bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polycythemia Vera with Multiple Clones Carrying Different Mutations (L611V, V617F, L611V/V617F) in Exon 14 of JAK2.
Authors: Cleyrat Cédric , Jelinek Jaroslav , Girodon François , Boissinot Marjorie , Ponge Thierry , Harousseau Jean-Luc , Issa Jean-Pierre , Hermouet Sylvie , Hermouet Sylvie 
Published: October 2019

Abstract
Abstract 3908
Poster Board III-844

Background
In more than 95% of cases, Polycythemia vera (PV) is characterized by the presence of the V617F mutation of JAK2 (JAK2-V617F). We report on three cases of V617F-positive PV with an additional mutation of JAK2 changing leucine 611 for a valine (L611V).

Design and Methods
We used allele-specific quantitative PCRs and pyro-sequencing to detect the L611V and V617F JAK2 mutants in genomic DNA and in cloned PCR products. The consequences of the different mutations on the function of JAK2 were investigated using transient expression in BaF-3/EpoR cells.

Results
We found equal proportions of L611V and V617F alleles in granulocyte DNA (patients Na249, 19%; Na382, 26%; Di362, 27%) and established the presence of alleles with L611V and V617F in cis (L611V/V617F). The double JAK2-L611V/V617F mutant represented 15% to 26% of JAK2 alleles in granulocyte DNA. JAK2-L611V/V617F was detected in 12% to 30% of erythroid colonies, always in a heterozygous fashion. Low levels of single mutant alleles were also found, in 0.5% of cloned PCR products (JAK2-L611V, patient Na249) and in 1.5% of genomic DNA and cloned PCR products (JAK2-V617F, patient Na249), indicating that mutations in JAK2 occurred more than once. Functionally, JAK2-L611V was found comparable to wild-type JAK2, whereas JAK2-L611V/V617F displayed greater constitutive tyrosine phosphorylation of JAK2 and AKT than wild-type JAK2 and JAK2-V617F. The double JAK2-L611V/V617F mutant was associated with a high hematocrit and normal counts of leukocytes and platelets. Patients Na382 and Di362, but not patient Na249, carried the 46/1 haplotype of chromosome 9p associated with a reported pre-disposition to mutations of JAK2 on the same allele.

Conclusion
PV patients can harbor multiple clones carrying different mutations of JAK2, which may or may not increase JAK2 activity. Functionally silent mutations, not leading to clonal expansion, may remain undetected. The double JAK2-L611V/V617F mutated clone was predominant whereas single L611V and V617F JAK2 mutants represented <2% of JAK2 alleles. Thus presence of the activating JAK2-V617F mutation does not ensure clonal expansion, implying that other factors promote or repress expansion of JAK2-mutated progenitors. Lastly, mutations of JAK2 can occur more than once in patients negative for the pre-disposing 46/1 haplotype.

Disclosures:
No relevant conflicts of interest to declare.
The Overexpression of Enhancer of Zeste Homologue 2 Is Associated with Tumor Proliferation and Aggressive Behavior in Adult T-Cell Leukemia/Lymphoma.
Authors: Sasaki Daisuke , Imaizumi Yoshitaka , Hasegawa Hiroo , Osaka Akemi , Tsuruda Kazuto , Tsukasaki Kunihiro , Iwanaga Masako , Miyazaki Yasushi , Yanagihara Katsunori , Marquez Victor , Kamihira Shimeru , Yamada Yasuaki 
Published: October 2019

Abstract
Abstract 3959
Poster Board III-895

Background
Enhancer of zeste homologue 2 (EZH2) is a critical component of the Polycomb Repressive Complexes PRC2, which mediates epigenetic gene silencing by the trimethylation of Lys27 of histone 3 (H3K27). This regulation is not only involved in embryonic development and stem cell renewal, but also in tumor progression. Adult T-cell leukemia/lymphoma (ATLL) is a single disease entity etiologically associated with human T-cell leukemia virus type-1 (HTLV-1). Its clinical behavior, however, is quite diverse among patients and is thus subclassified into several subtypes. The prognosis of the aggressive subtypes is very poor based on standard chemotherapy, and so the development of a new therapeutic approach is urgent.

