bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Unexpected Role For αIIbβ3 In Immune Complex Disorders
Authors: Zhi Huiying , Wu Nannan , Dai Jing , Fang Juan , Liu Pu , Liu Junling , Hu Hu , Zhu Jieqing , Newman Debra K. , Newman Peter J. , Gao Cunji 
Published: October 2019

Abstract
IgG immune complexes contribute to the etiology and pathogenesis of a number of autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus (SLE), rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Human platelets express on their surface an Fc receptor, termed FcγRIIa or CD32a, that when exposed to immune complexes initiates a potent signal transduction cascade that results in platelet activation and granule secretion. Patients with immune complex-related disorders are known to be highly susceptible to thrombotic events, and microthrombi have been observed to colocalize histologically in patients with immune complex disorders. To better understand the contribution of platelet adhesion receptors and signaling molecules to IgG immune complex-mediated thrombotic complications, we incubated platelets in microtiter wells that had been precoated with immobilized IgG. Platelets quickly formed filopodia, and then adopted a fully-spread morphology over a 30 minute period of time. Cytosolic proteins known to be involved in platelet spreading, including FcγRIIa, Src, Syk, and pp125FAK also became rapidly tyrosine phosphorylated. Because the integrin αIIbβ3 employs each of these signaling molecules in platelet spreading on immobilized fibrinogen, we next evaluated its role in platelet spreading on immobilized IgG. Interestingly, Fibans - small molecule antagonists of αIIbβ3-fibrinogen interactions – also blocked (1) platelet spreading on immobilized IgG, (2) the associated phosphorylation events, and (3) platelet thrombus formation over immobilized IgG under conditions of flow. Human platelets from Glanzmann thrombasthenic individuals, or murine integrin β3-deficient platelets expressing a human FcγRIIa transgene, also failed to spread on immobilized IgG and form thrombi on immobilized IgG under conditions of flow. FcγRIIa-transgenic mice lacking the Src-family kinase Lyn – thought to be responsible for phosphorylating the ITAM tyrosines of FcγRIIa – also failed to spread or form thrombi over immobilized IgG. Finally, Chinese hamster ovary cells transfected with αIIbβ3 and FcγRIIa failed to spread on immobilized IgG unless small amounts of fibrinogen were added to the IgG preparation before plating. Taken together, our data suggest a complex functional interplay between FcγRIIa and αIIbβ3 in immune complex-mediated thrombotic disorders in which platelets encounter immobilized IgG and become activated to secrete α-granule fibrinogen. Secreted fibrinogen, in turn, then becomes a substrate for αIIbβ3-mediated platelet spreading and subsequent thrombus formation.

Disclosures:
No relevant conflicts of interest to declare.
Role Of B Cell Tolerance In PF4/Heparin Antibody Production
Authors: Zheng Yongwei , Wang Alexander W , Yu Mei , Padmanabhan Anand , Tourdot Benjamin E , Newman Debra K. , White Gilbert C. , Aster Richard H. , Wen Renren , Wang Demin 
Published: October 2019

Abstract
Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis.

Disclosures:
White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.
EVI1, A Potential Regulator Of CEBPA; A Contributory Mechanism In Leukemogenesis?
Authors: Wilson Michael , Zhang Yi , Cui Xiaohui , Wuertzer Charles A. , Perkins Archibald S. 
Published: October 2019

