bluuebunny/crossref_metadata_2025_split huggingface dataset:
GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.
Authors: Spaunhorst Britnie , Weiner George J 
Published: October 2019

Abstract
Abstract 2707
Poster Board II-683

Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC.
GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present.
Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors.
We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC.

Disclosures:
No relevant conflicts of interest to declare.
Induction of Noxa Via Generation of Reactive Oxygen Species Plays a Key Role in Induction of CLL Cell Apoptosis by Two Novel p53-Independent Cytotoxic Agents.
Authors: Steele Andrew , Prentice Archibald G , Chanalaris Anastasios , Hoffbrand A. Victor , Cwynarski Kate , Wickremasinghe Ranmohan 
Published: October 2019

Abstract
Abstract 2752
Poster Board II-728
Approximately 15% of CLL patients present with a deletion of chromosome 17p, resulting in the loss of the p53 gene. The percentage of 17p-deleted patients can increase to between 30–50% following treatment with cytotoxic agents which induce CLL apoptosis via upregulation of p53. Therefore, identification of novel agents which kill CLL cells independently of p53 is of crucial importance. We have recently shown that two chemically unrelated agents, 2-phenylacetylenesulfonamide (PAS) and the sesquiterpene lactone LC1 kill CLL cells regardless of the functional status of p53. Furthermore, in contrast to the conventional drugs chlorambucil and fludarabine, PAS and LC1 induce apoptosis in the absence of p53 elevation. However, the mechanisms of action by which these agents induce p53-independent apoptosis are unclear. We have previously shown that both PAS and LC1 initiated CLL cell apoptosis within 6-10h with, maximal killing by 48h. Here we show that treatment of CLL cells with either agent results in the generation of reactive oxygen species (ROS), the activating phosphorylation of the pro-apoptotic MAP kinase family member JNK (Fig 1), resulting in turn in upregulation of its downstream target, the transcription factor c-JUN (Fig 2). The BH3-only pro-apoptotic protein Noxa was originally described as a pro-apoptotic target for upregulation by p53. However, both PAS (Figs1 and 2) and LC1 upregulated Noxa in a p53-independent manner. Addition of N-acetylcysteine (NAC), a free radical scavenger, decreased ROS generation by PAS or LC1 and also prevented phosphorylation of JNK and Noxa upregulation (Fig 1) and also the upregulation of c-JUN. NAC also strikingly abrogated apoptosis induction by either agent, as shown by quantitation of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP; Fig 1). Taken together, the data suggest both PAS and LC1 induce p53-independent apoptosis via upregulation of ROS and the subsequent induction of Noxa. The data are also compatible with a role for JNK and c-JUN in the events leading to Noxa upregulation.

Disclosures:
No relevant conflicts of interest to declare.
KIT Inhibition Affects Heat Shock Protein (HSP) Activity: A Potential Rescue Mechanism towards KIT Tyrosine Kinase Inhibition and a Rationale for Dual KIT/HSP Inhibition.
Authors: Kampa Kerstin M , Grandl Tanja , Mueller Sandra , Normant Emmanuel , Walter Michael , Schuetz Wolfgang , Kanz Lothar , Heinrich Michael C , Schittenhelm Marcus M 
Published: October 2019

Abstract
Abstract 2753
Poster Board II-729
Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition.
To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI.
Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone.
In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy.

