bluuebunny/crossref_metadata_2025_split huggingface dataset:
SOCS3 Phosphorylation In Myeloproliferative Neoplasms
Authors: Arnold Claire , Elliott Joanne , Johnston James A , McMullin Mary Frances 
Published: October 2019

Abstract
Abstract 3083

Introduction:
Suppressor of cytokine signaling protein 3 (SOCS3) is an important regulator of the erythropoietin receptor and the receptor-associated JAK2 kinase. We hypothesize that the mechanism of JAK2 mutant-mediated transformation in myeloproliferative neoplasms (MPN) involves overcoming SOCS3 inhibition. In co-expression studies SOCS3 protein is stabilized and tyrosine phosphorylated in the presence of JAK2 V617F (Hookham et al., Blood 2007) and exon 12 mutations (Elliott et al., Haematologica 2009) but not wild-type JAK2, rendering SOCS3 unable to downregulate phosphorylation of these JAK2 mutants. The purpose of this study was to determine if these observations can be reproduced in vivo, by comparing SOCS3 expression and phosphorylation in MPN patients and healthy volunteers.

Methods:
Patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) were recruited from Belfast City Hospital. The mutational status of the JAK2 gene had been previously assessed. Granulocytes were isolated from peripheral blood of patients and healthy volunteers by Ficoll density gradient centrifugation, dextran sedimentation and hypotonic red cell lysis. Estimation of the JAK2 V617F/wild type allelic ratio was performed by pyrosequencing. SOCS3 expression levels were measured by quantitative real-time reverse transcriptase-PCR. Granulocyte whole cell lysate was immunoprecipitated with anti-SOCS3 and Western blots were probed with anti-phosphotyrosine. SOCS3 phosphorylation was estimated by densitometry.

Results:
To date, 63 MPN patients and 27 healthy volunteers have been recruited. This total is comprised of 47 patients with PV (JAK2 V617F n=38, JAK2 exon 12 n=4, unknown n=5) and 16 patients with ET (JAK2 V617F n=15). Median V617F allele burden was 45% in PV (range 0–99%) and 20% in ET (range 7–84%). In 67% of patients with PV, SOCS3 expression was greater than healthy volunteers. Compared to healthy volunteers, SOCS3 phosphorylation in PV patients was higher in 67%, equivalent in 17% and lower in 17% of cases. Similarly in ET, SOCS3 phosphorylation was higher in 73%, equivalent in 18% and lower in 9% of patients compared with healthy volunteers. SOCS3 tyrosine phosphorylation was observed in association with both JAK2 V617F and exon 12 mutants.

Conclusion:
The presence of aberrant tyrosine phosphorylation of SOCS3 protein observed in some PV and ET patients supports the hypothesis that this is a potential mechanism by which mutant JAK2 escapes inhibition and leads to constitutive downstream signaling in MPN.

Disclosures:
No relevant conflicts of interest to declare.
Retrospective Study of the Utility of Follicular Lymphoma International Prognostic Index (FLIPI) and FLIPI2 In Patients with Follicular Lymphoma Uniformly Treated with Rituximab, Cyclophosphamide, Doxorubicin, Vincristin, and Prednisone
Authors: Numata Ayumi , Fujimaki Katsumichi , Tomita Naoto , Tanaka Masatsugu , Hashimoto Chizuko , Oshima Rika , Matsumoto Kenji , Matsuura Shiro , Yamamoto Wataru , Motomura Shigeki , Ishigatsubo Yoshiaki 
Published: October 2019

