ALBERT

Description: The percentage of fluorescing, porphyrin rich normoblasts in the bone marrow of five bovine porphyrics increased after bleeding to a range of 66-91 per cent, as contrasted with 26-50 per cent in the same animals before bleeding. This is believed to be more in accord with a uni- than a bimodal distribution of normoblasts in respect to the genetic abnormality and the resultant overproduction of porphyrin.

Description: Introduction Multiple Myeloma (MM)is characterizedby heterogeneous clinical outcomes to existing therapies, which reflects the diverse genetic and molecular properties of tumor clones among patients. This intra-clonal heterogeneity may affect distinct molecular pathways within individual patients, contributing to reduced treatment efficacy over time and eventual relapse. In this work we investigate this problem by applying Bayesian network inference to develop high-dimensional network models of MM based on the Interim Analysis 9 (IA9) CoMMpass trial dataset (NCT0145429), an effort by the Multiple Myeloma Research Foundation (MMRF) to collect longitudinal data of newly-diagnosed patients from the United States, Canada and Europe. We demonstrate that our approach finds a number of known drug targets and identifies potentially novel ones. These targets, in our simulations, affect a number of treatment efficacy outcomes. Methods The IA9 dataset encompasses 645 patients with complete clinical and molecular data. We created an integrated table of clinical and genomic data including RNAseqmeasurements, somatic copy numbers, single nucleotide variants, and structural variants, for a combined input table of 30426 variables by 645 patients. We performed causal modeling using REFS™ Bayesian causal inference engine, constrained only by a minimal set of biological considerations but otherwise entirely de novo. The objective of modeling is to discover the causal mechanisms among variables and, in particular, with respect to the outcomes, by means of a set of Bayesian network models that are consistent with the observed disease biology. Such a model ensemble captures uncertainty in inference and highlights similarities among the models, allowing us to distinguish confident predictions from incidental ones. We investigated our model ensemble by means of systematic perturbations to model variables while observing effects upon treatment outcomes within specific patient backgrounds. Results Drivers of High Risk High riskwas definedas having disease progression before 18 months. The model uncovered a pathway involved in cell cycle regulation that leads to high risk when overexpressed. Specifically, the model identified CDK1, PKMY1, MELK, and NEK2 as the top drivers of the probability of high risk. These genes are "actionable", having drugsbeing investigatedclinically in the context of MM or in other cancers. Drivers of Durable Response Durable responsewas definedas a treatment response that lasts over a year before disease progression. The model was able to identify several novel pathways that appear to drive the probability of a durable response: a pathway of ribosomal genes (RPL6, RPL23, RPL12), a pathway of translation elongation factor EEF1A1 and associated pseudogenes, and a pathway of regulatory noncoding genes MIR1302-9, RP11-946L20.4, RP11-346D14.1, and RP11-506N2.1. Not much is known regarding the connection of these genes to MM. However, the ubiquitin-proteasome pathway, which is central to ribosomal biogenesis, is a major drug target in MM. Conclusions In this work we have developed a causal model of MM. In-silico perturbations of the model uncovered known and novel causal mechanisms for relevant endpoints, including a pathway involved in cell cycle that leads to high risk when dysregulated; a pathway involving ribosomal proteins and translation elongation factors that drives durable response; and several novel noncoding regulatory genes that are relevant to various measures of response. Beyond generating novel targets of immediate biological interest, our work demonstrates the promise of large-scale de novo network inference to this and similar problems in the future. Disclosures Gruber: GNS Healthcare: Employment. McBride:Instat: Employment. Runge:GNS Healthcare: Employment. Wuest:GNS Healthcare: Employment. Hadzi:GNS Healthcare: Employment. Lonial:BMS: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Janssen: Consultancy; BMS: Consultancy; Onyx: Consultancy. Khalil:GNS Healthcare: Employment. Hayete:GNS Healthcare: Employment.

Description: Hypoxia-inducible factor–2α (HIF-2α) is highly expressed in embryonic vascular endothelial cells (ECs) and activates the expression of target genes whose products modulate vascular function and angiogenesis. In this report, we describe a genetic model designed to test the physiologic consequences of deleting HIF-2α in murine endothelial cells. Surprisingly, mice with HIF-2α–deficient ECs developed normally but displayed a variety of phenotypes, including increased vessel permeability, aberrant endothelial cell ultrastructure, and pulmonary hypertension. Moreover, these animals exhibited defective tumor angiogenesis associated with increased hypoxic stress and tumor cell apoptosis. Immortalized HIF-2α–deficient ECs displayed decreased adhesion to extracellular matrix proteins and expressed reduced levels of transcripts encoding fibronectin, integrins, endothelin B receptor, angiopoietin 2, and delta-like ligand 4 (Dll4). Together, these data identify unique cell-autonomous functions for HIF-2α in vascular endothelial cells.

