bluuebunny/crossref_metadata_2025 huggingface dataset:
hsTRAIL/Apo2L Induces Apoptosis in Enteropathy-Associated T-Cell Lymphoma
Authors: Radersma Marijn , de Baaij Laura R , Hijmering Nathalie J , Ossenkoppele Gert , Meijer Chris JLM , Mulder Chris JJ , Cillessen Saskia AGM
Published: October 2019

Abstract 1665

EATL is an intestinal tumor of aberrant intraepithelial T-lymphocytes (IELs) and may be preceded by refractory celiac disease type II (RCD II). Current therapies include surgery, chemotherapy and autologous and/or allogeneic stem cell transplantation. Despite these therapies, the overall outcome of EATL is very poor with 1- and 5-year survival rates in the range of 31–39% and 8–20%, respectively. Therefore, new therapeutic options are needed. Human soluble tumor necrosis factor-related apoptosis-inducing ligand, hsTRAIL/Apo2L, a member of the TNF family, has proven to selectively kill tumor cells via an alternative, death-receptor mediated apoptosis pathway. In this present study we evaluated if hsTRAIL/Apo2L induces apoptosis in both isolated lymphoma cells of EATL biopsies and isolated cells of RCD II biopsies.

hsTRAIL/Apo2L induced apoptosis in isolated EATL lymphoma cells. RCD II cells were less sensitive to hsTRAIL/Apo2L compared to EATL cells. hsTRAIL/Apo2L induced apoptosis in EATL cells was caspase-9 dependent, but unexpectedly active caspase-8 involvement could not be detected. RT-MLPA analysis on EATL samples confirmed this observation by showing increased levels of c-Flip in EATL cells, which suggests a blockage in the extrinsic apoptosis pathway. Both EATL and RCDII cells showed expression of TRAIL receptors R1 and R2 and almost no expression of R3 and R4.

In conclusion, our study showed that hsTRAIL/Apo2L induces apoptosis in EATL cells through the intrinsic apoptosis pathway. Moreover, we showed that TRAIL receptor R1 and R2 are expressed in EATL cells. Based on these results, hsTRAIL/Apo2L may be a new therapeutic option for EATL patients.

Disclosures:

No relevant conflicts of interest to declare.
Antisense Oligonucleotide Mediated Depletion of Factor XI Prevents Vascular Occlusion in An Experimental Thrombosis Model in Primates
Authors: Crosby Jeff R. , Marzec Ulla M , Zhao Chenguang , Gao Dacao , Revenko Alexey S , Matafonov Anton , Gailani David , MacLeod Robert , Gruber Andras , Hanson Stephen , Monia Brett P.
Published: October 2019

Abstract 2250

It has recently been demonstrated that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents. Furthermore, FXI deficiency is not associated with spontaneous bleeding in humans, and deficiency of a contact factor does not cause abnormal bleeding. We have previously shown that treatment with FXI Antisense Oligonucleotides (ASOs) produces potent, dose-dependent antithrombotic activity in various venous and arterial murine thrombosis models. In the current study we characterize the antithrombotic effects of FXI ASOs in a non-human primate model. Subcutaneous administration of FXI ASOs in cynomologus monkeys resulted in a dose-and time-dependant decrease in FXI mRNA levels in liver (up to 90%), decreased FXI protein and activity levels measured in plasma, and prolonged activated partial thromboplastin times (aPTT). Importantly, FXI depletion in monkeys was not associated with an increased risk of bleeding. Recent studies demonstrated that complete inhibition (>99%) of FXI by a monoclonal antibody reduced thrombin generation and prevented vascular occlusion in a collagen-coated graft inserted into a baboon arteriovenous shunt (Tucker et al., Blood 2009). In the present study, we set out to determine the relationship between FXI levels and antithrombotic activity, and in particular, the minimal level of FXI reduction required to produce an antithrombotic effect in a baboon thrombosis model. Using the FXI monoclonal antibody, we demonstrate that ∼50% inhibition of FXI levels produces antithrombotic activity in baboons. Next we applied baboon-specific FXI ASOs and demonstrated reductions in FXI protein levels and activity, with corresponding increases in aPTT levels. Baboons were then treated with FXI ASOs in a manner that would produce ∼50% reduction of FXI protein levels and measured anti-thrombotic activity. Similar to the antibody approach, ASO-mediated reduction of FXI plasma levels ≥ 50% resulted in a potent antithrombotic effect. Furthermore, reductions in FXI levels do not increase bleeding times in baboons. These results further support the development of FXI ASOs for antithrombotic therapy with the potential for a superior safety profile compared to currently available anticoagulants.