Abstract
Ecotropic Viral Integration Site 1 (EVI1) is a zinc finger oncoprotein implicated in a subtype of acute myeloid leukemia associated with poor prognosis. Leukemic cells overexpressing EVI1 display a block in myeloid differentiation and resistance to apoptosis, both of which are reversed with EVI1 shRNA knockdown. Previous mutagenesis studies have shown that DNA binding via the first set of zinc fingers (ZF1) is critical for leukemic transformation by EVI1. The mediators between EVI1 DNA-binding and leukemogenesis are mostly unknown. Using chromatin immunoprecipitation and sequencing (ChIP-Seq) in myeloid leukemic cells, we have recently identified an evolutionarily conserved EVI1 binding site 35 kilobases downstream of Cebpa, encoding the CCAAT enhancer-binding protein alpha, a master regulator of early granulopoiesis. In order to obtain a better understanding of the potential regulatory role of this region in leukemogenesis, we used genome database analysis to see what other transcription factors or epigenetic marks are present. ChIP-Seq data deposited in the UCSC databank indicates the presence of CTCF, GATA2 and CEBPβ, as well as TAL1, p300, and GATA1 in leukemic cells, the latter three of which are known to bind together in a complex. This data suggests that the Cebpa +35 region serves an important role in regulating hematopoietic development and Cebpa transcription. In order to characterize the binding of EVI1 ZF1 to the Cebpa +35 site, we performed methylation interference assays and oligonucleotide mutagenesis assays. EVI1 ZF1 binds within this +35kb control region to the sequence TGACAGTGACGG with 133% affinity relative to the canonical motif. In an effort to assess any biological effects linked to EVI1 binding at the Cebpa +35 motif, we conducted EVI1 overexpression studies in the EML hematopoietic progenitor cell line. While control EML cells exhibited marked upregulation of Cebpa upon induction, EVI1-transduced cells displayed a significant suppression of Cebpa transcript. To determine if this suppression requires DNA binding, we tested a mutant of EVI1 that is defective in DNA binding (EVI1R205N); this was unable to suppress Cebpa induction. To test whether EVI1 can bind to the +35 KB site, we performed ChIP-PCR, which indeed showed EVI1 occupancy at the +35 KB site. In conclusion, we have shown evidence that EVI1 suppresses Cebpa transcription in early hematopoietic progenitor cells, and that the EVI1 binding site at Cebpa +35 serves an important function in myeloid maturation. This regulatory node represents a possible therapeutic target for the treatment of acute myeloid leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Pre-Clinical Development Of a Novel Chimerical Antigen Receptor Targeting High-Risk Pediatric ALL Over-Expressing Tslpr
Authors: Qin Haying , Monica Cho , Haso Waleed , Zhang Ling , Barrett David M. , Orentas Rimas J , Grupp Stephan A. , Fry Terry J 
Published: October 2019

Abstract

Background
While current upfront therapy will cure over 80% of patients, B-cell precursor acute lymphoblastic leukemia ALL (BCP-ALL) remains a leading cause of cancer-related death in children, and outcomes for those patients who relapse have not been changed substantially over recent decades. Immunotherapy with a chimeric antigen receptor (CAR) targeting CD19 has resulted in impressive clinical responses, but alternative CAR targets are needed as not all patients respond and CD19 negative relapses have been observed. Thymic stromal lymphoprotein receptor (TSLPR) is coded by the CRLF2 gene. CRLF2 genetic alterations occur in 5-16% of pediatric and adult BCP-ALL and result in TSLPR over-expression that is associated with poor prognosis. Furthermore, emerging data suggests that TSLPR overexpression is functionally important for leukemic blasts. Thus, TSLPR is an ideal immunotherapeutic target. We constructed TSLPR-specific CARs and tested their therapeutic efficacy in vitro and in vivo.

Methods
Hybridomas producing TSLPR-specific antibodies were used to clone variable regions of the heavy and light chains for constructing TSLPR-specific scFv. The scFv was linked with the CD8α hinge and transmembrane domain, 4-1BB intracellular domain and CD3ζ chain, and then cloned into a 3rd generation lentiviral vector. For optimization of the CAR configuration, an IgG heavy chain constant domain (CH2CH3) spacer was fused to scFv to generate a “long” TSLPR CAR. Human T cells were transduced with CAR vectors and the transduced cells were evaluated in vitro and in vivo against TSLPR-overexpressing ALL, including a TSLPR-transduced REH cell line (REH-TSLPR) and patient-derived xenografts naturally overexpressing TSLPR.

Results
Both long and short TSLPR CARs could be efficiently transduced (approximately 60-80%) into human T cells using a lentiviral system. T cells expressing both long and short TSLPR CARs produced high levels of cytolytic cytokines upon co-culture with of TSLPR-overexpressing ALL. However, only the short TSLPR CAR demonstrated effective killing against TSLPR-expression ALL cells in vitro. Short TSLPR-expressing T cells completely eradicated leukemia in NSG xenografts mice bearing REH-TSLPR (figure 1) as well as TSLPR-expressing patient xenografts. The long TSLPR CAR did not show any activity in vivo.

