased by fV Leiden, while wild type fV enhances aPC's beneficial actions on innate immune cells. This explains why heterozygous carriers are protected from sepsis mortality by aPC, whereas homozygous Leiden carriers do not respond to endogenous or therapeutically administered aPC. FV Leiden hence emerges as a unique example of a balancing and evolutionary selectable polymorphism, whose balanced nature is the consequence of beneficial, cooperative interactions of the variant fV Leiden allele that augments generation of aPC and the normal fV allele that is necessary for the protective effects of aPC.

Disclosures:

Weiler: BloodCenter of Wisconsin: Patents & Royalties.
FXII Promotes Coagulation in a FXI and FIX Independent Manner
Authors: Puy Cristina , Wong Zoë C , Tucker Erik I , Gruber Andras , Gailani David , Smith Stephanie A. , Choi Sharon H. , Morrissey James H. , McCarty Owen J.T.
Published: October 2019

Abstract 3362

Activation of coagulation factors (F) XII and XI support thrombogenesis through multiple pathways. FXII-deficient mice are more resistant to FeCl3-induced arterial occlusion than either FIX or FXI deficient mice, suggesting that the resistance of FXII-deficient mice to experimental thrombosis is not completely explained by the FXII-FXI-FIX pathway, suggesting the existence of a pathological FXII by-pass, in vivo.

The APTT of FXII deficient plasma is longer than the APTT of FXI, FIX, or FX deficient plasmas. We found that addition of 150 nM activated FXII (FXIIa) decreased the recalcification time of FXI or FIX-deficient plasma by up to 85%. In a purified system FXIIa could activate prothrombin but not FX. Addition of rivaroxaban, a FXa inhibitor, to FXI or FIX-deficient plasma blocked the observed procoagulant effect of FXIIa, suggesting that FXIIa promotes the activation of FX independent of FXI or FIX, but the ability of FXIIa alone to induce coagulation is insufficient in plasma, in vitro.

Addition of long polyphosphate (polyP), typically found in bacteria, but not short polyP, which is secreted by activated platelets, decreased the recalcification time of FXI or FIX-deficient plasma. The presence of either corn trypsin inhibitor (CTI), that inhibits FXIIa, or rivaroxaban blocked the procoagulant effect of long polyP, suggesting that the activation of FXII by long polyP promotes coagulation in an FXI- and FIX-independent manner.

Addition of CTI or an antibody that inhibits FIX activation by FXIa, but not addition of an antibody that inhibits activation of FXI by FXIIa, increased the time of occlusive thrombus formation in recalcified human blood that was driven through collagen and tissue factor (TF)-coated capillary tubes, consistent with the thrombogenic roles of FXIIa and feedback activation of FXI. Only CTI inhibited the prothrombotic effect of long polyP, also suggesting that FXIIa could be thrombogenic independent of FXI and FIX.

In summary, we propose that pathological FXII activation, e.g., by foreign surfaces or long polyP, is thrombogenic both in FXI/FIX-dependent and -independent manners. Provided that FXII has no significant physiological function in humans, our data support the hypothesis that inhibition of FXII activity or activation may have safe antithrombotic effects.

Disclosures:

Morrissey: No organization, but the speaker is co-inventor on pending patent applications on the medical uses of polyphosphate: Patents & Royalties.
Circulating Nucleosomes and Neutrophil Activation As Risk Factors for Deep Vein Thrombosis
Authors: van Montfoort Maurits L , Stephan Femke , Lauw Mandy N , Hutten Barbara A , van Mierlo Gerard J , Solati Shabnam , Middeldorp Saskia , Meijers Joost CM , Zeerleder Sacha S
Published: October 2019

Abstract 3387

Objective:

The formation of neutrophil extracellular traps (NETs) and the exposure of nucleosomes on these NETs contribute to coagulation activation and the propagation of deep vein thrombosis (DVT) in animal models. However, no data is available on the role of NETs or nucleosomes in patients with thrombosis.

Methods and results:

We conducted a case-control study, in which levels of circulating nucleosomes and neutrophil elastase–α1-antitrypsin (EA) complexes were assessed in plasma from 150 patients with objectified symptomatic DVT (cases) and compared with 195 patients with a clinical suspicion of DVT, but in whom DVT was excluded (controls). We explored the association between both nucleosomes and EA complexes, and the presence of DVT by calculating the odds ratio (OR) with corresponding 95% confidence intervals (CI). Elevated levels of both circulating nucleosomes and EA complexes were associated with a 3-fold risk of DVT, and the associations remained similar after adjustment for potential confounders (malignancy, smoking, recent immobilization, recent hospitalization). The risk increased with higher nucleosome and EA complex levels, suggesting a dose-dependent relationship between circulating nucleosomes, activated neutrophils and DVT.

Conclusions:

Circulating nucleosomes and activated neutrophils contribute to the development of DVT in humans and might have implications for treatment and prevention.