Abstract
Abstract 3100
The Follicular Lymphoma International Prognostic Index (FLIPI) is the most useful prognostic index for follicular lymphoma (FL). FLIPI was developed from retrospective data, which was based on treatment without rituximab (R) therapy and did not contain β2-microglobulin data of some cases. Recently the working group that had suggested FLIPI proposed FLIPI2, based on prospectively collected data. However, FLIPI2 contains data on several different treatment methods, such as treatment with or without R therapy. The present study aimed to retrospectively analyze the prognosis of FL uniformly treated with the combination of R, cyclophosphamide, doxorubicin, vincristin, and prednisone (R-CHOP). This study involved 114 patients consecutively diagnosed with FL who were treated with R-CHOP and were registered in the data-base of the Yokohama City University Hematology Group between January 2001 and October 2009. All the parameters required for FLIPI and FLIPI2 calculation were present in 108 patients (men, 53; women, 55, median age; 57 years [34- 78 years]). The median follow-up period was 31.7 months (0.8- 97.5 months). According to the FLIPI score, 50 patients (46.3%) were in the high-risk group (HR); 31 patients (28.7%), the intermediate-risk group (IR); and 27 patients (25.0%), the low-risk group (LR). On the basis of the FLIPI2 score, 45 patients (41.7%) were in HR; 52 patients (48.1%), IR; and 11 patients (10.2%), LR. According to FLIPI, the 5-year overall survival (5yOS) was 74.1% (95% CI, 58.1– 94.4%) for HR, 89.5% (95%, CI, 76.6– 100%) for IR, and 100% for LR (p = 0.0049); the 5-year time to failure (5yTTF) was 42.3% (95% CI, 26.0– 68.9%) for HR, 39.1% (95% CI, 22.7– 67.3%) for IR, 66.4% (95% CI, 47.2– 93.4%) for LR (p = 0.244). According to the FLIPI2 score, 5yOS was 71.3% (95% CI, 53.2– 95.6%) for HR, 85.6 % (95% CI, 72.0– 100%) for IR, and 100% for LR (p = 0.197), the 5yTTF was 35.8% (95% CI, 20.0– 64.2%) for HR, 46.7% (95% CI, 31.1– 70.3%) for IR, and 76.2% (95% CI, 52.1– 100%) for LR (p = 0.075). In conclusion, in FL patients treated with R-CHOP, FLIPI provided a more accurate OS than that provided by FLIPI2; however, FLIPI2 provided a more accurate TTF than that provided by FLIPI.

Disclosures:
No relevant conflicts of interest to declare.
MicroRNA Profiling In Activated B-Cell and Germinal Center B-Cell Diffuse Large B-Cell Lymphoma Using Formalin Fixed, Paraffin Embedded Patient Biopsies
Authors: Glinsmann-Gibson Betty J. , McMillan Dennis E , Wilkinson Sarah , Teruya-Feldstein Julie , Rimsza Lisa M. 
Published: October 2019

Abstract
Abstract 3111
Diffuse Large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. The Leukemia Lymphoma Molecular Profiling Project (LLMPP) collaboration has done extensive work regarding this disease including gene expression profiling to show that the cell of origin impacts the prognosis of the patient. Those cases which arise from an activated B cell (ABC) have a worse prognosis than those cases which arise from a germinal center B cell (GCB). We have used a subset of cases from our institution which have had gene expression profiling performed on frozen material and assigned ABC (n=18) or GCB (n=24) status (Rosenwald et al NEJM 2002, 346:1937). We have used matched formalin fixed paraffin embedded (FFPE) tissue block from these cases to obtain a microRNA profile utilizing the qNPA technology (High ThroughPut Genomics) as previously published (Roberts et al Lab Invest 2007, 87:979). This novel FFPE based RNAase protection assay measured 688 human microRNAs. Each microRNA was represented twice on the array and the values averaged for a signal value. The data were normalized to the total signal of the microarray. We were able to define a signature of increased microRNA for each DLBCL subtype as shown in Table 1.
ABC subtype GCB subtype hsa-miR-155 hsa-miR-28-5p hsa-miR-196a hsa-miR-138 hsa-miR-501-3p hsa-miR-151-3p hsa-miR-656 hsa-miR-151-5p hsa-miR-1247 hsa-miR-182 hsa-miR-210 hsa-miR-613
The hsa-miR-155 is of importance in lymphoma as it regulates the generation of immunoglobulin class-switch in plasma cells (Turner et al Immunity 2007, 27:847) and was previously identified as a characteristic of ABC cell lines (Lossos et al Blood 2009,113:3754). The hsa-miR-210 has been shown to modulate the MYC antagonist MNT which allows for MYC expression (Grandori et all Cell Cycle 2009, 8:2756). This microRNA profile provides an opportunity to further explore the role microRNA plays in lymphoma biology and in particular, in DLBCL subtype determination. The success of the technique in FFPE tissue holds promise for further microRNA studies on archival material.