Conclusion
TSLPR-targeted CAR T cells is promising therapeutic approach in high risk BCP-ALL and represents the first example of an oncogene-specific cellular therapy in BCP-ALL.

Disclosures:
Grupp: Novatis: Research Funding.
Preclinical Testing Of a PI3K Inhibitor In T Lineage Leukemia: Target Validation and Notch1/Myc Down-Regulation In Drug Resistant Clones
Authors: Dail Monique , Wong Jason , Lawrence Jessica , O'Connor Daniel , Nakitandwe Joy , Chen Shann-Ching , Akagi Keiko , Lee Leslie , Aster Jon C. , Pear Warren S. , Sampath Deepak , Downing James R. , Shannon Kevin 
Published: October 2019

Abstract
Mutations that deregulate Notch1 and Ras/PI3K/Akt signaling are common in T-ALL and often coexist. Thus, inhibiting these pathways alone and in combination has been advocated as a rational therapeutic strategy. GDC-0941 is a pan-PI3K inhibitor that is advancing in clinical development that we evaluated for efficacy and mechanisms of drug resistance in T-ALL cell lines and primary murine T-ALLs. Cell lines are uniformly sensitive to GDC-0941; however gradual dose escalation results in resistant lines. By contrast, primary leukemia cells display variable in vivo sensitivity to treatment with GDC-0941 +/- a MEK inhibitor. Importantly, mice invariably relapse with drug resistant clones. A majority of GDC-0941-resistant T-ALL cell lines and primary leukemias unexpectedly showed reduced levels of activated Notch1 protein, down-regulate many Notch1 target genes including Myc, and display cross-resistance to gamma secretase inhibitors (GSIs). In multiple cases, Notch1 mutations that were present in drug-sensitive parental leukemias were absent at relapse. Importantly, resistant clones that emerge both in vitro and in vivo up-regulate PI3K signaling indicating an “on pathway” mechanism of resistance. Consistent with these data, inhibition of Notch1 signaling promotes GDC-0941 resistance and enhances PI3K signaling, whereas expression of activated Notch1 increases the GDC-0941 sensitivity of mouse and human T-ALL cell lines. Thus, oncogenic Notch1 mutations that promote clonal outgrowth during malignant transformation unexpectedly undergo negative selection during treatment with GDC-0941. Together, these in vivo studies: (1) validate PI3K as an important therapeutic target in T-ALL; (2) demonstrate that active Notch1 and elevated Myc expression are dispensable for T-ALL growth in vivo; and, (3) indicate that therapeutic PI3K inhibition selects for the outgrowth of leukemia cells with reduced Notch1 signaling. Importantly, our data also raise the possibility that simultaneously administering Notch1 and PI3K inhibitors will accelerate drug resistance in T-ALL, and support alternative approaches for deploying combination regimens against these dominant oncogenic “driver” pathways.

Disclosures:
Lee: Genentech: Employment. Aster:Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria. Sampath:Genentech: Employment, Equity Ownership.
Vgamma9Vdelta2 T Cells-Based Immunotherapy Targeting BTN3 Molecules in Acute Myeloid Leukemia
Authors: Olive Daniel , Benyamine Audrey , Le Roy Aude , Castellano Rémy , Gertner-Dardenne Julie , Vey Norbert 
Published: October 2019

Abstract
As they can kill Acute Myeloid Leukemia (AML) blasts in vitro and in vivo, Vg9Vd2T cells are key players in the design of new strategies of immunotherapy. AminoBisphonates (NBP) can enhance their activation in vitro and in vivo. Their combination with low-dose IL2 has shown promising results in 2 patients with AML who underwent partial remission. NBP treatment of blasts inhibits the Mevalonate pathway. The subsequent accumulation of Isopentenyl Diphosphate sensitize AML blasts to Vg9Vd2T cells killing but some AML cell lines blasts are resistant to this TCR mediated-lysis. Butyrophilin 3 A1 (BTN3A1) has been shown to be involved in IPP recognition and Vg9Vd2 T cells activation. Agonist monoclonal antibodies (mAb) recognizing the 3 isoforms of BTN3, can trigger BTN3 on tumor cell lines and sensitize them to Vg9Vd2 T cells lysis.
We show that primary AML blasts from patient at diagnosis are heterogeneously killed by allogenic-IL-2-NBP-expanded Vg9Vd2 T. Some are resistant to this lysis and/or poorly sensitized by NBP. BTN3 molecules are highly expressed by blasts of AML cell lines and primary AML samples. We show that treatment of primary AML blasts with agonist anti-BTN3 mAb can overcome the resistance to Vg9Vd2 cells lysis in vitro. We assess this effect in vivo, showing that the addition of agonist anti-BTN3 mAb to Vg9Vd2 cells infusion decreased the tumor burden and increased the survival of NOG mice xenografted with luciferase-transduced U937 cell line. We confirm this effect in a model of mice xenografted with primary AML blasts, showing that treatment with anti-BTN3 mAb added to Vg9Vd2 cells infusion can decrease the number of blastic cells in the spleen, bone marrow and the blood, without requiring additional cytokine infusion.
This drastic effect on sensitization of primary AML blasts to Vg9Vd2T cells killing could be of great interest especially in cases of refractory or relapsing AML.

