bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythrocyte Advanced Glycation End-Products as Novel Mediators of Endothelial Dysfunction Following Transfusion
Authors: Mangalmurti Nilam S. , Friedman Jessica , Siegel Don L. , Lee Janet , Albelda Steven M. 
Published: October 2019

Abstract
Abstract SCI-47
Red cell transfusions are associated with the development of acute lung injury in critically ill patients, yet the mechanisms for this association remain unknown. We have previously shown that stored red blood cells (RBCs) express the advanced glycation end-product Nε-carboxymethyl lysine (Nε-CML) (1). Advanced glycation end-products (AGEs), a heterogeneous group of adducts formed during states of increased oxidative stress or hyperglycemia, are thought to exert their effects by binding to membrane receptors, including RAGE (the receptor for advanced glycation-end products). RAGE is a multiligand pattern-recognition receptor implicated in vascular disease whose function in lung endothelium remains unknown. Our previous data demonstrate that RAGE ligands on stored erythrocytes can induce reactive oxygen species in lung endothelial cells. We therefore hypothesized that RAGE ligands on the surface of stored RBCs exacerbate lung inflammation by interacting with the pulmonary endothelium, thus possibly participating in the pathogenesis of lung injury following transfusion. We first examined lung endothelial cells at baseline using multiple techniques and observed low basal expression of RAGE. Because RAGE ligands can induce the expression of RAGE, and stored red blood cells (RBCs) express the RAGE ligand Nε-carboxymethyl lysine (Nε-CML), we investigated whether RBC transfusion would augment RAGE expression on lung endothelium utilizing a syngeneic model of RBC transfusion. Increased lung endothelial RAGE expression and enhanced lung inflammation and endothelial activation was observed, as evidenced by increases in lung high mobility group box 1 (HMGB1) and vascular cell adhesion molecule (VCAM-1) expression following transfusion. These effects were mediated by RAGE, as endothelial activation was absent in RBC-transfused Rage knockout mice. Therefore, functional consequences of RBC transfusion are augmented RAGE expression, increased RAGE ligation, and endothelial activation, thus linking RAGE activation to lung inflammation following RBC transfusion.

Disclosures:
No relevant conflicts of interest to declare.
Expression Proteomics As Surrogate Biomarkers For Early Treatment Response In Chronic Myeloid Leukemia (CML) Patients
Authors: Alaiya Ayodele , Chaudhri Naeem , Owaidah Tarek , Fox Jonathan , Shinwari Zakia , Barhoush Eman , Alsharif Fahad , Mohamed Said Y , Rasheed Walid , Alfraih Feras , Aljurf Mahmoud 
Published: October 2019

Abstract

Purpose
Despite advancements in diagnosis and treatment modalities for hematological malignancies, presently, there are no reliable blood tests for disease prognosis or prediction of patient’s response to particular treatments. Identification of such biomarkers for diagnostic or therapeutic response in clinical monitoring of hematologic diseases such as Chronic Myeloid Leukemia (CML) would be very useful.

Experimental Design
The ability to identify biomarkers in body fluids (i.e., plasma or serum) will not only involve minimally invasive procedures, but also more importantly, the potential for monitoring disease progression and effective response to therapy in relatively early stages. In this study, global proteome analysis of serum, plasma, and bone marrow samples from 28 patients with newly diagnosed chronic–phase CML were analyzed by using expression proteomics technology (label free quantitative liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The goal was to evaluate for early treatment response and discovery disease–specific /disease-associated proteins for prognostic monitoring and patient’s response to therapy at protein level.

Results
We identified a panel of 15 differentially expressed proteins (> 2- °- fold change, p< 0.001) to accurately discriminate between CML patients that achieved Major Molecular Response (MMR) vs. No-Major Molecular Response (NoMMR) at 6 months (Figure 1 & Table 1). Six (6) of the 15 identified proteins were filtered and mapped as potential biomarkers using Ingenuity Pathway Analysis. Among the identified proteins implicated in hematological diseases include Group-specific component (vitamin D binding protein), haptoglobin and vitronectin. Others were inter-alpha-trypsin inhibitor heavy chain 1, leucine-rich alpha-2-glycoprotein 1 and metallophosphoesterase 1. We also observed most of these proteins to be located in extracellular space, acts as transporters and have been implicated as markers of hematological disease and inflammatory response. Our data indicates that multivariate analysis of quantitative proteome data can potentially be useful as a means of unsupervised artificial intelligence algorithm for classification and stratification of clinical CML patients.