Description: CoMMpass [NCT0145429], a study by the Multiple Myeloma Research Foundation (MMRF), collects longitudinal data of newly diagnosed patients' responses to treatment in the context of their genetic and genomic profiles. The rapidly shifting treatment landscape highlights the importance of a deeper understanding of drug response pathways in order to enable better drug targeting and guide drug development. CoMMpass Interim Analysis 7 (IA7) dataset provides extensive genetic and genomic data on a population of almost 800 enrolled patients. Taking advantage of the rich dataset, we apply REFS™ state-of-the-art Bayesian causal inference engine to reverse-engineer the molecular pathways that most likely affect treatment outcomes in the CoMMpass population and to assess their significance in treatment response. The CoMMpass IA7 dataset, after reducing to patients with reasonably complete clinical and molecular data, comprises 452 patients. We aggregated genetic variables into gene region burden scores, drop mRNA and miRNA variables with excessive zero-inflation, and collate a final model data frame of 28200 variables by 452 patients. We then performed causal modeling constrained only by a minimal set of biological considerations but otherwise entirely de novo. The objective of the modeling is to explain the variability in the dataset and, in particular, in the outcomes, by a set of models that are all consistent with the observed disease biology. Such model ensemble captures uncertainty in inference and highlights similarities among the models, allowing us to distinguish confident predictions from incidental ones. For this study, we constructed an ensemble of 256 models (Figure 1). We investigated our model ensemble by the means of systematic perturbations to model variables, observing effects from such perturbations upon treatment outcomes, an approach that we have successfully applied elsewhere. Thus we are able to assess a prospective effect upon an outcome arising from an in vitro intervention, an adjuvant drug treatment, or an imposition of an enrollment criterion upon a clinical study. Assessing the effects of these in silico perturbations upon clinical outcomes, we conclude that key drivers of clinical outcomes, as defined by outcomes' sensitivity to perturbation, fall into broad categories of known response drivers, drivers in known pathways, and potential novel biology, or false positives. All three groups are broadly represented among top results (Table 1). The broad range of known disease modifiers, biomarkers, and drivers, such as stem cell transplant, Ig light chain, or RN7SK, identified as such by the REFS™ model de novo, leads us to have a higher confidence in the importance of the other predicted drivers of clinical outcomes, whether partially known (e.g., DNAH5, FAT1) or novel (e.g., mir3648-1, C19orf68). Furthermore, it will be possible to use future CoMMpass Interim Analyses to validate the significance of the predicted novel drivers and to help improve the quality of the causal model, promoting the discovery of further drivers of clinical outcomes. Figure 1. (a) A REFS model consists of an ensemble of causal networks (b) Consensus (average) causal network topology for the CoMMpass IA7 model, with key outcome variables highlighted Figure 1. (a) A REFS model consists of an ensemble of causal networks (b) Consensus (average) causal network topology for the CoMMpass IA7 model, with key outcome variables highlighted Figure 2. Predicted drivers of clinical outcomes Figure 2. Predicted drivers of clinical outcomes Disclosures Gruber: GNS Healthcare: Employment. Hayete:GNS Healthcare: Employment. Keats:Translational Genomic Research Institute: Employment. Karl:GNS Healthcare: Employment. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Khalil:GNS Healthcare: Employment.

