bluuebunny/crossref_metadata_2025 huggingface dataset:
Patients with Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia (ALL) Have High Levels of Minimal Residual Disease (MRD) at the End of induction—A Children's Oncology Group (COG) Study.
Authors: Wood Brent , Winter Stuart , Dunsmore Kimberly , Raetz Elizabeth , Borowitz Michael J. , Devidas Meenakshi , Winick Naomi J. , Carroll William L. , Hunger Stephen P , Loh Mignon L.
Published: October 2019

Abstract 9

Current risk stratification algorithms in children with B-lineage ALL have facilitated the identification of subgroups with adverse outcomes that benefit from intensified therapy. However, many of the features that are highly prognostic of outcome in B-precursor ALL, such as the combination of age and white blood cell count (WBC) at diagnosis, have limited ability to predict outcome in T-ALL. In contrast, early response to induction therapy, as measured by bone marrow (BM) morphology at days 8, 15 or 29, and day 29 (end induction) BM MRD are highly predictive of outcome in both B-precursor and T-ALL. Coustan-Smith and colleagues (Lancet Oncology 10:147-156 2009) have recently identified a subset of children with ETP ALL that have an extremely high risk of induction failure or relapse. The ETP ALL blasts exhibit stem cell-like features as defined by the presence of antigens seen on early progenitors including CD117, CD34, HLA-DR and/or CD13/33, the absence of antigens seen on later stage immature T cells including CD1a and CD8, and decreased to absent expression of the early T cell antigen CD5.

We analyzed patients enrolled in current generation COG ALL trials, including patients with B-precursor ALL treated on AALL0232 (high risk, n=2129) or AALL0331 (standard risk, n=3747), and 416 patients with T-ALL enrolled on AALL03B1. Clinical features including age, WBC, gender, and CNS status were compared between ETP-positive (n=25) and –negative (n=391) T-ALL patients. We also compared response to four weeks of induction therapy (prednisone, vincristine, PEG-asparaginase, and daunorubicin) as measured by day 15 and 29 BM morphology and day 29 BM MRD determined by flow cytometry. Twenty-five of 416 (6%) T-ALL patients had an ETP phenotype. The ETP-positive patients were older and presented with a lower white blood cell count than the ETP-negative patients (see Table). There was no difference in the gender distribution or the presence of CNS disease at diagnosis between the two groups. Strikingly, the ETP-positive patients had a dramatically inferior early response to therapy with 100% having ≥ 0.01% day 29 MRD (vs. 46% of ETP-negative T-ALL and 23.4% of precursor B-ALL) and 74% having ≥ 1% day 29 MRD (vs. 21% of ETP-negative T-ALL and 5% of precursor B-ALL). These results confirm the poor response of ETP T-ALL and suggest that novel treatment strategies are warranted in this patient subgroup.

Disclosures:

No relevant conflicts of interest to declare.
Induction with Velcade®/Dexamethasone Partially Overcomes the Poor Prognosis of t(4;14), but Not That of Del(17p), in Young Patients with Multiple Myeloma.
Authors: Avet Loiseau Hervé , Moreau Philippe , Mathiot Claire , Charbonnel Catherine , Caillot Denis , Facon Thierry , Attal Michel , Benboubker Lotfi , Hulin Cyrille , Marit Gerald , Minvielle Stéphane , Harousseau Jean-Luc
Published: October 2019