Results
In a comparative microarray analysis of primary ATLL samples, the acute type showed significantly higher EZH2, YY1, and RYBP (RING1 and YY1 binding protein) expressions compared with the chronic type. Further analysis employing real-time quantitative RT-PCR of various Polycomb group (PcG)-related proteins, including the ones mentioned above, revealed that the mRNAs of two other PRC2 components, EED and RBBP4, were also significantly elevated in ATLL, irrespective of the subtypes, compared with lymphocytes from HTLV-1 carriers. These results suggest that the dysregulation of PRC2 is a key event in the development and progression of ATLL. In the immunohistochemical analysis of ATLL lymph nodes, the nuclei of over 90% of ATLL cells were positive for EZH2, which in clear contrast to reactive or indolent B-lymphoma lymph nodes in which a few cells were positive. It has been shown that activated Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity. Western blotting of EZH2 showed that EZH2 of primary ATLL cells was not phospholyrated and remained in its active form. Indeed, the trimethylation of H3K27 in ATLL cells was confirmed by Western blotting and immunohistochemistry, whereas it was completely absent in lymphocytes from HTLV-1 carriers. Finally, in studies using ATLL cell lines, the knockdown of EZH2 by siRNA caused decrease in cell proliferation. Moreover, ATLL cell lines showed high sensitivities to histone deacetylase (HDAC) inhibitors as LBH589, and LBH589 decreased EZH2 expression. EZH2 inhibitor 3-deazaneplancin A (DZNeP) also reduced cell growth, not only in primary ATLL cells, but also in ATLL cells lines. These results strongly support the rationale for developing molecular targeting therapies against EZH2 in ATLL.

Disclosures:
No relevant conflicts of interest to declare.
Oncogenic Pim Kinase Activity Provides Resistance to Mtor Inhibition in Vitro and In Vivo.
Authors: Schatz Jonathan H , Wendel Hans-Guido 
Published: October 2019

Abstract
Abstract 3974
Poster Board III-910
The PI3K/Akt/mTor pathway is among the most frequently deregulated in human cancers, including many leukemias and lymphomas, and inhibitors targeting it at multiple levels are either clinically available or under development. The Pim family proteins are oncogenic serine/threonine kinases expressed in many malignancies and that have numerous overlapping downstream targets and functional consequences with PI3K/Akt/mTor. We here investigate the role of Pim proteins in oncogenesis and their ability in particular to mediate resistance to mTor inhibition. We find over-expression and genomic amplification of Pim1 and Pim2 in tumor samples and cell lines derived from patients with multiple B lymphomas. We show that both Pim1 and Pim2 powerfully mediate resistance to apoptosis in the murine pro-B cell line FL5-12 when withdrawn from IL-3. In murine tumor cells with constitutive activation of mTor, Pim1 and Pim2 provide resistance to treatment with the mTor inhibitor rapamycin. In vivo, we find that murine Pim2 (mPim2) accelerates tumorigenesis in the Eu-Myc transgenic model of Burkitt's lymphoma in a manner highly similar to Akt. However, unlike Akt, mPim2 leads to tumors that resist sensitization to chemotherapy by co-administration of rapamycin. We also investigate Pim1's role as a target of aberrant somatic hypermutation (ASHM), the process by which the enzyme activation induced deamidase (AID) introduces mutations into the coding and non-coding regions of the Pim1 locus and other proto-oncogenes in many B-cell malignancies. We find that structural Pim1 mutants found in patient tumors samples retain their ability to accelerate tumorigenesis in the Eu-Myc model in vivo and to mediate resistance to apoptosis and rapamycin in vitro. In conclusion, Pim activity is clinically significant, highly oncogenic, and provides resistance to mTor inhibition. Pim inhibition is therefore an attractive therapeutic approach, especially in combination with PI3K/Akt/mTor inhibition, even as ASHM provides a potential mechanism for B-cell tumors to escape it.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Significance of Lupus Anticoagulant and Anti-Beta2 Glycoprotein I Antibodies in a Pediatric Cohort.
Authors: Cook Rebecca P. , Marques Marisa , Oster Robert 
Published: October 2019

