bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Investigational Aurora A Kinase Inhibitor MLN8237 in Combination with Vincristine Demonstrates Robust Anti-Tumor Activity in Preclinical B-Cell Non-Hodgkin's Lymphoma Models
Authors: Zhang Mengkun , Huck Jessica , Hyer Marc , Kannan Karuppian , Shyu Wen Chyi , Chen Susan , Manfredi Mark , Ecsedy Jeffrey 
Published: October 2019

Abstract
Abstract 2733
Aurora A kinase (AAK) is a serine/threonine protein kinase that is essential for normal mitotic progression. MLN8237, an investigational, selective inhibitor of AAK, is being evaluated in multiple Phase 1 and 2 clinical studies in patients with solid cancers and hematological malignancies. In early clinical trials in heme-lymphatic malignancy, MLN8237 dosed as a single agent has demonstrated antitumor activity (Padmanabhan ASH 2010; Goldberg ASH 2010). In order to identify potential combination partners for MLN8237 for clinical evaluation, previous preclinical efforts demonstrated that MLN8237 combined with rituximab leads to significant antitumor activity in a panel of in vivo diffuse large B-cell lymphoma (DLBCL) models (Huck et. al. ASH 2008; Zhang et, al. AACR 2009). In the present preclinical studies, a novel combination treatment of MLN8237 and vincristine was tested in two xenograft models of DLBCL, WSU and OCI-LY19. Mice were dosed for three weeks with MLN8237 orally daily (QD) and with vincristine intravenously weekly (QW). In both tumor models, single agent dose dependent antitumor activity was observed with MLN8237 and vincristine; however, tumors gradually re-grew after the treatment. Combined MLN8237 and vincristine treatment led to additive or synergistic antitumor activity and resulted in significant tumor growth delay even after discontinuing treatment. In the OCI-Ly19 model, MLN8237 (20 mg/kg) with vincristine (0.5mg/kg or 1 mg/kg) resulted in complete cures with no tumor re-growth out to >120 day. In the WSU model, MLN8237 (20mg/kg) combined with vincristine (1mg/kg) led to tumor regression during the treatment period and significant tumor growth delay (TGD=38). Body weight loss (BWL) was observed in the combination arms in a dose dependent manner in an acceptable range (maximum ∼10%); however body weight recovered quickly after treatment termination. The plasma and tumor pharmacokinetic profiles of MLN8237 (20 and 3 mg/kg) and vincristine (1 mg/kg) in the WSU model demonstrated decreased MLN8237 exposure when co-administered with vincristine. Vincristine exposures were not affected by co-administration with MLN8237. Additional preclinical studies are planned to evaluate the antitumor activity of MLN8237 combined with vincristine and rituximab. Preclinical data showing the antitumor activity of the combination of MLN8237 and vincristine provides the basis for its clinical evaluation as a treatment option for aggressive DLBCL.

Disclosures:
No relevant conflicts of interest to declare.
The F-BAR Protein PACSIN 2 Specifically Coats the Anastomosing Intracellular Membrane Systems of Platelets and Megakaryocytes,
Authors: Jónsson Terese , Begonja Antonija Jurak , Kormann Jan , Merkl Barbara , Pluthero Fred , Kahr Walter H. , Hartwig John H. , Plomann Markus , Falet Hervé 
Published: October 2019

Abstract
Abstract 3261
Megakaryocyte (MK) maturation and platelet formation are associated with extensive intracellular membrane rearrangements. However, the cellular proteins and mechanisms responsible for the formation of the platelet open canalicular system (OCS) are unknown. PACSIN 1–3 belong to the novel family of F-BAR domain-containing proteins, which bind and deform membranes, promoting tubular invaginations. Blood platelets and MKs contain the ubiquitously expressed PACSIN 2, but not PACSIN 1 or 3, as evidenced by intracellular flow cytometry and immunoblot analysis. Spinning disk laser fluorescence confocal microscopy revealed a specific association between PACSIN 2 and tubular structures in human platelets. PACSIN 2 did not colocalize with granule or lysosome markers, i.e. CD62P, CD63 and LAMP1, suggesting that it coats the OCS. In mouse MKs, PACSIN 2 was distributed through the cell body and colocalized with the membrane marker, CD61. Immunoprecipitation of PACSIN 2 from human platelet lysates pulled down filamin A (FlnA), a major platelet cytoskeletal protein. The interaction required PACSIN 2 F-BAR domain, as evidenced by in vitro binding assays using recombinant proteins. Mice lacking FlnA in the MK lineage have a severe thrombocytopenia with large platelets (Falet et al, J Exp Med 2010; Jurak Begonja et al, Blood 2011). In contrast to wild type platelets, FlnA-null platelets had diffuse PACSIN 2 staining and abnormal intracellular membrane structures, suggesting that the PACSIN 2-FlnA interaction is necessary for the integrity of the platelet intracellular membrane systems. PACSIN 2-null mice had a mild thrombocytopenia, enlarged spleens and increased spleen megakaryopoiesis. Together, the data shows that PACSIN 2 is expressed in platelets and MKs, where it specifically coats the anastomosing intracellular membrane systems in cooperation with FlnA.