Abstract 957

Translocation t(4;14)(p16;q32) has been associated with a poor outcome in multiple myeloma. This poor prognosis has been identified both in patients treated with melphalan-prednisone (MP) and in those treated with high-dose melphalan after a VAD induction. For instance, in 100 patients with t(4;14) treated with VAD and MEL200, the median PFS and OS were 21 months and 41 months, respectively, as compared to 37 months and 65 months for patients lacking the t(4;14) (Moreau et al., Leukemia 2007). Some preliminary studies have suggested that bortezomib (Velcade®) was able to overcome the poor prognosis of the translocation in elderly patients treated with MP-Velcade® (San Miguel et al., NEJM 2008). In order to address this important question, we analyzed 436 patients treated in the IFM, according to the IFM-2005-01 trial, arm B: induction with 4 cycles of Velcade®/Dexamethasone (VD), followed by one or two courses of high-dose melphalan (MEL200). A translocation t(4;14) was observed in 67 of these 436 patients treated with VD (15%), whereas del(17p) was found in 51 patients (11%). Of note, 10 patients presented both the t(4;14) and the del(17p). The median PFS was 25 and 36 months, in patients with or without the t(4;14), respectively (p=0.006). At 3 years, 76% of the patients with t(4;14) were still alive, as compared to 88% of the patients lacking the translocation (p=.003). For comparison, the OS results were respectively 62% (patients with t(4;14)) and 73% (patients lacking the translocation) in patients treated with a VAD induction. Thus, it seems that VD is able to partially overcome the poor prognosis of t(4;14). We also looked at the prognostic value of del(17p) in this series of patients treated with VD. In contrast to the t(4;14) situation, VD was enable to rescue patients with del(17p) (same PFS and OS for patients treated with VD than for those treated with a VAD induction). Thus, this study (by far the largest so far reported) shows that VD as induction before intensification is able to improve the prognosis of patients with t(4;14), but not of those with del(17p).

Disclosures:

Avet Loiseau: Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Moreau:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Facon:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Attal:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Harousseau:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees.
Snake Venom and Receptor Crosslinking.
Authors: Clemetson Kenneth J.
Published: October 2019

Abstract SCI-37

Nearly all poisonous snakes venoms contain proteins including snaclecs (snake C-type lectins) and metalloproteases, which influence haemostasis by interacting with coagulation factors or platelets. Like other venom constituents they weaken the prey and make it easier to swallow and digest. Although some of those that affect platelets inhibit receptors, particularly integrins, many activate platelets by clustering receptors. The main receptors involved are GPIb and GPVI and the snaclecs and metalloproteases activate platelets in the same way as their natural ligands, von Willebrand factor (VWF) and collagen. Both VWF and collagen are thought to activate platelets by clustering receptors acting in somewhat parallel ways. In both cases a mainly signalling receptor, GPIb or GPVI, respectively, is clustered to activate an integrin, aIIbb3 or a1b2, respectively. The first step can be simulated by agents that bind to and cross-link GPIb or GPVI. These include antibodies, synthetic peptides or snake venom proteins. Many of the activating snaclecs belong to the tetrameric heterodimer group such as flavocetin, convulxin and its homologues, and some use both receptors. There remain however many unanswered questions about the non-covalent structure of these molecules and how they interact with receptors on, possibly, more than one platelet to induce activation even at very low concentrations (pM). A second class of snaclecs, including aggretin (rhodocytin), which has CLEC-2 as receptor, and purpureotin that acts via GPIb, are non-covalent dimers of heterodimers but may reorganise to form higher non-covalent multimers. These non-covalent interactions are still poorly understood and may be responsible for the low activity of recombinant snaclecs. Several activating snaclecs are glycosylated. Although this may simply enhance solubility, interactions with prey molecules can not yet be excluded. Many simple heterodimeric snaclecs are superficially inhibitory of washed platelets but can be converted to activators by cross-linking, either naturally or artificially. Finally, a major problem in snake venom genomics is ascribing function, such as target molecules, to novel snaclecs identified by DNA sequence alone. Although modelling has made great progress there are still too many unknown factors.