Disclosures:
No relevant conflicts of interest to declare.
TWIST2 Was Deregulated In Human Acute Myeloid Leukemia and Might Play a Tumor Suppressor Role
Authors: Zhao Yun , Zhang Xiuyan , Zhu Yongjin , Cui Jiangxia , Shan Lidong , Yao Hong , Zhu Xiaosu , Zhu Li 
Published: October 2019

Abstract
Abstract 3141
TWIST encodes a typical basic helix-loop-helix transcription factor. Twist-2, as a member of this family was recently identified as a negative regulator of normal murine myelopoiesis (Sharabi AB. et al. PLoS Biol. 2008, 6:2786-2800), however the expression and functional role of TWIST2 in normal and leukemic human myeloid cells is poorly understood. To address this, we detected the transcript expression of TWIST2 from CD34+ cells of 19 AML patients compared with those of their normal counterparts from 6 healthy donors, and found that the expression of this transcript was 5-fold decreased in the patients. A lentiviral vector, which could only express YFP was used as control; and then the other lentiviral vector to express TWIST2 plus YFP was constructed and validated to efficiently infect various human AML cell lines including THP-1, Dami, HEL, HL60, NB4 and SHI-1. Using all these cell lines, we found that averagely the control cells could expand in the liquid culture 46-fold within 6 days, however the TWIST2 overexpressed cells could only increase 7-fold; moreover in the colony-forming cell (CFC) assays with both Dami and THP-1 cells, the TWIST2 overexpressed cells lost nearly 80% CFC capacities compared with those of control cells (n=3, P<0.05). Next we found that more TWIST2 overexpressed cells could reside in the G0/G1 phase compared with control cells (61% vs. 44%, n=2, P<0.05) and accordingly less TWIST2 overexpressed cells were present in the G2/S/M phase compared with control cells (39% vs. 56%, n=2, P<0.05). In addition, the control and TWIST2 viruses infected Dami (2×106 cells/mouse) and THP-1 cells (4×106 cells/mouse) were subcutaneously injected into nude mice without irradiation respectively, and the control cells could generated tumors (7/7 mice with Dami cells and 2/8 mice with THP-1 cells) but not the TWIST2 overexpressed cells (0/8 mice with Dami cells and 0/6 mice with THP-1 cells) within 4 weeks. To obtain molecular insights of how TWIST2 suppressed the tumourgenesis capacity of these cells, microarray analysis (Agilent 44K) were used to assess the differentially expressed transcripts between control and TWIST2 overexpressed THP-1 cells (from 3 independent experiments). Some examples of the differentially expressed genes included CCND1, RBBP9 and EPHB4, which were down-regulated in the TWIST2 overexpressed cells compared with control cells; in the other hand CDKN1A (p21Cip1), TP53INP1, DDI1 and AKTIP, which were up-regulated in the TWIST2 overexpressed cells compared with control cells. Taken together, these data demonstrated that the TWIST2 was deregulated in human AML patients, and suggested it might play a tumor suppressor role in these cells.

Disclosures:
No relevant conflicts of interest to declare.
Pathway Dependence on the Tyrosine Kinase TYK2 and Its Mediator STAT1 In T-Cell Acute Lymphoblastic Leukemia
Authors: Sanda Takaomi , Tyner Jeffrey W , Gutierrez Alejandro , Ngo Vu N , Moriggl Richard , Ahn Yebin , Glover Jason M , Chang Bill H , Willis Stephanie G , Staudt Louis M. , Druker Brian J , Look A. Thomas 
Published: October 2019

Abstract
Abstract 3155
The often aggressive and unpredictable behavior of T-cell lymphoblastic malignancies continues to pose both major clinical challenges in children and adults. To discover oncogenic pathways downstream of critical genetic abnormalities that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), and to identify novel molecular targets for anti-leukemic agents with T-cell specificity, we carried out a genome-wide functional screen in T-ALL cell lines using a retroviral library of inducible short-hairpin RNAs (shRNAs). Among the genes that are required for the growth of T-ALL cells, we found that loss of TYK2, a JAK family tyrosine kinase, was specifically lethal in each of three T-ALL cell lines that we tested in this screen. By contrast, TYK2 knock-down did not affect the growth of cell lines from diffuse large B-cell lymphoma or multiple myeloma, indicating that TYK2 is specifically required for the growth of T-ALL cells. We confirmed by knock-down with multiple independent shRNAs that the loss of TYK2 induces apoptosis in T-ALL, whereas knock-down of other JAK proteins (JAK1, JAK2 or JAK3) had no effect. We found that the TYK2 protein is constitutively phosphorylated in many T-ALL cell lines, and that these cells are sensitive to small molecule JAK/TYK2 inhibitors, including JAK inhibitor-I, AG-490 and CEP-701. To identify upstream receptors involved in TYK2 activation, we knocked down IFNAR, IFNGR, IL10R and IL12RB, and found that loss of IFNAR1 and IFNAR2 specifically inhibit the growth of T-ALL cells, as strongly as TYK2 knock-down. In addition, targeted knock-down analysis of downstream STAT proteins revealed that loss of STAT1 also inhibits the growth of T-ALL cells, indicating that this transcription factor is involved in the TYK2 pathway and required for cell survival. In fact, STAT1 protein was constitutively phosphorylated in many T-ALL cell lines and this phosphorylation was inhibited by both TYK2 knock-down and treatment with JAK/TYK2 inhibitors. Although interferon-mediated pathways are cytostatic in many cell types, our results indicate a requirement for the IFNAR-TYK2-STAT1 pathway in promoting the growth and survival of T-ALL cells. Dependence on this pathway confers unique sensitivity of T-ALL cells to TYK2 inhibition by small molecule inhibitors, thus providing a novel therapeutic target for clinical testing in patients with this disease.