Disclosures:
No relevant conflicts of interest to declare.
Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,
Authors: Brophy Teresa M , Ahamed Jasimuddin , Coller Barry S. 
Published: October 2019

Abstract
Abstract 3271
Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation.

Disclosures:
Coller: Centocor/Accumetrics/Rockefeller University:.
JAK2 (V617F) Mutation and Cerebral Venous Thrombosis,
Authors: Martinelli Ida , Passamonti Serena Maria , Biguzzi Eugenia , Franchi Franca , Gianniello Francesca , Bucciarelli Paolo , Pietra Daniela , Mannucci Pier Mannuccio , Cazzola Mario 
Published: October 2019

Abstract
Abstract 3339

Background.
Whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm is currently unclear.

Methods.
Patients with cerebral venous thrombosis were tested for the JAK2 (V617F) mutation within one year from the onset of thrombosis and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up.

Results.
Ten of 152 patients (6.6%) carried the JAK2 (V617F) mutation. Three of them had known acquired risk factors for thrombosis and 5 had thrombophilia. The median duration of follow-up was 7.8 years (6 months to 21.3 years). Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, while three additional patients developed the disease during the follow-up, for an annual incidence of 0.26% patient-years (95% CI 0.05–0.64). The last patient has no evidence of disease after three years of follow-up. Patients without the JAK2 (V617F) mutation at the time of cerebral venous thrombosis were re-tested at the end of the follow-up and remained negative, with normal whole blood counts [log-rank test c2: 159 (p<0.0001)]. Hence, a myeloproliferative neoplasm was diagnosed in 90% of patients with the JAK2 (V617F) mutation and in none of those without (Fisher's exact test p<0.0001).

Conclusions.
Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Thus, patients with cerebral venous thrombosis should be tested for the JAK2 (V617F) mutation, irrespective of whole blood counts and the presence of other risk factors for thrombosis.

Disclosures:
No relevant conflicts of interest to declare.
Measurement of Dabigatran in Serum and Urine and As Point of Care Method in Urine,
Authors: Harenberg Job , Hagedorn Antje , Giese Christina , Kraemer Roland 
Published: October 2019

Abstract
Abstract 3352
The oral direct thrombin inhibitor dabigatran prevents development of arterial and venous thromboembolism. Dabigatran is determined in plasma by coagulation and chromogenic methods. Renal excretion plays an important role in the metabolism. We aimed to determine dabigatran in urine by means of a quantitative measurement in serum and urine and as qualitative determination in urine as point of care method.
Serum and urine (patent pending) from healthy donors were spiked with dabigatran (50ng/ml to 1000 ng). Thrombin inhibition was determined by means of a chromogenic substrate method comparing the results obtained from plasma. The reagents were lyophilized on mini-strips and incubated with urine (patent pending). The concentration of dabigatran was determined from plasma, serum, and urine and qualitatively by the mini-strips from 15 patients on treatment with dabigatran.
The concentrations of dabigatran spiked to plasma, serum and urine were independent of the medium (correlation between r=0.80 and 0.93). Patients on treatment with dabigatran showed a high correlation of plasma and serum values (r=0.9). The correlation to urine samples was lower do to the time dependence of drug intake. The qualitative mini-strips detected dabigatran in all samples independently of drug intake. There were no false positive or false negative detections of dabigatran by the mini-strips.
Detection of dabigatran in serum avoids separate blood sampling to obtain plasma. Detection in urine avoids blood sampling at all and allows point of care testing also by the patient him/herself. Control of intake and early detection of low urinary excretion leading to increased plasma levels of dabigatran may improve and facilitate patient care.