Disclosures

No relevant conflicts of interest to declare.
Drug Inhibition of GPIb.
Authors: Deckmyn Hans , De Meyer Simon F. , Vanhoorelbeke Karen
Published: October 2019

Abstract SCI-38

The platelet-specific glycoprotein (GP)Ib, member of the GPIb/IX/V complex, is the main receptor for von Willebrand Factor (VWF), but also for a growing list of other ligands such as thrombin, coagulation factor XI and XII, Mac-1 and P-selectin. For VWF and GPIb to interact, VWF needs to undergo a conformational change. In the circulation, this is induced by shear forces that stretch VWF, especially when bound onto collagen at vascular lesions. As the contribution of VWF to platelet adhesion becomes more important with increasing shear forces, the idea emerged that inhibition of the GPIb-VWF interaction could actually result in an antithrombotic effect targeted to high shear arterioles or stenosed arteries, leaving hemostasis virtually unaffected in vessel beds with slower circulation. And in the mean time, a growing amount of evidence suggests that this could lead to a lower bleeding risk than what is seen with currently available antithrombotic agents. Several compounds have been tested in the past such as fragments of VWF, snake venoms and antibodies. We developed an anti-human GPIb monoclonal antibody, 6B4, that competes directly for the binding site of VWF. The humanized 6B4 Fab-fragment effectively prevented repetitive occlusions of the damaged and stenosed femoral artery in a baboon thrombosis model. Interestingly, only little prolongation of the template bleeding time and minimal increase in blood loss from a standardized incision was observed. This apparent larger therapeutic window of GPIb inhibitors could be particularly useful in ischemic stroke, where the utility of current platelet inhibitors and anticoagulants is significantly counterbalanced by the risk of intracerebral bleeding complications. Recent promising results in a mouse model of cerebral ischemia/reperfusion indeed show a significantly less severe stroke outcome using a GPIb-inhibitor, either administered before or, importantly, after cerebral artery occlusion. No increase in intracerebral hemorrhages was detected, in contrast to what is seen with e.g. GPIIb/IIIa inhibition. Finally, as antibodies may be useful for acute treatments but not for chronic applications, the identification of a GPIb-inhibiting nonamer peptide raises hopes towards development of orally available small molecule inhibitors.

Disclosures

No relevant conflicts of interest to declare.
TRALI Pathophysiology.
Authors: Stroncek David
Published: October 2019

Abstract SCI-48

Transfusion related acute lung injury (TRALI) is clinically defined as the new onset of acute lung injury within 6 hours of a transfusion. In TRALI a transfusion activates neutrophils leading to pulmonary leukostasis, endothelial damage, capillary leak and pulmonary edema. A number of elements (bioactive lipids, sCD40L, and leukocyte antibodies) found in blood products can active neutrophils and are risk factors for TRALI. Bioactive lipids and sCD40L accumulate in both stored red cell and platelet components. Leukocyte antibodies are most often found in blood components collected from women alloimmunized during pregnancy. A number of animal and in vitro models have shown that neutrophils activated by leukocyte antibodies cause pulmonary leukostatsis, endothelial damage and capillary leak. Far more blood components contain these TRALI factors than cause TRALI suggesting that additional patient, clinical, or blood component factors are required for the development of TRALI. For example, neutrophil-specific antibodies cause reactions in, at most, 25% of transfusion recipients. Animal models and in vitro studies have found that blood component factors are more likely to induce lung injury if the neutrophils and/or pulmonary endothelial cells are primed or activated. Endothelial cells can be primed by endotoxin and neutrophils can be primed by bioactive lipids and sCD40L. The priming of neutrophils and /or endothelium results in the tight adhesion of neutrophils to endothelial cells and enhances endothelial cell injury Between neutrophil and HLA Class I and II antibodies, neutrophil antibodies are most potent at initiating TRALI and HLA Class I antibodies are least potent. HLA Class I antigens are expressed by platelets, lymphocytes, monocytes and soluble HLA Class I antigens are present in plasma. These sources of Class I antigens likely compete with neutrophils for transfused Class I antibodies and may render them less effective at initiating TRALI than neutrophil-specific or HLA Class II antibodies. Some evidence suggests that HLA Class I antibodies induce TRALI by binding to pulmonary endothelium and activating neutrophils through their Fc portion. Class II antibodies may initiate TRALI by binding to monocytes and stimulating cytokine release. In summary, TRALI is the result of patient and blood component factors which lead to neutrophil activation and endothelial cell damage and capillary leak. Most cases likely require the confluence of multiple factors to form a “perfect inflammatory storm” which leads to significant lung injury.