Disclosures:
Druker: Molecular MD: Equity Ownership.
MicroRNA Mir-125b Causes Leukemia
Authors: Bousquet Marina , Harris Marian , Zhou Beiyan , Fleming Mark D. , Lodish Harvey 
Published: October 2019

Abstract
Abstract 3158
MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Symptomatic and Incidental Thromboses Are Both Associated with Mortality In Pancreatic Cancer
Authors: Menapace Laurel A. , Peterson Derick R. , Berry Andrea , Sousou Tarek , Khorana Alok A. 
Published: October 2019

Abstract
Abstract 3167

Background:
Incidentally diagnosed venous thromboembolism (VTE) is a growing clinical problem. Although pancreatic cancer is well-known to be associated with VTE, contemporary rates of incidental and symptomatic VTE events and their association with mortality are incompletely understood.

Methods:
We conducted a retrospective cohort study of consecutive pancreatic adenocarcinoma patients seen at the University of Rochester from 2006–2009. Radiologic reports were reviewed for presence of pulmonary embolism (PE), deep venous thrombosis (DVT), and visceral vein thrombosis. Multiple clinical variables and mortality outcomes were collected. Data were analyzed using a multivariate Cox proportional hazards model.

Results:
A total of 1151 radiologic exams for 135 patients were included. Forty-seven patients (34.8%) experienced at least one VTE event. There were 12 PEs (n=12 patients, 8.9%), 34 DVTs (n=17 patients, 12.6%), 47 visceral vein (n=31 patients, 22.9%) and 2 arterial (n=2 patients, 1.5%) events. Twenty-one patients (15.5%) experienced more than one event. Incidental events comprised 33.3% (n=4) of PEs, 17.6% (n=6) of DVTs and 100% (n=47) of visceral VTE. Median survival for the study population was 237 (95% CI 199–277) days. Patients with VTE had significantly reduced survival (73 vs. 233 days at 3 months post-diagnosis; 66 vs. 245 days at 6 months post-diagnosis). There was no significant difference between asymptomatic and symptomatic events in terms of conditional median survival at 3 months-, 6 months- or 1 year-post diagnosis. In multivariate analysis, occurrence of either DVT (HR 7.4 95% CI 3.8–14.6, P<0.0001) or visceral asymptomatic events (HR 2.5 95% CI 1.6–3.8, P=0.0001) was significantly associated with mortality along with advanced stage.

Conclusions:
VTE occurs in over one-third of pancreatic cancer patients, including a significant proportion with incidentally discovered events. Patients with visceral vein events are generally not anticoagulated but these findings suggest a similar association with mortality as symptomatic DVT. Our findings require reconsideration of prognosis and anticoagulation options in pancreatic cancer patients with both incidental and symptomatic VTE.