Disclosures:
No relevant conflicts of interest to declare.
Repression of Mir-495, a Microrna Associated with Favorable Outcome of Acute Myeloid Leukemia Patients, Is Required for the MLL-Associated Leukemogenesis,
Authors: Jiang Xi , Huang Hao , Li Zejuan , Li Yuanyuan , Chen Ping , He Chunjiang , Arnovitz Stephen , Neilly Mary Beth , Rowley Janet D. , Chen Jianjun 
Published: October 2019

Abstract
Abstract 3462
Acute myeloid leukemia (AML) bearing MLL (mixed lineage leukemia) translocations are associated with poor survival, and only fewer than 50% of the patients survive longer than 5 years. Thus, an improved strategy leading to a higher cure rate is urgently needed to treat MLL-associated AML. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been postulated to be important gene expression regulators in all biology including human leukemia. Through large-scale, genome-wide miRNA expression profiling assays, we determined that miR-495 is significantly down-regulated in the majority of human AML samples, particularly, in those with MLL rearrangements. More interestingly, through correlating the expression signature of miR-495 with clinical outcome of AML patients, we revealed that a low expression level of miR-495 is a predictor of poor prognosis in most AML patients. Our further qPCR assays confirmed that the expression of miR-495 is even more significantly downregulated in MLL-rearranged AML primary patient samples and cell lines. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we found that forced expression of miR-495 significantly inhibits the capacity of the MLL-AF9 fusion gene to support colony formation in mouse bone marrow progenitor cells in vitro and to induce leukemia in vivo. In leukemia cell lines, overexpression of miR-495 greatly inhibits the viability of the cells, while increasing apoptosis. Furthermore, by using 3 algorithms for miR-495 3'UTR binding sites, we identified several well-known MLL leukemia-related genes, e. g. BMI1, MEF2C, BID and MEIS1, as potential targets of miR-495. Results of qPCR revealed that forced expression of miR-495 significantly inhibits the expression levels of these genes in leukemia cell lines, mouse bone marrow progenitor cells, as well as mouse peripheral blood cells with MLL fusion genes. Therefore we hypothesize that miR-495 may function as a tumor suppressor in AML with MLL rearrangements by targeting essential tumor-related genes. Further studies will focus on: 1) effects of miR-495 on the functions of target genes studied in vitro and in vivo; 2) the epigenetic mechanisms and the signaling pathways involved in regulating the expression level of miR-495 in human leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Effectiveness of Transcriptional CDK Inhibitors in Targeting Non-Proliferating CD34+CD38- AML Cells,
Authors: Pallis Monica , Burrows Francis , Whittall Abigail , Seedhouse Claire , Boddy Nicholas , Russell Nigel H. 
Published: October 2019

Abstract
Abstract 3482
Quiescent cells conserve energy and are characterised by low RNA synthesis. In contrast, cancer cells are thought to be addicted to high RNA synthesis, particularly synthesis of survival molecules. We hypothesised that quiescent cancer cells, already low in RNA, would be sensitive to apoptosis induced by transcriptional cyclin-dependent kinase (CDK) inhibitors that further deplete RNA. We cultured the CD34+CD38- KG1a cell line continuously in the presence of an mTOR inhibitor, which maintained excellent viability and enriched the cells for quiescent stem cell features including low RNA content, low metabolism, low reactive oxygen species production and decreased size. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that the basal mitochondrial apoptotic machinery is neither impaired nor improved. We treated quiescence-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against transcriptional CDKs - flavopiridol, roscovotine and TG02. All of the agents used showed increased kill in the unmanipulated compared to the quiescence-enriched cells, emphasising the chemoresistant nature of quiescent cells. To put a value on the difficulty of eradicating the leukamic clone, we used the parameter 2 X IC50 (which theoretically would kill 100% cells in a totally homogeneous population). In quiescence-enriched cells, the percentage of cells killed at 2 X IC50 was roscovotine - 80%, TG02 – 72%, flavopiridol - 62%, 5-azacydidine - 61%, daunorubicin, clofarabine and irinotecan - 60%, etoposide - 58%, and for araC no IC50 for quiescence-enriched cells was reached at 20 times the IC50 of proliferating cells. The data show conclusively that, in this model, transcriptional CDK inhibitors outperformed the agents conventionally used in AML chemotherapy. We also showed that transcriptional CDK inhibitors induced serine 2 dephosphorylation of RNA polymerase II in equal measure in proliferating and quiescence- enriched cells. We have already shown that TGO2 targets primary CD34+CD38- cells in vitro (ASH 2010, abstract 1823). Here we also show that in four primary AML samples sensitive to roscovotine in vitro, the median percentage kill is greater in the CD34+CD38- subset, (which consists largely of quiescent cells and tends to contain the leukaemic stem cells,) than in the bulk cells (40.5% versus 19.5% kill at 2 micromolar roscovotine). We suggest that transcriptional CDK inhibitors may be a useful class of agent for targetting the occult quiescent CD34+CD38- cells thought to contribute to relapse in AML.