Disclosures:
Normant: Infinity: Employment.
Consequences of 4-1BB-4-1BB Ligand Interaction for NK Cell Anti-Tumor Immunity: Opposite Effects of Seemingly Analogue Molecules in Mice and Men.
Authors: Baessler Tina , Charton Jean E , Buechele Corina , Schmiedel Benjamin J , Gruenebach Frank , Krusch Matthias , Wacker Alexander , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abstract 279
In mouse models, stimulation of the TNF receptor family member 4-1BB/CD137 on cytotoxic lymphocytes by agonistic antibodies potently stimulated anti-tumor immunity, which has led to the development of humanized agonistic 4-1BB mAb that are presently being evaluated in clinical phase I/II studies (www.clinicaltrials.gov, keyword: CD137). However, seemingly analogue immunoregulatory molecules may mediate different effects in mice and men, and in humans yet nothing is known regarding the role of 4-1BB in NK cell reactivity. NK cells play an important role in tumor immunosurveillance, and largely contribute to the success of therapeutic strategies like (haploidentical) stem cell transplantation (SCT) or application of monoclonal antibodies like Rituximab by mediating graft versus leukemia activity and performing antibody-dependent cellular cytotoxicity (ADCC). Here we analyzed the expression of 4-1BB in human NK cells and compared the effect of 4-1BB signaling on effector functions of NK cells in mice and men. While being absent in resting state, both human and mouse NK cells acquire expression of 4-1BB at similar levels with comparable kinetics upon activation. Next we cultured human and mouse NK cells with target cells transfected with human and mouse 4-1BB ligand (4-1BBL) and analyzed the effect of 4-1BB on NK cell effector functions. 4-1BB – 4-1BBL interaction enhanced effector functions of mouse NK cells, while degranulation, cytotoxicity and cytokine production of human NK cells were substantially impaired (all p<0.01, Student's T-test). In line, employing primary leukemia cells from patients with AML (n= 65) and CLL (n=49), which were found to express 4-1BBL in 35% and 100%, respectively, we found that blocking 4-1BB-4-1BBL interaction markedly enhanced granule mobilization, cytotoxicity and interferon-g production of allogenic and autologous human NK cells in response to leukemia cells. In CLL, this inhibitory effect of 4-1BB-4-1BBL interaction on NK cell reactivity was observed both with regard to direct and Rituximab-induced cytotoxicity and cytokine production. Together, our data implicate that one should exercise caution in applying anti-4-1BB therapeutically, underline the necessity of detailed analyses regarding the function of seemingly analogue immunoregulatory molecules in mice compared to men and demonstrate that blocking 4-1BBL-4-1BB interaction may serve to enhance NK cell reactivity in therapeutic settings like allogenic SCT and antibody-treatment of malignancies in humans.

Disclosures:
No relevant conflicts of interest to declare.
Nuclear Expression of the Sox 11 Transcription Factor in Mantle Cell Lymphoma, and Cytoplasmic Expression in Follicular Lymphoma and Multiple Myeloma: Pathogenetic Implications.
Authors: Williams Michael E , Rutherford Sarah , Tang Yunjia , Cousar John 
Published: October 2019

Abstract
Abstract 2925
Poster Board II-901
Mantle cell lymphoma (MCL) is characterized by nuclear cyclin D1 expression resulting from the t(11;14)(q13;q32). As cyclin D1 overexpression alone is insufficient for B-cell transformation, we have investigated other potentially contributing mutations. Sox11 is a member of a large family of transcription factors containing a DNA-binding high-mobility group (HMG) domain and shares homology with Sox4, which is involved in lymphopoiesis. Recent reports have identified Sox11 expression in the majority of MCL, suggesting it may contribute to pathogenesis (Ek et al, Blood 2008;111:800; Wang et al, Br J Haematol 2008;143:248).

Methods:
Patients with MCL diagnosed at the Univ. of Virginia from 1997-2008 and for whom nodal, marrow or spleen tissue blocks were available were identified from the Pathology database. Follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL) and multiple myeloma (MM) were also analyzed. Formalin-fixed, paraffin-embedded samples were stained by immunohistochemistry for cyclinD1, Sox11 (rabbit a/Sox11, 1:100, Sigma) and Ki-67. Nuclear and/or cytoplasmic expression was determined by two of us (YT, JC).

Results:
All MCL samples revealed nuclear cyclin D1 expression. Nuclear but not cytoplasmic Sox11 expression was identified in all 24 non-blastoid and in 5/6 blastoid samples; 1 blastoid sample showed only cytoplasmic Sox11 staining (Table). Four of 5 HCL expressed cyclin D1 but were negative for Sox11; 4/5 MM also were cyclin D1-positive, with 3 positive for cytoplasmic Sox11 including 2 of the cyclin D1-positive cases. All 5 FL showed cytoplasmic Sox11 positivity, whereas all MZL and SLL were negative for cytoplasmic or nuclear staining.

Conclusions:
Nuclear Sox11 expression was uniformly identified in MCL, with the exception of cytoplasmic expression in a single blastoid case. No nuclear Sox11 was present in HCL or MM despite the expression of cyclin D1, although 3 MM showed cytoplasmic Sox11 staining. These findings suggest that nuclear Sox11 overexpression is not a direct result of dysregulated cyclin D1 signaling but instead occurs by alternative mechanisms. The significance of cytoplasmic staining remains uncertain. A potential pathogenetic role for Sox11 and possibly other Sox family transcription factors warrants further investigation in MCL and other lymphoproliferative neoplasms for diagnostic use and therapeutic targeting.

Disclosures:
No relevant conflicts of interest to declare.
Incidence and Management of Isolated Subsegmental Pulmonary Embolism.
Authors: Shen Yi-Hao , Wells Phil , Dennie Carole , Carrier Marc 
Published: October 2019

Abstract
Abstract 2989
Poster Board II-965

Introduction:
The use of computed tomographic pulmonary angiography (CTPA) in patients with suspected pulmonary embolism (PE) has improved the visualization of subsegmental pulmonary arteries. However, the clinical significance of subsegmental PE is unclear. In the PIOPED Study, PE limited to subsegmental pulmonary arteries was most prevalent among patients with low-probability ventilation/perfusion (V/Q) scans. Patients with non-diagnostic (low or intermediate) V/Q scans can be safely managed without anticoagulation. The incidence and clinical management of subsegmental PE remains uncertain.