Conclusion
Our results highlight the power of proteomics in the discovery of biomarkers for more accurate prediction of prognosis and monitoring treatment response in CML patients. These proteins might be valuable, once validated, to complement the currently existing parameters for reliable and objective prediction of disease progression, monitoring treatment response and clinical outcome of CML patients.

Disclosures:
No relevant conflicts of interest to declare.
Pharmacokinetic (PK) and Pharmacodynamic (PD) Study Of Dasatinib In Chronic Phase Of Newly Diagnosed Chronic Myeloid Leukemia (CML-CP)
Authors: Ishida Yoji , Murai Kazunori , Yamaguchi Kohei , Miyagishima Takuo , Shindo Motohiro , Ogawa Kazuei , Nagashima Takahiro , Sato Shinji , Watanabe Reiko , Yamamoto Satoshi , Hirose Takaaki , Kato Yuichi , Yonezumi Masakatsu , Kondo Takeshi , Mochizuki Nobuo , Ohno Keiko , Kishino Satoshi , Ito Shigeki , Kubo Komei , Saito Sohichi 
Published: October 2019

Abstract

Purpose
Dasatinib is a novel kinase inhibitor of BCR-ABL and SRC family kinases. Dasatinib has shown promising anti-leukemic activity in chronic myeloid leukemi (CML). Tounderstand how to administer dasatinib with best efficacy and less adverse events, the pharmacokinetic (PK) properties of dasatinib and the relationship of PK and pharmacodynamic (PD) characteristics were investigated in newly diagnosed CML-chronic phase (CP) patients.

Methods and Materials
The PK analysis of dasatinib: The plasma concentrations of dasatinib (1, 2, 4 hr after taking dasatinib post 28 days) were determined by Francia's method using high performance liquid chromatography with mass spectrometry. PK analysis was performed using Phoenixâ NLMEâ1.2. The maximum plasma concentration (Cmax) was determined by visually inspecting the profiles of plasma drug levels.
PD analysis of dasatinib: Phospho-CrkL in CD 34 positive cells was analyzed by flow cytometry using anti-phospho-CrkL (Tyr207) antibody after incubation of bone marrow mononuclear cells in the presence of dasatinib for 2 hours.
PK/PD analysis: Area under the curve (AUC) and time above IC50 were calculated using Phonix WinNonlin 6.3.

Results
Twenty-eight newly diagnosed CML-CP patients were included. The correlation between the efficacy and PK/PD parameters were analyzed using Peason's correlation coefficient test (Table1). The efficacy(expression of bcr/abl at 1 month) was not correlated with AUC, Cmax, AUC/IC50 and Cmax/IC50 but significantly correlated with TAIC50 (r=0.4763, p=0.0104). The reduction rate at 1 month was correlated significantly with only TAIC50(r=0.5136, p=0.0052)(Figure 2). Eight cases reached MMR at 3 month among 15, whose TAIC50s were more than 12 hrs(53.3%), while only 2 cases reached MMR among 13, whose those were less than 12 hrs(15.4%).

Conclusion
Dasatinib has anti-leukemic activity in a time dependent manner. Exposure more than 12 hrs at TAIC50 will get benefits of better prognosis.