Disclosures:
Burrows: Tragara Pharmaceuticals: Employment.
Preclinical Assessment of a Novel CDC7 Inhibitor: Genomewide RNAi Screening Identifies Unique Synergetic and Resistance Genes,
Authors: Liu-Sullivan Nancy , Bhinder Bhavneet , Shum David , Ramirez Christina , Radu Constantin , Djaballah Hakim , Frattini Mark G. 
Published: October 2019

Abstract
Abstract 3570
Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems. While insufficient drug-gene engagement leads to drug failure, de novo escape mutations give rise to patients relapsing, calling the need for systemic studies on how genes influence drug responsiveness. Towards this end, we have explored a functional short hairpin RNA (shRNA) based genomic screening platform aimed at interrogating drug-gene engagement and assessing its consequences on signaling pathways. We propose this concept as a novel way to evaluate drug candidates prior to clinical trials enabling liability assessment and predicting clinical outcome. We took advantage of the arrayed shRNA library produced in lentiviral particles and characterized by several obvious advantageous features including shRNA targeting one hairpin at a time and on the fly high content whole well microscopy imaging analysis. We carried out three parallel genomewide shRNA screens in the absence or presence of the novel CDC7 kinase inhibitor (MSK-777) at its IC20 and IC50 and have identified several gene candidates that influence MSK-777 sensitivity and resistance. These include synergizers that enhance MSK-777 sensitivity and rescuers that confer MSK-777 resistance. IPA analysis mapped clusters of these hits to multiple major pathways among them were the NF-kB pathway, the ubiquitin-proteasome pathway, DNA replication, and several epigenetic regulatory genes. We will present and discuss this concept together with the emerging pathways as a means to identify both key therapeutic targets and biomarkers of sensitivity and resistance. Thus, allowing for not only a broader applicability of assessing candidate genes that modulate specific drug agents, but also for the identification of a tailored and more efficacious therapeutic regimen to treat cancer.

Disclosures:
No relevant conflicts of interest to declare.
Erythrocytes and Platelets May Contribute to Hypercoagulability In Nephrotic Syndrome Through Enhanced Phosphatidylserine Exposure and Microparticles Release
Authors: Gao Chunyan , Xie Rui , Wang Jing , Yu Chengyuan , Tan Xiaoyan , Li Jianan , Xie Rujuan , Zhou Jin , Gilbert Gary E. , Shi Jialan 
Published: October 2019

Abstract
Abstract 36

Background.
Nephrotic syndrome (NS) is frequently accompanied by thromboembolic complications such as renal vein thrombosis, deep vein thrombosis and pulmonary embolism. However, the cause of the hypercoagulable state in NS patients is not understood. In contrast to quiescent cells, membrane-derived microparticles have exposed phosphatidylserine which may support procoagulant enzyme complexes in thrombosis.

Objectives.
The aim of this work was to measure the type and quantity of MPs that expose PS in NS patients, and to evaluate the associated procoagulant activity (PCA).

Methods.
The subjects with membranous nephropathy (MN) or minimal change nephrotic syndrome (MCNS) were compared to healthy controls. Flow cytometry and confocal microscopy was used to evaluate microparticles. PCA was determined by clotting time and purified coagulation complex assays.

Results.
We found that the number of lactadherin+ MPs was significantly higher in each NS group MCNS (3230 ± 536)/MN (4642 ± 697) than that in the controls (1748 ± 239), furthermore, MPs in MN patients are significantly higher than ones in MCNS (P < 0.05; Table 1), which mostly derived from RBC and platelet membranes. The percentage of lactadherin+ RBCs was also significantly increased in each NS group, MN (9.7 ± 3.2%)/MCNS (5.1 ± 2.4%) patients compared to the controls (0.5 ± 0.2%). In addition, the mean percentage of lactadherin+ RBCs in MN showed significantly higher than that in MCNS (P < 0.05). In both NS groups, the percentage of lactadherin+ platelets was significantly higher than that in healthy control subjects (4.1 ± 1.1%) (P < 0.05 for both). Furthermore, patients in MN (11.5 ± 3.1%) also had significantly increased lactadherin+ platelets than in MCNS (7.3 ± 2.3%) (P < 0.05). By confocal laser-scanning microscope, nearly no staining by Alexa Fluro 488-lactadherin could be detected on RBCs or platelets membranes in healthy subjects, whereas a light green fluorescence on RBC and platelet with lactadherin accompanying vesiculation were observed in NS patients. PCA of RBCs/platelets from healthy individuals and NS patients was assessed by recalcification time assays, intrinsic, extrinsic FXa and prothrombinase assays. MPs shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients and blockade of PS with lactadherin inhibited over 90% of PCA. However, an anti-TF antibody had no significant inhibition effect (Figure 1).

Conclusions.
This is the first study to show that loss of RBCs/platelets membrane phospholipid asymmetry with increased PS exposure and MPs release may contribute to the hypercoagulable state of NS patients. The extent of PS exposure on cells and MPs may be a marker of thromboembolic risk in these patients.

Disclosures:
No relevant conflicts of interest to declare.