Secretome-Based Screening in Target Discovery

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function

The remarkable ability to generate unlimited numbers of cardiomyocytes and other differentiated cell types, from any person, and to edit the genome to introduce or correct disease-causing mutations, creates unprecedented opportunities for drug discovery. The new technologies have the potential to revolutionize the drug development pipeline, from delineating disease mechanisms and discovery of therapeutic targets to library screening and validation of therapeutic strategies. Moreover, since in vitro phenotypes reflect patient genetics and might predict outcomes, patient induced pluripotent stem cell (iPSCs) might eventually contribute to patient selection for clinical trials and inform individual patient treatment. This chapter focuses on the application of iPSC-derived cardiomyocytes in large-scale applications relevant to discovery of disease mechanisms and therapeutic targets for heart disease and for assessing the cardiomyopathic and proarrhythmic risk of drugs. We review the current status of large-scale screening of iPSC-derived cardiomyocyte disease models and explore new advances in cell culture, three-dimensional engineered tissues, and instrumentation that might address current weaknesses in the iPSC-cardiomyocyte technology. Our philosophy is that advancing iPSC-derived cardiomyocyte models that faithfully recapitulate disease and enable large-scale chemical or functional genomics screening could shift the paradigm of drug discovery by introducing human disease phenotype into the early stages of the development process, with the potential for increasing the safety and efficacy of new medicines.

Biocontrol agents are living organisms with the potential to suppress populations of plant pathogens or pests in a cropping system. The complex interplay between the different players and the changing environment, results in a combination of different modes of action. Here, we applied an mRNA-based systems approach to gain insight into the antagonist-pathogen-host interaction of Golubevia sp. isolates BC0812 and BC0850 with the causal agent of wheat powdery mildew, Blumeria graminis f. sp. tritici, in planta over time. Bioassays were performed on potted wheat plants (water-treated control, antagonist, pathogen, antagonist+pathogen) under controlled conditions. A significantly higher percentage of mildew conidia were parasitized after treatment with Golubevia sp. BC0812 with 26% and BC0850 with 16% compared to the water control with 1%. Differential gene expression analysis of antagonists, pathogen and host 5, 6, 7, and 11 days after inoculation (dai) with the antagonist pointed to a combination of different modes of action: An interplay of modulating plant defense responses, impairing conidiogenesis of the pathogen by scavenging H2O2, facultative hyperparasitism and nitrogen competition. Microscopic observations supported the suggested hyperparasitism as thin mycelium could be observed on Bgt conidia at 6 dai and later.Taken together the results allowed the formulation of new hypothesis regarding modes of action and the interplay between antagonist, pathogen and host. It showed that a solid molecular understanding of the antagonist-pathogen relationship over time is essential for less biased mode of action studies. Understanding this complex interplay is the basis for targeted optimization strategies and allows discovery of new potential targets and markers for future biocontrol development.

BACKGROUND: Success in precision medicine depends on the ability to accurately and rapidly identify master regulators of critical gene subnetworks. To achieve this goal requires a seamless computational target discovery platform that integrates global network visualization with identification of master regulatory subnetworks (MRS) and their key influencers. METHODS: We have created NetZen - a computer cluster-capable computational suite allowing for 1) robust network generation with million edges; 2) network-based MRS identification; 3) hierarchical analysis with automated annotations; and 4) whole-network 3D visualization of all 20,000 genes. To identify cancer MRS, we applied NetZen to expression datasets of tumors and normal tissues, tumor-initiating cells (TICs), non-TIC cancer cells, and normal stem and non-stem cells from TCGA, GEO and internal sources. For validation, we introduced these subnetworks in normal cells to recapitulate TIC phenotypes in vivo or disrupted them in patients-derived TICs and mouse xenografts (PDX). To identify MRS of mortality, we applied NetZen to survival data in TCGA. RESULTS: Networks of 28 human cancers were generated, integrating transcriptomics, microRNAs, proteomics, and clinical data. For cancers with TIC datasets, we identified TIC-specific MRS with clear-cut hierarchy controlling immune surveillance, EMT and developmental functions. Interestingly, in some cancers, the immune subnetwork including immune checkpoints was entirely encompassed in the developmental subnetwork. We present representative validation on glioblastoma TIC (GIC) MRS with features common in other cancers’ TICs. Exogenous introduction of this subnetwork in normal astrocytes forced them to become GICs and form tumors in mice. Disrupting this subnetwork in 10 independent PDXs profoundly blunted tumor growth and prolonged survival. Lastly, we present detailed MRS of mortality analysis of multiple cancer types and identify common pathways directly influencing outcomes. DISCUSSION: NetZen provides a complete solution to big data analysis by effortlessly processing million-edge hierarchical networks with biological insights through automated annotations. The findings that the immune and developmental subnetworks were intimately linked suggest that immune escape by TICs is intrinsic of cellular transformation rather than a product of selection. Lastly, detailed network hierarchy provides a logical roadmap for cancer therapeutic development. Citation Format: Son Le, Alberto Riva, Changwang Deng, David D. Tran. NetZen: A comprehensive network-based pathway and target discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2456.

The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.

The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.