Disclosures:

Crosby: Isis Pharmaceuticals: Employment. Zhao:Isis Pharmaceuticals: Employment. Gao:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Gruber:Aronora, LLC: Consultancy, Equity Ownership. Monia:Isis Pharmaceuticals: Employment.
Family History of Venous Thromboembolism (VTE) and the Risk of VTE Recurrence in Patients with a First Unprovoked VTE: A Multicenter Prospective Cohort Study
Authors: Gauthier Karine , Sabri Elham , Kahn Susan R. , Wells Philip S , Anderson David , Gal Gregoire Le , Solymoss Susan , Crowther Mark A. , Perrier Arnaud , White Richard H. , Kovacs Michael J. , Carrier Marc , Rodger Marc A.
Published: October 2019

Abstract 2299

Introduction:

The duration of anticoagulation after unprovoked venous thromboembolism (VTE) has been characterized as the most important unanswered question in clinical thrombosis management. This has led to research to identify predictors of recurrent VTE to identify high-risk patients who might warrant indefinite anticoagulation. Many clinicians assume that a family history of VTE is a predictor of recurrent VTE. This study aims to assess the value of family history as a predictor for recurrent VTE.

Methods:

Prospective multi-center multi-national cohort study recruited patients with a first objectively proven unprovoked VTE who completed 5 to 7 months of anticoagulation therapy. A detailed family history of VTE was completed for every subject. The information recorded included the number of affected relatives, whether they were first or second degree relatives and if the VTE was unprovoked or secondary. Patients were then followed for recurrent VTE.

Results:

664 subjects were enrolled between October 2001 and March 2006, 649 subjects were followed for a mean duration of 3.8 years (3.6–3.98 95% C.I.). The mean age of subjects in this cohort was 53 years (min-max 18–95) and 49% of subjects were females. A family history of VTE in at least 1 first-degree relative was recorded for 112 (17.3%) subjects. A total of 142 (21.9%) suspected VTE events were adjudicated as recurrences. The recurrence rate was 5.94% (4.89–7.15 95% C.I.) per patient-year for patients without any family history of VTE, and it was 4.82% (3.02–7.30 95% C.I.) per patient-year in patients with a family history of VTE in at least 1 first-degree relative. In secondary analyses, neither a family history of unprovoked VTE, multiple unprovoked VTE, in first-degree nor second-degree relatives was a predictor of recurrent VTE. A multivariate analysis was performed to adjust for known risk factors for VTE recurrence, but the adjusted hazard ratios were again not significantly different.

Conclusion:

A family history of VTE is not a predictor for recurrent VTE, and therefore should not be used to segregate unprovoked VTE patients in high- and low-risk categories.

Disclosures:

No relevant conflicts of interest to declare.
Unlike Heparins Newer Oral Anticoagulants Do Not Interact with HIT Antibodies and Maybe Useful in the Longterm Anticoagulant Management of Heparin Compromised Patients
Authors: Lewis Bruce E , Aranda Christopher , Lewis Mary , Hoppensteadt Debra , Walenga Jeanine M. , Fareed Jawed
Published: October 2019

Abstract 2317

Introduction:

Heparin and low molecular weight heparins such as enoxaparin and dlateparin are widely used in the management of thrombosis and cardiovascular disorders. However these anticoagulants are capable of producing the generation of HIT antibodies and their use is not recommended in patients with previous history of HIT. More recently several new oral anticoagulants have been approved around the world. These include the oral anti-Xa agents namely, Apixaban (Bristol-Myers Squibb), Rivaroxaban(Bayer Healthcare) and anti-IIa agents, dabigatran (Boehringer-Ingelheim). All of these represent synthetic low molecular weight compounds. Apixaban and Rivaroxaban are active agents where as Dabigatran requires endogenous activation. The purpose of this investigation was to determine the relative effects of the newer anticoagulants and enoxaparin on HIT antibody mediated platelet aggregation and other interactions with platelets.

Materials:

Rivaroxaban was obtained in powdered form from Bayer Healthcare (Wuppertal, Germany) Apixaban and Dabigatran were of synthetic origin. Enoxaparin was obtained from Sanofi-Aventis (paris, France). All drugs were dissolved in appropriate solutions and a working solution of 100 ug/ml was prepared in buffered saline. Pooled HIT sera was prepared by pooling clinically symptomatic HIT patients with high titers of anti-heparin platelet factor 4 antibodies.

Method:

Whole blood samples drawn from normal healthy volunteers were supplemented with each of these agents at a graded concentration of 0–100 ug/ml for 60 minutes to determine the relative release of platelet factor 4. To test the interaction of HIT antibody with each of these agents 50 ul of PRP samples were mixed with 150 ul HIT positive heat inactivated sera/plasma or plasmapheresis fliud (collected from symptomatic HIT patients). Graded amounts of each of these new anticoagulants at 0.1, 1.0 and 10 ug/ml were added to test the platelet aggregation response at a period of 60 minutes.

Results:

In contrast to enoxaparin which produced an increase in the platelet factor 4 release upon 60 minute incubation 25.6+3.1 ng/ml), none of the new oral anticoagulants produced an increase in the PF4 <15.0 ng/ml. Enoxaparin also produced a strong HIT antibody mediated response, whereas none of these newer oral agents produced any aggregation responses.

Conclusion:

These studies demonstrate that –enoxaparin the newer oral anticoagulant drugs do not interact with HIT antibody to mediate plarelet aggregation responses. Moreover, these newer agents do not promote platelet factor 4 release. Thus, these agents can be used in the long term anticoagulant management of heparin compromised patients.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Implications of Ventx in Acute Lymphocytic Leukemia
Authors: Gao Hong (Jenny) , Wu Xiaoming , Zhu Zhenglun (Jerry)
Published: October 2019

Abstract 2457

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, representing about a third of all pediatric cancers. Despite significant progress in ALL treatment over the past decades, relapse occurs in about 20% cases, which are often resistant to conventional chemotherapy. The canonical Wnt/b-catenin signaling has been implicated in pathogenesis of ALL, and has been a target of intensive investigation for its potential application in cancer prevention and treatment. During our recent studies, using methods of reverse genetics and developmental modeling of Xenopus embryogenesis, we identified VentX as a novel Wnt antagonist and a putative tumor suppressor in lymphocyte leukemia. On the basis of our prior findings that VentX inhibit proliferation of lymphoblastic leukemic cells, our current studies are designed to explore the potential role of VentX as a novel therapeutic target of ALL and its underlying mechanisms.

Using lymphoblastic cell model and real-time RT-PCR technique, we found that VentX expression can be induced by chemotherapeutic agents. We found that elevated expression of VentX caused a senescence phenotype in lymphoblastic leukemic cells and importantly, down-regulation of VentX expression by RNA interference technique led to strong attenuation of the cytotoxic effects of chemotherapeutic agents. Mechanistically, we found that VentX binds to the promoter and p53 and p16 and transactivates the p53/p21 and the p16/pRb senescence pathways, which have been implicated in pathogenesis of ALL. We found further that VentX control these two senescence pathways in primary B lymphocytes and that inactivation of both pathways rather than one is required to abolish the inhibitory effects of VentX on the proliferation of lymphoblastic leukemia cells.