Disclosures:
No relevant conflicts of interest to declare.
Mapping the Interface Between Protease Activated Receptor 1 (PAR1) and PAR4 Heterodimers
Authors: Noble Daniel N. , de la Fuente Maria , Nieman Marvin T. 
Published: October 2019

Abstract
Abstract 3195
Antiplatelet therapy is a key component of the treatment regimen for acute coronary syndrome and stroke. A major obstacle in developing antiplatelet agents is for agents to be effective without causing bleeding complications. Protease activated receptor 4 (PAR4) is a potential attractive target for antiplatelet therapies because of its requirement for stable clot formation and limited tissue distribution. The purpose of the current study is to determine how PAR4 interacts with other proteins on the surface of platelets and how these interactions influence activation and signaling of PAR4. We have demonstrated that PAR4 forms homodimers using two techniques, bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). These interactions are specific, as PAR4 does not interact with two unrelated G-protein coupled receptors (GPCRs) rhodopsin and dopamine D2 receptor. In addition to homodimers, PAR4 forms heterodimers with PAR1. The heterodimers are also specific since PAR1 also does not interact with other GPCRs. PAR1 does not interact with rhodopsin; therefore we generated a series of rhodopsin-PAR4 chimeras to determine the region on PAR4 that interacts with PAR1. We have mapped the region on PAR4 required for heterodimerization to transmembrane helix 4 (TM4). When the rhodopsin-PAR4 junction is at the beginning of TM2, TM3 or TM4, the chimera interacts with PAR1 in a manner indistinguishable from PAR4-wt. However, when the junction is moved to the beginning of TM5, TM6 or TM7 the chimera does not interact with PAR1-wt. Molecular modeling of PAR1 and PAR4 has identified residues within TM4 that are exposed and are directed away from the other TM helices creating a potential interaction interface. Co-expression of PAR1 with PAR4 increased the efficiency of PAR4 activation by thrombin ∼10-fold indicating a functional significance for PAR1-PAR4 interactions. The results from these studies provide detailed map of the interactions between two GPCRs on the cell surface and may provide insights for developing PAR antagonists. The current strategy of developing PAR antagonists is to target the individual receptors. However our data show that the functional unit is the PAR1-4 heterodimer and, as such, this may the relevant target to disrupt thrombin activation of platelets. Antagonists to PAR1 or PAR4 need to developed and studied in the context of these interactions, not in isolation.

Disclosures:
No relevant conflicts of interest to declare.
Critical Role of CalDAG-GEFI In FCγRIIa-Dependent Platelet Activation and Thrombosis
Authors: Stolla Moritz , Stefanini Lucia , Ouellette Timothy Daniel , Ahmad Firdos , Reilly Michael P , McKenzie Steven , Bergmeier Wolfgang 
Published: October 2019

Abstract
Abstract 3196
Immune-mediated thrombosis is a major cause of death in autoimmune diseases and contributes to complications in drug treatments (e.g. Heparin Induced Thrombocytopenia). The major receptor on platelets for immunoglobulin-mediated activation is FcγRIIA. FcγRIIA signals through an immunoreceptor tyrosine-based activation motif (ITAM) that leads to phospholipase C activation, which induces the release of calcium and diacylglycerol (DAG). In our previous work, we identified CalDAG-GEFI (calcium and DAG regulated guanine nucleotide exchange factor I) as a key component of collagen/ITAM-mediated platelet activation. In the current study, we evaluated if CalDAG-GEFI is a potential target for the intervention with FcγRIIA receptor dependent, immune-mediated thrombosis.
Mice transgenic for the human FcγRIIA (hFCR) and deficient for CalDAG-GEFI-/- (hFCR/CDGFI-/-) were generated. In vitro, aggregation of hFCR/CDGFI-/- platelets required 50–100-fold higher concentrations of anti-CD9 antibodies than hFCR/WT controls. In comparison, inhibition of P2Y12, the target of clopidogrel, shifted the dose response curve for anti-CD9 in hFCR/WT platelets by only ∼2-fold. In addition to their aggregation defect, hFCR/CDGFI-/- platelets were characterized by markedly impaired Rap1 activation.
To assess the role of CalDAG-GEFI in FcγRIIA -mediated thrombosis in vivo, we developed a model of antibody-mediated thrombosis, were we injected mice with an Alexa-750 labeled antibody against GPIX and extracted the lungs to visualize pulmonary thrombosis on a LICOR scanner. Anti-GPIX induced pulmonary thrombosis in hFCR mice but not in WT animals. Pretreatment with clopidogrel did not provide a substantial protection from thrombosis in hFCR mice. In contrast, no pulmonary thrombi were observed in hFCR/CDGFI-/- mice.
Together, our studies are the first to highlight the importance of CalDAG-GEFI downstream of platelet Fc-receptor/ITAM signaling and suggest CalDAG-GEFI as a powerful new target in the intervention of immune-mediated thrombosis.

Disclosures:
No relevant conflicts of interest to declare.