Abstract
Abstract 3989
Poster Board III-925

Introduction
Antiphospholipid antibodies (APAs) (lupus anticoagulant (LA), anti-beta2 glycoprotein I (anti-B2GPI) and anti-cardiolipin) are seen in a variety of conditions, yet their clinical implication varies. In adults, some APAs cause thrombosis and/or recurrent pregnancy loss. LAs with specificity for anti-B2GPI appear to be more closely associated with thrombosis. In children, APAs are often transient after viral or bacterial illnesses and little is known about their pathologic potential. In this study, we sought to determine the clinical associations of positive LAs with and without anti-B2GPI activity. Patients and Methods: We tested remnant plasma from 115 pediatric patients with positive LA result (according to the International Society on Thrombosis and Haemostasis guidelines) for anti-B2GPI IgG, IgM, and IgA using an indirect EIA supplied by REAADS (Corgenix Inc, Broomfield, CO) and reviewed the patient's medical records to correlate with the results.

Results
There were 61 females (53%) and 54 males (47%) with the average age of 10.6 years (range: 0.5 to 19.5 years). An underlying medical condition such as malignancy, autoimmune disorder, infection/sepsis, or concurrent thrombosis was observed in 97 patients (84%). Eighteen patients (16%) were healthy children and LA was detected incidentally during workup for a prolonged PTT. The mean age of the group with positive medical history (11.5 years; range: 0.5 to 13 years) was statistically different than that of the healthy children group which was 5.8 years (range: 0.5 to 3.75 years) (P<0.001). Of the 115 patients, 37 (32%) had thrombosis at the time of LA testing; only 6 patients had known inherited hypercoagulability. Thirty-four of the 37 thrombotic episodes (92%) were venous and 3 were arterial (8%). An autoimmune disorder was present in 46 patients (40%) and 10 developed a thrombosis. Forty-one patients (36%) had active infection/sepsis, and 15 had thrombosis. Of 12 patients with malignancies (10%), 4 had thrombosis. The average age of those with thrombosis, 13.3 years (range: 0.5 to 15 years) was higher than that of children without thrombosis, 10.3 years (range: 0.6 to 10.5 years) (P=0.018). Only 12 of 115 patients (10%) were positive for anti-B2GPI and 3 of them had venous thrombosis. Of the anti-B2GPI positive patients, 8 had underlying autoimmune conditions, 2 had no medical history but had current thrombosis, one had a malignancy with thrombosis, and another had an acute infection. Conclusions: In children, APAs occur in a variety of conditions, but may also be found incidentally. Perhaps surprising, LA was associated with thrombosis in 32% of patients. However, only 10% tested positive for anti-B2GPI, with an even smaller percentage having thrombosis (3%). These results suggest that anti-B2GPI is not necessary for the development of thrombosis in children with LA.

Disclosures:
No relevant conflicts of interest to declare.
Increased Sperm Count Maintains High Population Frequency of Factor V Leiden.
Authors: Cohn Danny M , Repping Sjoerd , Büller Harry R , Meijers Joost CM , Middeldorp Saskia 
Published: October 2019

Abstract
Abstract 3994
Poster Board III-930

Introduction
Factor V Leiden (FVL) increases the risk of venous thrombosis and pregnancy loss in carriers. Nevertheless, this relatively old mutation is still prevalent in Caucasion populations, which could be explained by positive selection pressure. Men with FVL have previously been found to have higher fecundity (the time between marriage and first pregnancy). Whether this is caused by increased fertility is unknown. We therefore aimed to assess whether men with FVL have increased fertility.