Disclosures

No relevant conflicts of interest to declare.
Foxn1 and the Rise and Fall of the Thymic Microenvironment.
Authors: Manley Nancy R.
Published: October 2019

Abstract SCI-50

The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). There is abundant and growing evidence that the cellular and compartmental organization of the thymus is crucial for optimal organ function. Changes in the organization and stromal composition of the thymus as a result of genetic changes or during aging-associated involution can negatively impact both T cell production and function. The TEC-specific transcription factor Foxn1 is a critical regulator of these processes. I will present data from our Foxn1 allelic series showing that Foxn1 regulates TEC proliferation and differentiation at both fetal and adult stages, and that Foxn1-dependent TEC differentiation is required to assemble the multiple cell types that make up the complex structure of the functional thymus. Thus, Foxn1 is a central regulator throughout the life cycle of the thymus, and orchestrates both fetal organ formation and maintenance of the postnatal thymus. In vivo analysis of thymic function and stromal phenotypes is made difficult by the cellular complexity of the thymic microenvironment. I will also present data from a new quantitative, theoretical approach we are using to generate a “geographical map” of the cellular organization of the thymus. This approach will allow us to determine the spatial relationships of identifiable cell types, allowing us to generate new hypotheses about functional parameters of tissue organization at the cellular level. Thus, these results will provide a novel approach to generating quantitative and predictive models of the spatial and functional architecture of the thymic microenvironment.

Disclosures

No relevant conflicts of interest to declare.
Transcriptional Regulation of Erythropoiesis.
Authors: Weiss Mitchell J.
Published: October 2019

Abstract SCI-7

Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas.

Disclosures

No relevant conflicts of interest to declare.
The Reduced and Altered Activities of PAX5 Are Linked to the Protein-Protein Interaction Motif (coiled-coil domain) of the PAX5-PML Fusion Protein In t(9;15)-Associated Acute Lymphocytic Leukemia.
Authors: Qiu Jihui , Chu Huimin , Dong Shuo
Published: October 2019

Abstract 1022

The paired box domain of PAX5 was reported to fuse with the sequence of promylocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia (B-ALL). In the present studies, we found, by gel-shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction (RQ-PCR) analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil protein interaction domain located within PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared to wild-type PAX5. Furthermore, the dimerization domain (coiled-coil) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the coiled-coil domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins.

Disclosures:

No relevant conflicts of interest to declare.
Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.
Authors: Lehotzky Erica A. , Chiang Mark Y.
Published: October 2019

Abstract 1026

Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
SOCS1 Cooperates with FLT3-ITD In the Development of Myeloproliferative Disease by Promoting the Escape From External Cytokine Control.
Authors: Brandts Christian H. , Reddy Pavankumar , Sargin Bülent , Choudhary Chunaram , Muller-Tidow Carsten , Berdel Wolfgang E. , Serve Hubert
Published: October 2019

Abstract 1054

Activating mutations of FLT3 such as FLT3-ITD are frequently found in AML patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control, which regulates normal hematopoiesis. We have recently demonstrated that FLT3-ITD, when localized to the endoplasmic reticulum, aberrantly activates STAT5. Here we show that one of the target genes induced by STAT5 is SOCS1, a potent negative regulator of cytokine signaling and known tumor suppressor gene. Importantly, a significantly increased SOCS1 expression was found in FLT3-ITD+ AML. While SOCS1 expression in murine bone marrow severely impaired cytokineinduced colony growth, it failed to inhibit FLT3-ITD supported colony growth, indicating resistance of FLT3-ITD to SOCS1. Furthermore, SOCS1 co-expression inhibited interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-γ mediated growth inhibitory effects. In a murine bone marrow transplantation model, the co-expression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared to FLT3-ITD alone (p<0.01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting malignant transformation. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
Effectiveness of a Short Course of Complex Lymphedema Therapy or Graduated Compression Stocking Therapy In the Treatment of Post-Thrombotic Syndrome (PTS): A Randomized Control Trial.
Authors: Holmes Chris E. , Bambace Nadia M. , Lewis Patricia , Callas Peter , Cushman Mary
Published: October 2019