Description: Background Recombinant FVIIa (NovoSeven®) is used for the treatment of spontaneous bleeds in haemophilia patients with inhibitory antibodies against FVIII or FIX. Two theories have been proposed to describe the mechanism of action of FVIIa (van't Veer C et. al., (2000) Blood 95:1330-5, Monroe DM et. al., (1998) Blood Coagulation Fibrinolysis 9(S1):S15-S20); a) competition with zymogen FVII to bind to surface exposed TF at site of injury, and b) activation of FX by FVIIa bound to phospholipids exposed on activated platelets. This study aims at generation and characterization of a novel reagent that will provide new insights into the extent of TF influence in vascular injury. Aim To engineer and characterize FVIIa variant with abrogated TF binding ability and to address influence of TF on FVIIa activity in bleeding models (in whole blood clotting). Methods Based on the x-ray crystal structure (PDB: 1DAN), the binding interface between soluble TF (sTF) and FVIIa on the FVIIa light chain was targeted for engineering a new N-glycan. FVIIa variant I69N/F71T was expressed in BHK cells. Majority of the FVIIa variant was additionally glycosylated as assessed by PNGase treatment and SDS-PAGE analysis. Low-levels of non-glycosylated FVIIa variant were removed by passing the FVIIa variant through a sTF column. The resulting FVIIa preparation was homogenous as assessed by HPLC analysis and was characterized as reported earlier (Persson E et al., (2009) FEBS J. 276:3099-109). Results We have successfully introduced a new N-glycan in FVIIa by engineering an N-glycosylation site Asn-X-Ser/Thr in the FVIIa light chain that provides steric hindrance to sTF. WT-FVIIa binds to sTF with Kd of 6 nM, I69N/F71T-FVIIa displays abrogated sTF binding ability up to 10 µM sTF. At higher concentrations, sTF may interact with FVIIa protease domain directly. Proteolytic and amidolytic activity of WT and FVIIa variant are comparable in the absence of sTF, suggesting that the additional N-glycan does not perturb the protease domain. Data from FX activation and ATIII inhibition assays show virtually no effect of sTF on the functional properties of I69N/F71T-FVIIa in presence or absence of phospholipids. Thrombelastography (TEG) assay initiated by kaolin in haemophilia like human whole blood, in the presence of a TF specific inhibitory antibody, revealed no significant differences in clotting time (R-time) and maximum thrombus generation (MTG) for WT and I69N/F71T FVIIa variants. Similar data when TEG assay was initiated by re-lipidated TF (Innovin®) showed marked differences between WT and I69N/F71T FVIIa variants suggesting loss in the TF binding ability of the FVIIa variant. Conclusions Present work demonstrates that, using exclusive mutagenesis strategy, it is possible to engineer FVIIa variant with minimal changes in the primary sequence that displays abrogated TF binding ability but intact amidolytic and proteolytic activity. Functional properties of this variant were characterized by in vitro biochemical and TEG assays. Such a variant should prove useful to delineate involvement of TF in FVIIa activity in bleeding models. Disclosures Gandhi: Novo Nordisk A/S: Employment. Grøn:Novo Nordisk A/S: Employment. Petersen:Novo Nordisk A/S: Employment. Reedtz-Runge:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Østergaard:Novo Nordisk A/S: Employment.

Description: We have evaluated a method for quantitation of eosinophil migration to stimuli in vivo. Upon transfusion into normal syngeneic mice, 111In- labeled eosinophils had an intravascular half-life of 9.5 hr and distributed predominantly into spleen, bone marrow, and liver. In either Schistosoma mansoni-infected mice or recipients of lymphoid cells from infected mice, intradermal (ear pinna) injection of the schistosomal egg antigenic preparation (SEA) elicited time-dependent accumulation of 111In-labeled eosinophils detectable by either gamma scintillation counting of tissue samples or by nuclear medicine external imaging. Intradermal administration of a lymphokine fraction (containing eosinophil stimulation promoter activity) similarly caused accumulation of 111In-labeled eosinophils. Both reactions depended on the concentration of stimulus (SEA or lymphokine). 111In-labeled neutrophils or macrophages or 125I-albumin did not preferentially accumulate at the reactions examined to the extent found with 111In- labeled eosinophils, indicating that localization of label depends on an active process and is due to eosinophils rather than a contaminating cell type. The method was used to estimate how long eosinotactic lymphokine remained at dermal sites: 60% of initial activity was present 12 hr after injection. The model is discussed with regard to the role of lymphokines in hypersensitivity reactions with eosinophil involvement, such as the granulomatous response to S. mansoni eggs.

Description: Mammalian platelets are small, anuclear circulating cells that form tightly adherent, shear-resistant thrombi to prevent blood loss after vessel injury. Platelet thrombi that form in coronary and carotid arteries also underlie common vascular diseases such as myocardial infarction and stroke and are the target of drugs used to treat these diseases. Birds have high-pressure cardiovascular systems like mammals but generate nucleated thrombocytes rather than platelets. Here, we show that avian thrombocytes respond to many of the same activating stimuli as mammalian platelets but are unable to form shear-resistant aggregates ex vivo. Avian thrombocytes are larger than mammalian platelets, spread less efficiently on collagen, and express much lower levels of the α2bβ3 integrin required for aggregate formation, features predicted to make thrombocyte aggregates less resistant than platelets are to the high fluid shear forces of the arterial vasculature. In vivo carotid vessel injury stimulates the formation of occlusive platelet thrombi in mice but not in the size- and flow-matched carotid artery of the Australian budgerigar. These studies indicate that unique physical and molecular features of mammalian platelets enable them to form shear-resistant arterial thrombi, an essential element in the pathogenesis of human cardiovascular diseases.