Patients and methods
We performed a pilot study in 19 men with FVL. For confirmation, we performed a cohort study among 961 consecutively included male partners of couples presenting at our university hospital for fertility workup between January 2000 and July 2007. We genotyped all participants and compared the outcome of the fertility workup between FVL carriers and non-carriers.

Results
The pilot study showed increased sperm concentration and total sperm counts in the 19 FVL carriers as compared to men from the general population. In the cohort study, we identified 37 FVL carriers and 921 non-carriers. FVL carriers had higher total sperm counts and total motile sperm counts than non-carriers: 236 ×106 (95% CI 158-292 ×106) versus 163 ×106 (95% CI 147-178 ×106) and 81 ×106 (95% CI 54-105 ×106) versus 52 ×106 (95% CI 48-57 ×106), respectively.

Conclusions
Our results provide a possible explanation for the high prevalence of FVL among Caucasians. To our knowledge, this is the first study that indicates that an increased prevalence of a genotype is controlled by increased sperm counts.

Disclosures:
No relevant conflicts of interest to declare.
A Chemical Genetic Analysis of Platelet Activation.
Authors: Flaumenhaft Robert , VerPlank Lynn , Dilks James R. , Blair Price S. , Mairuhu Albert , Negri Joseph , Burbank Jason , Walzer Gil , Aldredge Tyler , Dockendorff Chris , Gould Robert , MacPherson Lawrence , Palmer Michelle 
Published: October 2019

Abstract
Abstract 4009
Poster Board III-945
Platelets are anucleate cells that are not amenable to traditional forward genetic analysis. In collaboration with the Broad Institute Probe Development Center, we have performed a chemical genetic analysis of platelet activation. Chemical genetics involves exposure of cells to a library of small molecules, identification of compounds that produce a phenotype of interest, and determination of the target of these small molecules. We have used an assay designed both to identify allosteric inhibitors of Protease Activated Receptor-1 (PAR1) and to find inhibitors that selectively target granule release. This assay monitors dense granule secretion mediated by SFLLRN, a PAR1-specific agonist, using a luciferase-based assay system to detect ADP/ATP release. For primary screening, over 300,000 compounds were assayed in duplicate using freshly outdated platelet-rich plasma supplied by several blood banks across the United States. Computational analyses of the primary data demonstrated that approximately 0.2% of compounds showed ≥50% inhibition relative to maximally inhibitory concentrations of the known antiplatelet agent, cilostazol. Secondary screening using 8-point dose response curves were performed on the 629 inhibitory compounds, 742 compounds with inconclusive activity (e.g., ambiguous duplicates in primary screening), and 213 structural analogs of active compounds. These assays identified 367 active compounds with IC50s ≤10 micromolar. Counter screening to exclude luciferase inhibitors demonstrated 137 small molecules that inhibited PAR1-mediated ATP/ADP release without significant inhibition of luciferase. Database mining using PubChem and CAS search engines was performed to assess the selectivity of active compounds. Twenty eight compounds were selected for further testing based on their IC50s in confirmatory assays, lack of activity in unrelated bioassays, and chemical structure. Known platelet inhibitors were excluded. Of the 28 compounds, 16 compounds potently inhibited SFLLRN-induced alpha-granule release from washed platelets, as monitored by P-selectin expression. IC50s for these compounds ranged from <0.3 to 1 micromolar. None of the selected compounds that failed to inhibit alpha-granule release demonstrated significant inhibition of SFLLRN-induced 14C-serotonin release at 10 micromolar. Ongoing studies are directed at selecting best candidates from among the 16 confirmed inhibitory compounds to develop as biological probes that target either PAR1 activation or distal steps in granule secretion.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Platelet Small-Molecule Agonist Targeting Glycoprotein Ibalpha.
Authors: Yang Jianfeng , Chen Zhi , Zhu Weiliang , Ruan Changgeng 
Published: October 2019

Abstract
Abstract 4010
Poster Board III-946

Introduction
Interaction of glycoprotein (GP) Ibα with Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction for αIIbβ3 activation under condition of high shear stress.