Abstract 1087

Background:

Investigations of treatment options for established PTS are limited. Graduated compression stocking therapy (GCS) is often used in clinical practice despite limited research on effectiveness. We postulated that the venous hypertension seen in PTS leads to lymphatic overload and secondary lymphedema. Therefore, in patients with established PTS, we compared the efficacy of GCS alone to complex lymphedema therapy, a treatment that includes compression stocking use, exercise, patient education, skin care and manual lymphatic drainage.

Methods:

In an investigator-blind randomized controlled trial of 31 patients with a clinical diagnosis of PTS and prior history of DVT, we compared GCS alone using 30–40 mm Hg compression to complex lymphedema therapy. Primary outcomes were the 1- and 3-month changes in PTS severity by the Villalta score (higher score indicates more severe PTS) and disease-specific quality of life by the VEINES QOL score (high score indicates better QOL). Analysis was by intent-to-treat.

Results:

Overall, 39% of patients enrolled in the study had moderate or severe PTS: baseline mean PTS severity score was 9.9 (SD 7.1) in the lymphedema group and 10.9 (SD 5.3) in the GCS group (p=0.66). The complex lymphedema therapy group had a reduction in mean PTS severity score of -2.4 (p=0.02) at 1 month and -2.3 (p=0.05) at 3 months follow-up. The GCS group had a reduction in PTS severity score of -2.1 (p=0.03) at 1 month and -3.3 (p=0.03) at 3 months. The improvement in PTS severity score did not differ significantly between treatment groups (p=0.61 at 3 months). Neither group had a significant change in VEINES QOL score. Patients not routinely using GCS prior to trial entry derived a greater benefit from either intervention with a decrease in PTS severity score (-8.8 in nonusers vs -1.5 in those using GCS at baseline, p=0.07) and an increase in VEINES QOL score (+1.9 vs -2.0, p=0.02). At 3 months, average self-reported GCS use was 6.7 days/wk (lymphedema therapy) and 5.9 days/wk (GCS) with 78% of patients in the lymphedema therapy group receiving manual lymphatic drainage (home or therapist). Four patients experienced recurrent thrombosis on the trial (3 in GCS group; 1 in lymphedema therapy group); 1 stocking allergy and ulcer formation was also seen in the GCS group with no cardiopulmonary complications reported in either group.

Conclusion:

In this first clinical trial of complex lymphedema therapy for patients with established PTS, a short course of lymphedema therapy was safe and had a similar effect on PTS severity as compression stocking use.

Disclosures:

No relevant conflicts of interest to declare.
Increased Platelet Binding of NN1731, a Factor VIIa Variant with Enhanced Tissue Factor-Independent Activity.
Authors: Hoffman Maureane R. , Persson Egon , Ezban Mirella , Monroe Dougald M.
Published: October 2019

Abstract 1133

Introduction:

Platelet surface activity plays an important role in the efficacy of FVIIa as a bypassing agent in hemophilia. An analog of FVIIa with increased tissue factor (TF) independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain: V158D, E296V, and M298Q. Modeling suggests that Gln in position 298 and Asp in position 158 form a hydrogen bond network with a water molecule that stabilizes the proteolytically active conformation of the molecule. The Val introduced at position 296 avoids electrostatic repulsion between a native Glu residue and the introduced Asp at 158. These changes result in a FVIIa variant that partially mimics the conformation of TF-bound FVIIa. However, in the presence of TF the activity of the wild type FVIIa and NN1731 are indistinguishable. Previous studies suggested that the increased intrinsic activity of NN1731 was not directly reflected in the level of activity on the platelet surface. The goal of the current work was to compare the platelet binding and platelet surface activity of NN1731 and wild type FVIIa.