In summary, the results of our investigation suggest that VentX is a novel therapeutic target of ALL. We found that VentX poised as a pivotal link between the oncogenic Wnt signaling pathway and the tumor suppressing senescence pathway. Given the implications of Wnt and senescence pathway in leukemia stem cells, a resource of chemo-resistance and relapse, targeting VentX may present novel opportunities for managing refractory cases of ALL and the eventual cure of the diseases.

Disclosures:

No relevant conflicts of interest to declare.
The t(14;18)(q32;q21) Confers Resistance to Apoptosis-Induced by Smoothened Inhibitors In Diffuse Large B-Cell Lymphoma That Can Be Restored by Functional Inhibition of BCL2
Authors: Kunkalla Kranthi , Qu Changju , Liu Yadong , Singh Rajesh , Vega Francisco
Published: October 2019

Abstract 2479

Generally in cancers, high expression of anti-apoptotic proteins induced by oncogenic signaling pathways is a major cause for treatment failure. Previously, we have demonstrated that Hedgehog (Hh) signaling is activated and functional in diffuse large B-cell lymphoma (DLBCL) and that its inhibition, using the Smoothened (SMO) inhibitor cyclopamine-KAAD, induces apoptosis in cell lines of DLBCL of activated B-cell (ABC) subtype and cell cycle arrest (G1-phase) in those of germinal center (GC) subtype. Induction of apoptosis in ABC-DLBCL was due to decrease of BCL2 expression (protein and mRNA levels), a direct target of Hh signaling (Leukemia 2010;24:1025–36). We also found that BCL2 is upregulated in the tumor cells in the presence of stroma and that Hh inhibition abrogates this upregulation (Oncogene 2011, in press). Here, we explored the possibility of overcoming resistance to apoptosis-induced by Hh inhibition in GC-DLBCL using a combination of the SMO inhibitor “Hh antagonist” (Genentech Inc) with the BH3 mimetic ABT-737 (Abbot laboratories) in a panel of 5 GC type DLBCL cell lines, 4 of them with the t(14:18)(q32;q21) (DOHH2, SUDHL4, Toledo and OCI-LY19) and one without the translocation (HT). ABT-737 is a functional inhibitor of three major anti-apoptotic proteins of the BCL2 family (BCL2, BCL-xL and BCL-W). Combinatorial treatments were performed with increasing concentrations of the Hh antagonist (2.5, 5.0 and 7.5 μM) and with variable but minimal lethal doses of ABT-737 (inducing cell death less than 20%). Using cell viability, apoptosis (annexin-V) and cell cycle based assays, we found that the combined treatments resulted in a additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatment with the Hh antagonist alone. In contrast to ABC type DLBCL cell lines, in DLBCL cells with t(14;18), Hh antagonist did not result in decrease expression of BCL2, BCL-xL and BCL-W indicating that the additive effect of ABT-737 was due to their functional inhibition and not by suppressing expression levels of these proteins. On the other hand, treatments with the Hh antagonist resulted in a concentration dependent decrease of BCL2 protein levels in HT cells. In summary, our data confirm that in the presence the t(14;18), inhibition of SMO does not result in decrease of BCL2 expression. In these cases, concomitant inhibition of BCL2 function and SMO is needed to induce apoptosis. Combined inhibition of Hh signaling and BCL2 function may have a therapeutic value for lymphomas with the t(14;18)(q32;q21).