Description: We utilized a kaolin-activated partial thromboplastin time (APTT) using rabbit brain phospholipid, in which the capacity of a fourfold increased “high” phospholipid concentration (PC) to normalize the abnormal “standard” PC-APTT in patients with lupus anticoagulants is assessed. This system was also used to measure factors VIIIC, IX, and XI. The tissue thromboplastin inhibition test (TTI), a prothrombin time system in which the activity of a lupus anticoagulant is unmasked by the use of dilute thromboplastin, was simultaneously evaluated. Test sensitivity was defined by results on 31 consecutive patients with standard PC-APTT inhibitors and no bleeding tendency. Specificity was based on 94 patients with various other coagulopathies, including coagulation factor inhibitors, severe congenital factor deficiencies, hepatic insufficiency, and warfarin and heparin treatment. Twenty-one patients with lupus erythematosus and standard PC-APTT results within normal limits were also tested. Sensitivity of the APTT system was superior to that of the TTI (97% v 58%); high PC normalized clotting time ratios and factor levels. Positive results were common with both assays in the group of 20 heparinized patients. The APTT system had superior specificity in remaining cases; there were no positive tests among 74 patients. The lupus erythematosus group had a significant decrease in the clotting time ratio with high PC, indicating that low- level lupus anticoagulants are quite prevalent in this group. The kaolin clotting time using rabbit brain phospholipid in standard and high concentrations is a simple, sensitive, and specific technique for diagnosis of lupus anticoagulants.

Description: Introduction Regardless of significant advances in the therapy of multiple myeloma (MM) there is still a lack of effective treatment options for patients with high-risk disease. In this context, we recently developed a network of high-risk disease based on more than 30 000 genomic and clinical variables from 645 patients of the CoMMpass dataset (Gruber et al., ASH 2016). Validation of these findings has been performed in the IFM/DFCI 2009 trial dataset (Furchtgott et al., ASH 2017). This comprehensive computational approach revealed a network of 17 genes driving high-risk (defined as progression or myeloma-related death within 18 months). Here, we performed preclinical validation of potential novel drug targets to confirm the utility of in silico guided target discovery in high-risk MM. Methods TTK (CFI402257, BAY-1217389), PLK4 (CFI400945, Centrinone), MELK (OTSSP167) and CDK1 (CPG71514) inhibitors were studied in a panel of human MM cell lines (n=11) for their activity in cell viability, cell growth, cell cycle, apoptosis, colony formation, drug combination and co-culture experiments. PKMYT1, TTK and PLK4 were targeted with doxycycline-inducible shRNAs. Analysis of gene expression (GEP) data (GSE24080) was used to link candidate genes to certain MM subgroups. Results The network of 17 genes driving high-risk disease contained eight kinases that serve as attractive drug targets (AURKA, NEK2, CDK1, BUB1B, MELK, TTK, PKMYT1, and PLK4), all of them involved in cell cycle regulation. Accordingly, expression levels of all kinases (except PKMYT1) were enriched in the GEP-defined proliferation associated subgroup of MM and thus linked to poor outcome. To study the interconnectedness of the individual network genes we first investigated the impact of previously reported CDK1 and MELK inhibitors on other network members. This demonstrated rapid loss of CDK1, NEK2, MELK, PKMYT1 and FOXM1 protein levels. We then selected TTK and PLK4 as putative novel MM targets with available inhibitors undergoing clinical testing in solid tumors. Protein and mRNA expression of both genes was confirmed in all MM cell lines. Two selective compounds per gene were used for preclinical studies. All four inhibitors significantly reduced MM cell viability and single dose IC70 treatment impaired cell growth up to 10 days (60-98% reduction, P

Description: We utilized a kaolin-activated partial thromboplastin time (APTT) using rabbit brain phospholipid, in which the capacity of a fourfold increased “high” phospholipid concentration (PC) to normalize the abnormal “standard” PC-APTT in patients with lupus anticoagulants is assessed. This system was also used to measure factors VIIIC, IX, and XI. The tissue thromboplastin inhibition test (TTI), a prothrombin time system in which the activity of a lupus anticoagulant is unmasked by the use of dilute thromboplastin, was simultaneously evaluated. Test sensitivity was defined by results on 31 consecutive patients with standard PC-APTT inhibitors and no bleeding tendency. Specificity was based on 94 patients with various other coagulopathies, including coagulation factor inhibitors, severe congenital factor deficiencies, hepatic insufficiency, and warfarin and heparin treatment. Twenty-one patients with lupus erythematosus and standard PC-APTT results within normal limits were also tested. Sensitivity of the APTT system was superior to that of the TTI (97% v 58%); high PC normalized clotting time ratios and factor levels. Positive results were common with both assays in the group of 20 heparinized patients. The APTT system had superior specificity in remaining cases; there were no positive tests among 74 patients. The lupus erythematosus group had a significant decrease in the clotting time ratio with high PC, indicating that low- level lupus anticoagulants are quite prevalent in this group. The kaolin clotting time using rabbit brain phospholipid in standard and high concentrations is a simple, sensitive, and specific technique for diagnosis of lupus anticoagulants.