Methods:

Mutant FVIIa molecules were prepared as described previously (PNAS 98:13583-8, 2001). We assessed binding to purified human platelets by flow cytometry and activity by measuring FXa generation.

Results:

FVIIa and NN1731 had identical binding to phospholipid vesicles designed to have lipid composition similar to platelets. However, the two exhibited different binding to platelets. At all concentrations, more NN1731 bound to thrombin or thrombin-plus-convulxin activated platelets. This was primarily because NN1731 bound to a greater number of sites per platelet. It appears that NN1731 and FVIIa bind similarly to a higher affinity site, but NN1731 binds to an additional low-affinity site. Removal of the Gla domain abolished binding. Thus, the protease domain of NN1731 was insufficient to mediate binding to the low affinity site. Two additional FVIIa mutants were tested: the single M298Q (“Q”) variant, and the double E296V and M298Q (“VQ”) variant. These variants bound to platelets identically to FVIIa.

Conclusion:

While the Gla domain is essential for FVIIa binding to platelets, the three amino acid changes in the protease domain in NN1731 enhance platelet binding as well as proteolytic activity. Since NN1731 and FVIIa bind similarly to phospholipid vesicles, characteristics in addition to lipid composition likely contribute to platelet binding. The platelet surface components that contribute to NN1731 binding remain to be defined.

Disclosures:

Hoffman: Novo Nordisk: Research Funding. Persson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment. Monroe:Novo Nordisk: Research Funding.
Characterizing Membrane-Bound Forms of Coagulation Factors at Atomic Resolution: Exploring Differences In Membrane Affinity.
Authors: Arcario Mark J , Tajkhorshid Emad
Published: October 2019

Abstract 1139

Membrane binding constitutes a key step in the activation of coagulation factors. This process largely hinges on specialized “floating” domains, most prominently the GLA and the C1/C2 domains found in almost all coagulation proteins. Currently, the details of how these domains interact with the membrane are largely unknown, due primarily to the lack of atomic models describing the proteins' membrane-bound forms. In sharp contrast to integral membrane proteins, studying insertion of membrane floating proteins has proven extremely challenging, mainly due to the unknown depth of membrane penetration. Although several computational studies have attempted to study the process using conventional models of the membrane, these methods are often prohibitively costly and inefficient due, in part, to the slow diffusion of the lipid molecules, which is required to accommodate the insertion of the floating domain. Here, we present a study on the membrane-bound forms of several important hemostasis factors, namely, human factor IX (hfIX), human factor × (hfX), human protein C (hPrC), human protein S (hPrS), and human protein Z (hPrZ), which utilize the GLA domain for membrane binding, as well as human factor V (hfV) and human factor VIII (hfVIII) in which the C1/C2 domain plays this role. To make the binding event accessible to molecular dynamics (MD) simulations, we have developed a novel membrane mimetic system, composed of a highly mobile hydrophobic core and explicit head groups that allow one to study specific interactions between the floating domains and lipid molecules. These interactions determine not only the specificity of binding to certain biological membranes, but also the molecular basis of the variable binding affinity of coagulation factors. These two factors are of utmost importance in physiology of blood coagulation, in designing mutations of altered affinity, and in developing novel pharmacological agents for thrombotic disorders. The floating domains were placed in the membrane-mimetic system and simulated under physiological conditions. Given the efficiency of the method, we have been able to capture spontaneous binding of these coagulation factors to the membrane (Figure 1) multiple times, despite starting from various initial orientations ensuring that each coagulation factor converged to the same final, membrane-bound structure over multiple trials. From these simulations, we have been able to elucidate the residues important for association with the membrane, as well as several unique phosphatidylserine (PS) specific binding sites in hfIX, hfX, hPrC, hPrS, and hPrZ. Using the membrane-bound structures of these coagulation factors together with free energy calculations, we have been able to elucidate important differences between the binding of high-affinity and low-affinity coagulation factors to the membrane.