Disclosures:

No relevant conflicts of interest to declare.
The Prognosis of Children Acute Lymphoblastic Leukemia (ALL) with Minimal Residual Disease (MRD) ≥10-2 After Induction (TP1) Depends on the Prephase Response and on the Level of the MRD After Consolidation (TP2): Results of the 58951EORTC Trial
Authors: Bertrand Yves , Suciu Stefan , Grardel Nathalie , Bakkus Marleen , Mazingue Francoise , Yacouben Karima , Uyttebroeck Anne , Lutz Patrick , Plantaz Dominique , Girard Sandrine , Dastugue Nicole , Benoit Yves , Cavé Hélène
Published: October 2019

Abstract 2571

Background:

In our previous EORTC 58881 trial in childhood ALL, we have shown the prognostic impact of the MRD, quantitated by polymerase chain reaction assay. Patients with TP1 MRD ≥10–2 (D35), had a higher risk of relapse than those with TP1 MRD<10-2 (Cavé et al, NEJM, 1998). For these patients hematopoietic stem cell transplantation (HSCT) with an HLA matched donor was recommended in our subsequent EORTC 58951 trial. Here we report on the patients with MRD ≥10-2 (TP1) in the 58951 trial.

Methods:

Between December 1998 and July 2008, 1955 children (1y-18y) with ALL were enrolled in the 58951 trial, 1459 being evaluable for the MRD (TP1) by PCR (rearrangements of the T-cell receptor and immunoglobulin heavy chain genes). Seventy-nine patients with Phi-negative ALL had a TP1 MRD ≥10-2, and 69 of them achieved a complete hematological remission after induction. Here we report on the outcome of these 69 patients.

Results:

At a median follow-up of 7.6 years, the overall 5-years EFS and OS rates for the 69 patients were 56.1% (SE 6.0%) and 62.0% (SE 5.9%) respectively. Prephase good responders (PGR) (<1000 blasts/mm3 D8) (N=40) had a better OS rate as compared to prephase poor responders (PPR) (N=29): 72.2% (SE 7.1%) vs 48.0% (SE 9.3%) (hazard ratio=0.43, p = 0. 002). In B-lineage ALL patients the OS was similar between PGR (N=32) and PPR (N=6) subgroups, whereas in T-ALL patients there was a better OS for PGR (N=8) than for PPR (N=23) subgroup. Of note, all the patients with hyperdiploidy > 51 chromosomes were PGR.

TP2 MRD (after consolidation) was assessed in 59 pts. All patients but one (1/13) with TP2 MRD ≥10-2 died. In PGR patients, those with TP2 MRD < 10-3 (N=18) had a significant better OS rate at 5 years than those with TP2 MRD ≥10-3 (N=17): 88.9% (SE 7.4%) vs 52.3 % (SE 12.3%), hazard ratio=0.17, p=0.009. We observed no relapses among patients with hyperdiploidy > 51 chromosomes and TP2 <10-3. PPR patients had a poor 5-year OS, independent of TP2 level (MRD <10-3 (N=8) or MRD ≥10-3 (N=16)).

Conclusions:

The prognosis of children ALL with TP1 MRD ≥10-2 has improved with early intensification of the treatment as compared to the one observed in our previous 58881 study. However the outcome of these patients was still heterogeneous and the prephase response was still of prognostic importance. Patients with GPR and TP2 <10-3 had a favourable prognosis and for those patients HSCT is not mandatory.

For other patients with PPR and/or TP2 ≥10-3 the prognosis was worse and these patients may benefit from alternative therapy and/or intensification with HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Implications of PET-Negative Residual Disease At the Completion of Chemotherapy for Diffuse Large B-Cell Lymphoma
Authors: Dabaja Bouthaina S. , Phan Jack , Medeiros L. Jeffrey , Hagemeister F.B. , Chuang Hubert , Fayad Luis , Shihadeh Ferial , Allen Pamela , Wogan Christine , Rodriguez Maria A.
Published: October 2019

Abstract 2695

Purpose:

To evaluate potential differences in overall survival (OS) and progression-free survival (PFS) according to PET and CT disease status at completion of chemotherapy for patients with Diffuse Large B-cell lymphoma.

Patients and Methods:

Subjects were 303 patients with histologically confirmed DLBCL treated between January 2001 and December 2007; no patient received radiation therapy. We evaluated: age, sex, Ann Arbor stage, bulky disease, International Prognostic Index score, Ki-67 expression, PET standardized uptake values (SUVs), disease status after chemotherapy and at last follow-up.