Disclosures:
No relevant conflicts of interest to declare.
The Polymorphism Of Base Excision Repair Gene, XRCC1 Arg399Gln Is Associated With The Therapy-Related Myelodysplastic Syndromes (MDS)
Authors: Norjmaa Batchimeg , Saitoh Takayuki , Iwasaki Atsushi , Nitta Yasuhiro , Masuda Yuta , Suda Itsumi , Kaneko Ayaka , Shimizu Hiroaki , Handa Hiroshi , Yokohama Akihiko , Tsukamoto Norifumi , Sakura Toru , Murakami Hirokazu 
Published: October 2019

Abstract

Background
Base excision repair (BER) is critical for genome maintenance, and is mainly responsible for the correction of small base changes of DNA damage. BER pathway involved many enzymes including OGG1, XRCC1, APE1 and MUTYH. Single nucleotide polymorphisms (SNPs) in DNA repair genes result reduced DNA repair capacity, have been reported to be associated with an increased risk of various cancers including hematologic malignancies. However, it is unclear that these polymorphisms alter the susceptibility and clinical outcome of myelodysplastic syndromes (MDS) patients. The aim of this study is to evaluate the association of polymorphisms in gene encoding four key proteins of DNA BER: OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and MUTYHGln324His with the susceptibility and clinical features of MDS.

Methods
Our study included 113 MDS patients [median 68.3 years, range 17.1-86.5 years; male/female 76/37; RCUD (n=37), RARS (n=6), RCMD (n=21), MDS-u (n=11), RAEB-1(n=14), RAEB-2 (n=11), others (n=13)] and 192-health control group. Twenty four patients with MDS had the history of cancer. Genetic polymorphisms in BER pathway genes were examined using PCR and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between patients group and control group by using χ2-test. All patients and healthy controls received written information about the study. This study was approved by the International Research Board of Gunma University Hospital.

Results
There was no statistically significant difference in the allele and genotype frequencies of the OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and MUTYH Gln324His polymorphisms between the MDS patients and the control group. In the analysis of clinical characteristics, XRCC1 non Arg/Arg genotype (low DNA repair type) was significantly associated with lower Hb level (8.64±2.29g/dL vs. 9.96±2.08 g/dL, p<0.005) and higher frequency of the complex karyotype (14.9% vs. 2.8%, p=0.05). Furthermore, XRCC1 non Arg/Arg genotype was associated with therapy- related MDS (OR 3.15, 95% CI 1.24-7.98, p=0.02) and especially the past history of radiotherapy (14.3% vs. 0%, p<0.005). In contrast, the polymorphisms in OGG1 Ser326Cys, APE1 Asp148Glu, and MUTYH Gln324His were not involved in the clinical features of MDS.

Conclusion
The low DNA repair polymorphism, XRCC1 Arg399Gln is associated with the clinical features of MDS, including therapy- related MDS. Further investigation of BER polymorphisms will provide the understanding of pathogenesis of therapy- related MDS in a larger sample size analysis.

Disclosures:
No relevant conflicts of interest to declare.
Fibrotic Tissues In Human Primary Myelofibrosis With JAK2 V617F Are Developed Clonally From Malignant Hematopoietic Stem Cells
Authors: Yamauchi Takuji , Shima Takahiro , Takenaka Katsuto , Miyamoto Toshihiro , Akashi Koichi 
Published: October 2019

Abstract
Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies.

Disclosures:
Miyamoto: Kyushu University Hospital: Employment.
Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells
Authors: Uri Rozovski , Calin George , Setoyama Tetsoro , Lucilla Abundu , David Harris , Li Ping , Zhiming Lui , Srdana Grugurevic , Ferrajoli Alessandra , Faderl Stefan H. , Burger Jan A. , O'Brien Susan , Wierda William G. , Keating Michael J. , Estrov Zeev 
Published: October 2019

Abstract
MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL.

Disclosures:
No relevant conflicts of interest to declare.
Dnmt3a Deletion Predisposes Hematopoietic Stem Cells To Malignant Transformation
Authors: Mayle Allison , Yang Liubin , Challen Grant A , Zhou Ting , Rebel Vivienne I. , Goodell Margaret A. 
Published: October 2019