Objective:
To evaluate the incidence and clinical management of subsegmental PE in patients with suspected acute PE.

Methods:
This is a retrospective cohort study of consecutive patients with suspected acute PE undergoing CTPA at the Ottawa Hospital from Jan 1, 2007 to Dec 31, 2008. Subsegmental PE was defined as one or more pulmonary artery filling defects located in the subsegmental level, with no filling defects visualized at more proximal pulmonary artery levels. All patients were followed for a minimum of 6 months after the index PE.

Results:
A total of 78 (10.4%, 95% CI: 8.4 to 12.8%) cases of subsegmental PE were identified out of 748 cases of PE diagnosed by CTPA. Forty-three (77%) of these had a single isolated subsegmental filling defect. Data could be extracted in 56 (72%) of the 78 cases. Among these 56 patients, 18 (32%) had unprovoked PE. Further investigations were performed in 50 patients (Ultrasonography (U/S) of legs (n=38), U/S and V/Q scan (n=11) or V/Q scan alone (n=1)). Deep vein thrombosis was detected in 12 patients and two patients had a high probability V/Q scan. Forty-eight (86%) patients were anticoagulated. All patients with concurrent DVT or high probability V/Q scan were started on anticoagulation therapy. Two patients experienced a major bleeding episode after anticoagulation. Out of the 8 patients left untreated, there were no recurrent VTE.

Conclusion:
Isolated subsegmental PE represents approximately 10% of all acute PE diagnosed by CTPA. Patients with subsegmental PE diagnosed on CTPA are more commonly receiving anticoagulation than not. Further studies are needed to establish the risk benefit ratio of anticoagulation therapy in patients with subsegmental PE diagnosed on CTPA.

Disclosures:
No relevant conflicts of interest to declare.
Revised Model for Platelet Adhesion to Collagen.
Authors: Stefanini Lucia , Stolla Moritz , Maloney Sean F , Ouellette Timothy Daniel , Roden Claire , Diamond Scott L. , Bergmeier Wolfgang 
Published: October 2019

Abstract
Abstract 2999
Poster Board II-968
The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.
Authors: Wang Zhicheng , Li Suping , Liu Guanglei , Shi Quanwei , Yan Rong , Ruan Changgeng , Dai Kesheng 
Published: October 2019

Abstract
Abstract 3003
Poster Board II-980
Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis.

Disclosures:
No relevant conflicts of interest to declare.
Sialic Acid Deficiency Impairs GPVI-Mediated Platelet Activation in ST3Gal-IV−/− Mice.
Authors: Rumjantseva Viktoria , Sørensen Anne Louise , Hoffmeister Karin M , Falet Hervé 
Published: October 2019

Abstract
Abstract 3005
Poster Board II-972
Lack of terminal sialic acid residues on platelet surface glycoproteins results in rapid platelet clearance. Using null mice for the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice), we have recently identified galactose residues on the N-terminus of the platelet Von Willebrand Factor receptor GPIbαa as a major counter receptor for the lectin domain of the asialoglycoprotein receptor on both hepatocytes and liver Kupffer cells (Sørensen et al., Blood 2009). ST3Gal-IV−/− mice have increased tail bleeding time. However, the role of terminal sialic acid residues on platelet activation is unclear. We investigated here whether loss of sialylation affects platelet activation mediated through the collagen receptor GPVI or by thrombin. Platelets were isolated from ST3Gal-IV−/− and ST3Gal-IV+/+ mouse littermates, stimulated with collagen-related peptide (CRP), convulxin (CVX), or thrombin, and platelet activation was evaluated by flow cytometry using P-selectin expression, as a marker for αa-granule secretion, and fibrinogen binding, as a marker for integrin αaIIbβ3 activation. Stimulation of ST3Gal-IV−/− platelets with CRP and CVX revealed a profound activation defect, compared to ST3Gal-IV+/+ platelets. The defect was not due to loss of surface receptor expression since ST3Gal-IV−/− and ST3Gal-IV+/+ platelets had comparable GPVI expression, as evidenced by flow cytometry. By contrast, activation of ST3Gal-IV−/− platelets with thrombin was normal. The data show that terminal sialic acid residues on GPVI are required for maximal platelet activation by CRP and CVX.

Disclosures:
No relevant conflicts of interest to declare.