Disclosures:

No relevant conflicts of interest to declare.
Expansion of Liver-Infiltrating CTL Clone In Concert with the Development of Hepatitis-Associated AA.
Authors: Ikawa Yasuhiro , Nishimura Ryosei , Mase Shintaro , Kuroda Rie , Araki Raita , Maeba Hideaki , Shimizu Masaki , Wada Taizo , Toma Tomoko , Koizumi Shoichi , Yachie Akihiro
Published: October 2019

Abstract 1159

Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which an episode of hepatitis precedes AA, namely hepatitis/aplastic anemia syndrome (HAAS). In AA, cytotoxic T-lymphocytes (CTL) play a central role in bone marrow (BM) destruction, and oligoclonal expansion of CTL correlates with disease activity. Immune mediated mechanisms have also been postulated in the pathogenesis of fulminant hepatitis (FH). Therefore we hypothesized that common CTLs recognizing similar target antigens against both liver and BM cells exist during the early period of FH, and subsequently selective proliferation of clones that are highly tropic to BM cause HAA at later stages. To this end, T cell phenotypes infiltrating liver, BM and peripheral blood (PB) samples obtained from 1-year-old boy with HAAS over time were determined by complementarity-determing region 3 (CDR3) size distribution patterns by polymerase chain reaction. Subsequently, CDR3 nucleotide sequences of predominant products were determined. Extremely skewed pattern was observed in Vb23 in BM and PB specimens at the onset of HAA (3 months after FH). A Vb23 clone with identical CDR3 nucleotide sequence predominated (clone 1) in BM and PB (34% and 60% within Vb23, respectively). This clone constituted a minor, but definitive clone in liver at the onset of FH (16% within Vb23). In contrast, initially predominant clones in liver (clone 3; 47% within Vb23) contracted to a minor subpopulation in BM and PB when the patient developed HAA (17% and 10% within Vb23, respectively).

This patient had a very good response to immunosuppressive therapy (IST) using antithymocyte globulin, cyclosporine, and granulocyte-colony stimulating factor. Two months after starting IST, the pattern of CDR3 size distribution was just recovering to normal and initially predominant clones in both liver and BM almost disappeared in Vb23 in PB. However, extremely skewed pattern of CDR3 region was still observed and abnormal clone 1 had still occupied 71% within Vb23 in BM. As of 8 months after starting IST, CDR3 size distribution recovered to normal patterns (Gaussian distribution) and CTL clones against both liver and BM almost disappeared in both BM and PB. These suggest that it would take a relatively long time to recover immunological parameters completely even in IST good responders.

In conclusion, our data is the first report to suggest that oligoclonal CTL clones simultaneously directed against liver and BM are involved in the pathogenesis of HAAS.

Disclosures:

No relevant conflicts of interest to declare.
Binding of FVIII to Recombinant VWF
Authors: Schrenk Gerald , Varadi Katalin , Rottensteiner Hanspeter , Ehrlich Hartmut J. , Scheiflinger Friedrich , Turecek Peter
Published: October 2019

Abstract 1408

Von Willebrand factor (VWF) forms a non-covalent complex with FVIII in the circulation, which not only stabilizes the heterodimeric structure of FVIII but also protects this coagulation protein from early proteolytic degradation and cellular uptake. By convention, the physiological ratio in plasma between VWF and FVIII is 1 IU to 1 IU, corresponding to a stoichiometry of approximately 1 FVIII molecule bound per 50 VWF monomers. However, since each VWF monomer contains one FVIII binding site, this ratio does not seem to reflect the genuine binding capacity of VWF for FVIII.