Disclosures
No relevant conflicts of interest to declare.
a Randomized, Controlled, Prospective Study for Efficacy of Micafungin Versus Itraconazole As Empirical Antifungal Therapy for Febrile Neutropenic Patients with Acute Leukemia
Authors: Jeong Seong Hyun , Kim Dae Young , Jang Jun Ho , Mun Yeung-Chul , Kim Sung-Hyun , Choi Chul Won , Kim Jin Seok , Park Joon Seong 
Published: October 2019

Abstract
Background: Micafungin, one of the powerful ecchinocandins, needs to be clarified its efficacy and safety of empirical therapy on febrile neutropenic patients compared with other anti-fungal drugs such as azole compounds.
Methods: A randomized, multi-center, prospective study (age-, sex-, diagnosis-, risk-controlled) was conducted for a comparison of efficacy and safety between micafungin and intravenous itraconazole as an empirical therapy on febrile neutropenic patients with acute leukemia. Anti-fungal drugs (study drug, micafungin; control drug, itraconazole) were administrated for sustained high fever following appropriate empirical antibiotic therapy (°Ã 3 days of combination antibiotics). The overall success rate [if the patient meets all of the following criteria: (1) no breakthrough fungal infection; (2) survived for 7 days past the end of the therapy; (3) no premature discontinuation; (4) disappearance of fever during neutropenia; (5) successfully treated base-line fungal infection] and side effects were evaluated. And duration of fever and neutropenic fever were estimated and compared between two groups.
Results: A total of 134 patients with acute leukemia were enrolled in the study (86 patients with AML, 41 patients with ALL, 7 patients with secondary leukemia). The overall success rate of micafungin and itraconazole was 63.6% and 53.2%, respectively (p = NS) (Figure 2). However, duration of fever and duration of neutropenic fever were significantly shorter in micafungin group (p = 0.0007, p = 0.0118, respectively) and survival benefit was marginal (OS, median months, 10.93 vs. 4.77, p = NS) (Figure 1). Grade III-IV adverse events including hyperbilirubinemia (2 vs. 7), elevation of transaminase level (2 vs. 4), electrolytes imbalance (1 vs. 2), atrial fibrillation (1 vs. 0), and anaphylaxis (1 vs. 0) occurred at 7 and 13 patients in micafungin (10.4%) and itraconazole (18.8%) group, respectively. All adverse events were manageable however, 4 patients died of progression of infection in each group.
Conclusion: This randomized, controlled, prospective study documented that micafungin, when compared with itraconazole, showed significantly superior effect on shortening the duration of fever and neutropenic fever, resulting in marginal survival benefit on acute leukemia patients who are suffering from life-threatening neutropenic period under anti-leukemic therapy.

Disclosures
No relevant conflicts of interest to declare.
Preclinical Efficacy of MEK Inhibition in Nras Mutant Acute Myeloid Leukemia
Authors: Burgess Michael R. , Hwang Eugene , Firestone Ari J , Huang Tannie , Xu Jin , Zuber Johannes , Bohin Natacha , Wen Tiffany , Kogan Scott C. , Haigis Kevin M. , Lowe Scott W. , Shannon Kevin , Li Qing 
Published: October 2019

Abstract
Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations.

Disclosures
No relevant conflicts of interest to declare.