Methods
Based on the crystal structure of platelet GPIbα (PDB:1P9A), virtual screening was employed to identify active compounds. Compounds in SPECS database were docked to VWF binding site on the surface of GPIbα. The screening was carried out with the DOCK4.0 program. The 150 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbα were investigated the effect on platelet activation and aggregation.

Results
We found one compound, designated it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα (H1-V289) by ELISA method. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation, on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. A VWF A1 blocking antibody could not block platelet aggregation induced by YC148 despite it completely inhibited ristocetin-induced platelet agglutination. And YC148 also stimulated washed platelet aggregation where VWF was absent in the resuspension buffer. These indicated that the aggregation stimulated by YC148 could not the result from VWF binding. Flow cytomety also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. The platelet aggregation stimulated by YC148 was inhibited by anti-GPIbα monoclonal antibody AN51 and 6D1.

Conclusion
A novel exogenous small-molecule agonist was found to activate platelet through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction. The effect of YC148 on platelet from Bernard-Soulier syndrome or GPIbα N-terminal fragment deficient platelet after in vitro cleavage will be further investigated.

Disclosures:
No relevant conflicts of interest to declare.
Antagonism of P2y12 Receptor Results in Severe Impairment of Serum Thromboxane Levels. Can We Manage Patients without Combination Therapy with Aspirin?
Authors: Bhavaraju Kamala , Georgakis Alexander , Jin Jianguo , Tomiyama Yoshiaki , Nurden Alan T. , Nurden Paquita , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 4013
Poster Board III-949
Anti-platelet therapy is a widely accepted treatment regimen for the management of patients with cardiovascular risks. In clinics, many patients with cardiovascular diseases are managed on a combination therapy with Aspirin and Clopidogrel. Aspirin, being a COX-1 inhibitor blocks thromboxane generation and Clopidogrel metabolite antagonizes the P2Y12 receptor (ADP receptor). Thus these drugs block the two most important platelet positive feedback mediators and prevent amplification of platelet responses. The molecular mechanism of P2Y12 receptor antagonism on impairment of serum thromboxane is not understood to-date. Understanding this mechanism will clarify the molecular effects of P2Y12 antagonists when aspirin is clinically contraindicated in patients.

Hypothesis
We hypothesized that P2Y12 antagonism would lead to a decrease in serum thromboxane generation. We tested our hypothesis by employing both pharmacological and molecular genetic approaches.

Methods and Results
Serum thromboxane levels are a cogent marker for platelet hyperreactivity. It is produced when thrombin, which is generated during coagulation cascades, acts on PAR receptors on platelets. Serum was collected from non-anticoagulated blood after clot formation, following which serum thromboxane levels were measured with ELISA. The serum thromboxane levels in mice dosed with clopidogrel (n=4) (30 mg/kg body weight) were inhibited 88.04% compared to untreated mice. In human blood treated with the active metabolites of clopidogrel (R138727) or prasugrel (R361015) (n=3) were inhibited by 83.7% and 95.1%, respectively, compared to untreated human serum (100%). We also evaluated serum thromboxane levels in P2Y receptor null mice (n=4). Where as serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, those in the P2Y12 null mice were inhibited by 78.9%. Furthermore serum thromboxane levels in P2Y12 deficient patients previously described in France and Japan were reduced to 25.3% and 11.7%, respectively, compared to normal volunteers (100%). We had one patient who was only on clopidogrel whose serum thromboxane levels were inhibited by 92.1%, compared to normal volunteers. In a pilot study, we recruited healthy volunteers (n=6), who were given clopidogrel for a week. Serum thromboxane levels were measured before and after the administration of clopidogrel. On an average, the serum thromboxane levels in these volunteers were inhibited by 72.3% after receiving clopidogrel.

Conclusions
In conclusion, P2Y12 receptor antagonism alone could lead to decrease in serum thromboxane levels. Hence in future novel P2Y12 antagonists alone might offer better protection without an additional need for aspirin.

Disclosures:
No relevant conflicts of interest to declare.