We therefore investigated the interaction between FVIII and a recombinant VWF (rVWF) drug candidate under different conditions. The FVIII binding capacity of VWF (VWF:FVIIIB) was measured by an ELISA system, where the VWF-bound FVIII was quantified by a FVIII chromogenic assay. Under this condition, VWF:FVIIIB of rVWF was similar to that of VWF derived from human normal plasma. The binding affinity of VWF for FVIII was determined using a Biacore system in which VWF is immobilized onto the sensor chip surface. The FVIII binding affinities were similar for rVWF KD=0.50) and pdVWF (KD=0.49 nM) and agreed with data from the literature.

Plasma-derived VWF was reported to show increased FVIII binding when in solution (instead of being immobilized to a surface). To find out whether this also holds true for rVWF, we developed a new assay system based on Biacore technology which measures the binding of FVIII to VWF in solution without any surface binding step of the VWF-FVIII complex. A mixture of rVWF and rFVIII is thereby injected into the flow cell of a Biacore sensor chip with immobilized rVWF. In this indirect assay system, the rFVIII that forms a complex with rVWF in solution is not available for binding to the immobilized rVWF. Under these conditions, rVWF was endowed with a 30-times higher FVIII binding capacity than could be anticipated from the physiological ratio and similar results were obtained for pdVWF.

The combined results showed that the ability to serve as the physiological chaperone protein for FVIII is fully preserved in rVWF, which can therefore be expected to stabilize FVIII in the circulation in a similar manner to VWF in plasma. Results from an ongoing phase 1 study show the good FVIII stabilizing properties of rVWF also when administered in humans with type 3 VWD.

Disclosures:

Schrenk: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment.
A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.
Authors: Staber Janice M. , Burnight Erin R , Korsakov Pavel , Kaminski Joseph , Craig Nancy L , McCray Paul B
Published: October 2019

Abstract 1477

Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein.

Disclosures:

Staber: Bayer Healthcare: Research Funding.
Improvement In Survival of Patients with Multiple Myeloma: An Update of Population Based Survival Trends
Authors: Pulte Dianne , Gondos Adam , Brenner Hermann
Published: October 2019

Abstract 1510

Background:

Treatment options for multiple myeloma have changed significantly over the past decade with new treatment options demonstrating improved survival in multiple clinical trials. We have previously published data demonstrating that survival has improved on the population level (Brenner et al Blood 2008) and projections of survival expectations for patients recently diagnosed with multiple myeloma which predicted continued improvement in survival for patients diagnosed in the years 2006-10 (Brenner et al Haematologica 2009). Here, we provide updated estimates of survival in myeloma and compare the results to projected survival.

Methods:

Period analysis was used to calculate 5- and 10-year relative survival for patients diagnosed with multiple myeloma from 1997-01 and 2002-06, using data from the Surveillance, Epidemiology, and End Results (SEER) database. Survival estimates for single years were calculated as well to determine whether changes in survival were ongoing throughout the 2002-06 period.

Results:

Five and 10-year relative survival in 2002-06 was significantly higher than in earlier time periods and was similar to survival predicted for patients diagnosed 2006–2010 (see table). When individual years between 2002 and 2006 were evaluated, survival was higher in 2005 and 2006 as compared to 2002-4. Survival was higher for all age groups as compared to survival previously calculated by period analysis for 1997–2001 (see table).

Conclusions:

Survival for patients diagnosed with multiple myeloma is improving for all age groups at a rate faster than predicted by previous models of survival. This implies a greater than linear increase in survival over the early part of the 21st century.

Disclosures:

No relevant conflicts of interest to declare.
A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.
Authors: Shibayama Hirohiko , Saito Yuri , Tanimura Akira , Tanaka Hirokazu , Matsumura Itaru , Kanakura Yuzuru
Published: October 2019

Abstract 1552

[Background]

Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated.

[Methods & Results]

In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells.

[Conclusion]

P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions.

Disclosures:

No relevant conflicts of interest to declare.