Disclosures:

No relevant conflicts of interest to declare.
Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic STEM and Progenitor CELL Mutagenesis in Response to Irradiation
Authors: de Laval Bérengère , Pawlikowska Patrycja , Roch Benoit , Petit-Cocault Laurence , Bilhou-Nabera Chrystele , Souyri Michele , Gaudry Murielle , Pouzoulet Frédéric , Porteu Francoise
Published: October 2019

Abstract 3447

Radiation-induced double-strand breaks (DSBs) represent a serious threat to the preservation of genetic information when it reaches hematopoietic stem cells (HSCs). Residual loss of HSC functions and increased risk of developing hematopoietic malignancies are two concerning complications of anti-cancer radiotherapy. Management of acute myelosuppression following radio- or chemotherapy has been significantly improved in recent years by the use of growth factors. However, how cytokine/environmental signals integrate the DNA damage responses in HSCs and regulate the long-term residual HSC defects following radio-or chemotherapy is unknown. Notably, the contribution of cytokines regulating HSC functions to HSC intrinsic DNA damage repair processes remains to be delineated. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal, survival and expansion posttransplantation. In this study, we uncover an unknown and unique function for TPO/Mpl in the regulation the DNA damage response. We show that DSB repair, measured by both γH2Ax foci resolution and neutral comet assays, following γ-irradiation (IR) or topoisomerase II inhibitor treatments, is defective in Mpl−/− and Mpl+/− HS and progenitor cells (HSPCs). Similar defects were found in wild-type cells treated in the absence of TPO. This indicates that the impaired DNA repair of Mpl−/− and Mpl+/− cells results from a specific loss of TPO-mediated DNA damage response signaling at the time of IR rather than from intrinsic constitutive differences. TPO stimulates DNA repair by increasing IR-induced DNA-PK phosphorylation at Ser2056 and Thr2609 and non-homologous end joining (NHEJ) efficiency in both HSPCs and the human UT7-Mpl cell line. This is to our knowledge the first demonstration that a cytokine involved in the homeostatic maintenance of HSCs may also regulate their response to external DNA damaging insults by controlling the DSB repair machinery. Short TPO treatment in vitro or single TPO injection to TPO/Mpl proficient mice prior to sublethal total body IR reduced IR-induced HSC genomic instability and loss of long-term reconstitution ability. This may open new avenues for administration of TPO agonists before radiotherapy to minimize radiation-induced HSC injury and mutagenesis. In addition, since Mpl is haploinsufficient in the regulation of DNA damage repair, these data suggest that Mpl might also act as a tumor suppressor in response to radiotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Kruppel-Like Factor, Klf9, Promotes Proliferation of Notch-Independent Precursor T-Cell Lymphoblastic Lymphomas
Authors: Wong Peggy P. C. , Merico Daniele , Matei Irina , Ling Vicki , Bashir Shaheena , Chang Stephen G. , Cheung Queenie C. K. , Haw Robin A. , Stueker Oliver , Montpellier Bertrand , Bader Gary D. , Beyene Joseph , Danska Jayne S. , Guidos Cynthia J.
Published: October 2019

Abstract 3496

Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations.

Disclosures:

No relevant conflicts of interest to declare.
Dietary Restriction and Weight Loss Improves the Outcome of Acute Lymphoblastic Leukemia in Diet-Induced Obesity
Authors: Alhushki Waseem , Mittelman Steven , Sheng Xia , Ehsanipour Ehsan
Published: October 2019

Abstract 3570

Background:

Obesity increases the mortality of numerous types of cancer. Furthermore, children over 10 years of age who are obese at diagnosis of acute lymphoblastic leukemia (ALL) have twice the risk of relapse. We have found that obese mice transplanted with leukemia are more likely to relapse after treatment with chemotherapy. We further found that adipose tissue can protect ALL cells from chemotherapy in culture. But it is unclear whether a weight loss intervention after leukemia diagnosis will improve outcome.

Hypothesis:

A weight loss intervention by dietary modification in diet induced obese mice will decrease the ability of their adipose tissue explants to protect ALL cells from chemotherapy.

Methods:

Diet-induced obese C57Bl/6 mice (n=7) were switched to a low-fat diet either 3 or 21 days prior to sacrifice at 20 weeks of age. Adipose tissue explants (100 ± 5 mg) from perirenal, visceral, and subcutaneous sites were collected and used in transwell co-cultures with 250 X103 8093 murine pre-B ALL cells. Explants from non-dieted obese mice and lean mice were used as controls. Viable ALL cells were quantified using trypan blue exclusion after a 3-day exposure to 15 nM daunorubicin or 5 nM of vincristine.

Results:

Dietary intervention significantly reduced body weights in the short (39.2 ± 2.4 grams; P= 0.014) and long (32 ± 2.9 grams; P=0.041) diet groups compared to obese mice (44.5 grams ± 2.6).

Leukemia cells were partially protected from daunorubicin by both visceral (21.5±15.7×103 viable cells, p=0.01) and perirenal (28.0±20.7×103, p=0.013) fat explants from obese mice, compared to cultures without adipose tissue (9.4±8.6×103). Fat explants from mice dieted for 3 days prior to collection showed similar protection of ALL cells from daunorubicin (not shown); however, fat explants from mice dieted for 21 days did not protect ALL cells from daunorubicin (visceral: 9.4±8.0×103, p=0.009; perirenal: 7.8±6.3×103, p=0.066 vs. obese).

Similar findings were obtained with vincristine. Leukemia cells were protected from vincristine significantly by visceral fat from obese mice (50.8±5.9×103, p=0.01) and marginally by perirenal and subcutaneous fat (93.8±139.1×103, p=0.06 and 113.4±143.7×103, p=0.07 respectively) compared to cultures without adipose tissue (12.4±11.7×103). While short dieting did not restore sensitivity to vincristine, long dieting of obese mice reduced explants leukemia protection to vincristine significantly in visceral (21.8±20.2×103, p=0.004) and marginally in perirenal (34.9±43.3×103, p=0.06) explants compared to the obese group.

Conclusions:

Dietary intervention and weight loss in diet induced obese mice decreases the ability of adipose tissue ex vivo to protect ALL cells from daunorubicin and vincristine. These results could support a potential role for dietary intervention in improving leukemia outcome in obese patients.

Disclosures:

No relevant conflicts of interest to declare.