Results:

Median age was 61 years; 149 men; 81 (27%) had stage I-II, 242 (73%) stage III-IV. A total of 248 patients (82%) completed 6–8 cycles of doxorubicin-based therapy. On multivariate analysis, both OS and PFS were significantly influenced by: the presence of PET negative residual mass on CT at completion of therapy (P < 0.001 for OS and P < 0.001 for PFS) (Figure 1); number of cycles and type of chemotherapy (P < 0.001 for OS and P < 0.001 for PFS); Combined presence (p=0.01 for OS and P=0.003 for PFS) of high Ki 67, high PET SUV, and bulky disease; and IPI score (P = 0.001 for OS and P < 0.001 for PFS). Same factors remained significant when replacing response to therapy with size of the residual mass on CT (Figure 2), or number of residual sites (Figure 3) (p<0.0001 OS and p<0.0001 PFS).

Conclusion:

Presence of a residual mass on CT at the completion of chemotherapy has both prognostic and predictive value in patients with DLBCL. These patients should be considered for biopsy and further consolidative therapy.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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 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.
MicroRNA Analysis in the Cerebrospinal Fluid and Blood Serum of Lymphoma Patients At Diagnosis and in Response to Therapy,
Authors: Pospisil Vit , Mocikova Heidi , Jankovska Milada , Markova Jana , Gaherova Lubica , Kozak Tomas , Stopka Tomas
Published: October 2019

Abstract 3663

MicroRNAs are non-coding RNAs (19–23nt) that negatively regulate expression by interference with the translation or RNA stability of up to hundreds of targets. Tumors including leukemias and lymphomas are associated with deregulated expression of microRNAs (Volinia et al. 2010). Several microRNAs including miR-17-92 cluster members and miR-155 are overexpressed in lymphomas and showed promise in predicting relapse (Fabbri M et al. 2011). Unlike longer species of RNA, microRNAs are preserved and detectable in human fluids including serum, plasma and cerebrospinal fluid (CSF) (Baraniskin A. et al. 2011). We investigated abundance of microRNAs in the CSF and sera and tested whether their levels are associated with diagnosis or treatment of lymphomas. We studied patients with primary central nervous system lymphomas (PCNSL) (N=3) and systemic lymphomas either with CNS dissemination (N=3) or without (N=11). Histological type was predominantly diffuse large B cell lymphoma - DLBCL (N=13). Sera of a control group were also used. 200 uL of CSF and sera (both cell free) were used for total RNA isolation followed by PCR and the data were adjusted to levels of control microRNAs (miR-let-7a or miR-24). Our data demonstrate that the microRNAs of the miR-17-92 cluster (miR-19a, miR-20a, miR-92a) and miR-106b-25 cluster (miR-106b, miR-25) and miR-155 are detectable (unlike miR-106a, miR-18a) in both the CSF and sera. Particularly, miR-17-92 and miR-106b-25 are increased in CSF of the PCNSL and the systemic lymphomas with CNS involvement compared with systemic lymphomas without CNS involvement. Conversely, in the sera, expression of miR-17-92 is increased in systemic lymphomas compared to PCNSL and the control group. Next, we investigated the levels of these microRNAs in a patient with systemic DLBCL with CNS dissemination during the treatment (R-CHOP alternating with RMPV) resulting in complete remission. Analysis of the CSF and also of the sera (at 9 different time points within three months) revealed a gradual decrease (∼6 fold) of the levels of miR-19a, miR-20a, miR-92a, miR-106b, and miR-25. However, the levels of these microRNAs in the sera at the stage of complete remission were still 2 fold higher compared to controls. We conclude that measurement of microRNAs in both CSF and sera represents novel sensitive tool, additional to other approaches, for tumor detection and/or for estimation of the therapy efficacy. (Grants: NT10310-3/2009, MPO FR-TI2/509, NPVII 2B06077, MSM 0021620806, LC 06044, SVV-2011-262507)

Disclosures:

No relevant conflicts of interest to declare.