Abstract
DNA methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, is mutated in various hematological malignancies affecting both myeloid (20%), mixed (50%), and lymphoid (18%) malignancies and is associated with poor prognosis. The most frequently reported DNMT3A mutation is R882 in acute myeloid leukemia (AML), which results in altered enzyme activity, but various missense and nonsense mutations have also been found throughout the gene, suggesting that loss-of-function mutations in DNMT3A may also contribute to leukemogenesis. Our group recently showed that transplantation of HSCs from Dnmt3a knock-out (KO) mice led to increased hematopoietic stem cell (HSC) self-renewal and inhibition of differentiation, but was insufficient to cause transformation. However, in these experiments, Dnmt3a-KO HSCs were transplanted alongside wild-type whole bone marrow to quantitate HSC function, potentially protecting against malignant transformation.
We hypothesized that if Dnmt3a-KO HSCs were transplanted alone, a predisposition to transformation would be uncovered. We established a large non-competitive transplantation cohort receiving 500 control or Dnmt3a-KO HSCs and monitored the mice closely for disease. Strikingly, mice with Dnmt3a-KO HSCs had significantly shorter survival (246d vs 467d, p<0.0001, Figure 1). As mice succumbed to disease, we analyzed histological changes in hematopoietic organs and performed CBCs and immunophenotyping to diagnose the diseases. We identified multiple disease classes within the Dnmt3a-KO recipients, including T-cell acute lymphoblastic leukemia, myeloproliferative disease (MPD), myelofibrosis (MF), and myelodysplastic syndromes (MDS). The relatively long disease latency suggests that acquisition of secondary hits promotes disease; identification of these secondary mutations is ongoing.Figure 1Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignanciesFigure 1. Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignancies
Here, we show that Dnmt3a deletion in noncompetitive transplanted HSCs leads to an array of hematologic disorders that models the spectrum of disorders seen in human malignancies. Since DNMT3A mutations are known early genetic lesions in leukemia development, mutations that cooperate with DNMT3A might influence the type of disease developed. This mouse model serves to validate an important role for Dnmt3a in the development of hematologic malignancies, and is also valuable for the study of future targeted therapies.
Mice transplanted with Dnmt3a-KO HSCs died significantly earlier than mice transplanted with control HSCs (median survival 246 days and 467 days, p<0.0001). Fifty and 20 female mice were transplanted with 500 Dnmt3a-KO or control HSCs respectively, all at 6-8 weeks of age. Censored points indicate mice that were euthanized for unrelated reasons.

Disclosures:
No relevant conflicts of interest to declare.
Eptifibatide Reduced Cell Adhesion and Recruitment Of The Myeloid Sarcoma-Inducing Leukemia Cells
Authors: Fu Jen-Fen , Shih Lee-Yung 
Published: October 2019

AML patients with myeloid sarcoma (MS) usually had a poor outcome. Our clinical data revealed that AML patients harboring MLL/AF10 and RAS gene mutations were associated with MS formation. By using retroviral transduction/transplantation assay, we demonstrated that the mice transplanted with bone marrow (BM) cells carrying cooperating MLL/AF10(OM-LZ) and KRAS-G12C mutations induced MPD-like myeloid leukemia and MS. Gene expression analyses identified Gpr125, an adhesion G protein-coupled receptor, was up-regulated in the cells carrying cooperating mutations than the cells carrying MLL/AF10(OM-LZ) alone. Knockdown of Gpr125 by RNA interference reduced the number and the size of MS, suggesting that Gpr125 was involved in the MS formation. As Gpr125 contains a HormR domain with Lysine-Glycine-Aspartic acid (KGD) motif which is known to involve in the cell-extracellular matrix (ECM) and cell-cell adhesion, we investigated whether a cyclic RGD peptide drug, eptifibatide (Ep), could interfere MS formation. An in vitro cell-ECM binding assay showed that Gpr125 interacted with fibronectin. Ep reduced leukemia cell-fibronectin binding. Ep also reduced homotypic leukemia cell adhesion and leukemia cell-adipocyte adhesion. In vivo assay demonstrated that Ep reduced leukemia cells recruitment to the adipose tissues, spleen and bone marrow. Our results suggested that blocking Gpr125-mediated cell-ECM and cell-cell adhesion might be helpful to interfere MS formation and BM/spleen recruitment of leukemia cells.

Disclosures:
Off Label Use: Eptifibatide (Integrilin, Millennium Pharmaceuticals, also co-promoted by Schering-Plough/Essex), is an antiplatelet drug of the glycoprotein IIb/IIIa inhibitor class.