High-throughput Functional Screening Research Articles

MiRNA-519e promotes cardiomyocyte cell division accompanied by pro-angiogenic and anti-apoptotic effects

Abstract Background MicroRNAs have the therapeutic potential for heart repair after myocardial infarction(MI). Using a functional high-throughput screening approach, we identified microRNA-519e(miR-519e) as an inducer of cell-cycling in human-derived iPSC-cardiomyocytes(hiPSC-­CM). Purpose This study examines miR-519e for cell division in hiPSC-­CM, additional beneficial functions in heart-related non-CMs, and direct target identification and validation in vitro and in vivo. Methods MiR-519e-mimics or miR-scrambled(miR-scr) were transfected into hiPSC-­CM, human aortic endothelial cells(HAEC) and human cardiac fibroblasts(cFB). Proliferative markers (EdU(5-Ethynyl-2'- deoxyuridine), H3P(Phospho-Histone H3) and AURKB(Aurora B-kinase)) and apoptotic response using MTT assay after exposure to doxorubicin were assessed in hiPSC­-CMs. Angiogenic capacity of HAEC was evaluated by tube formation assay and fibrotic response of cFBs using EdU and α-SMA(smooth muscle actin) staining. In vivo, a mouse MI model with ligation of the left anterior descending followed by intramyocardial injection of miR-519e or miR-scr was used. Direct target identification of miR-519e was assessed using in silico analysis combined with RNA-Seq of miR-519e-mimic treated hiPSC-CMs and validated using Ago2-RNA immunoprecipitation(RIP) followed by qPCR. Results After transfection of miR-519e-mimics in hiPSC-­CM, a significant proliferative response indicative of cell division was observed in immunofluorescence staining and on protein level as compared with miR-scr treated hiPSC-CMs (EdU, H3P and AURKB, p<0.01). Apoptosis was blunted in miR-519e-treated hiPSC-CMs (p<0.05). MiR-519e-treatment of HAEC resulted in more meshes (p<0.01) and longer tube length (p<0.05), indicating enhanced pro-angiogenic capacity. Importantly, EdU-uptake and α-SMA were similar in miR-519e-treated cFB as compared to miR-scr, suggesting neutral effects of miR-519e on myofibroblast transition. In mice undergoing MI, injection of miR-519e-mimics significantly downregulated CDKN1A/P21(Cyclin dependent kinase inhibitor 1A) (p<0.05 vs. miR-scr), a direct target of miR-519e and negative master regulator of cell-cycling. For further novel target identification of miR-519e, we performed in silico analysis combined with RNA-Seq data from miR-519e-treated hiPSC-CM and validation using Ago2-RIP followed by qPCR. Herein, we identified PTEN(Phosphatase and tensin homolog) and TGFBR2(Transforming growth factor β receptor 2) that are involved in cardiomyocyte proliferation and hypoxic response, as direct targets of miR-519e. Conclusion Collectively, miR-519e is a potent inducer of cell division in human cardiomyocytes, with anti-apoptotic and pro-angiogenic capacity in the absence of pro-fibrotic effects. We show that miR-519e treatment downregulates key negative cell-cycle regulators in vivo and we identified novel direct targets of miR-519e that are involved in regulatory pathways for heart regeneration.

Identification and functional characterization of lncRNAs involved in human monocyte-to-macrophage differentiation

ABSTRACT Although long noncoding RNAs (lncRNAs) constitute the majority of the human transcriptome, the functional roles of most remain elusive. While protein-coding genes in macrophage biology have been extensively studied, the contribution of lncRNAs in this context is poorly understood. Given the vast number of lncRNAs (>20,000), identifying candidates for functional characterization poses a significant challenge. Here, we present two complementary approaches to pinpoint and investigate lncRNAs involved in monocyte-to-macrophage differentiation: RNA-seq for functional inference and a high-throughput functional screen. These strategies enabled us to identify four lncRNA regulators of monocyte differentiation: lincRNA-JADE1, lincRNA-ANXA3, GATA2-AS1, and PPP2R5C-AS1. Preliminary insights suggest these lncRNAs may act in cis through neighbouring protein-coding genes, although their precise mechanisms remain to be elucidated. We further discuss the strengths and weaknesses of these methodologies, along with validation pipelines crucial for establishing lncRNA functionality.

Single-cell multi-modal integrative analyses highlight functional dynamic gene regulatory networks directing human cardiac development.

Illuminating the precise stepwise genetic programs directing cardiac development provides insights into the mechanisms of congenital heart disease and strategies for cardiac regenerative therapies. Here, we integrate invitro and invivo human single-cell multi-omic studies with high-throughput functional genomic screening to reveal dynamic, cardiac-specific gene regulatory networks (GRNs) and transcriptional regulators during human cardiomyocyte development. Interrogating developmental trajectories reconstructed from single-cell data unexpectedly reveal divergent cardiomyocyte lineages with distinct gene programs based on developmental signaling pathways. High-throughput functional genomic screens identify key transcription factors from inferred GRNs that are functionally relevant for cardiomyocyte lineages derived from each pathway. Notably, we discover a critical heat shock transcription factor 1 (HSF1)-mediated cardiometabolic GRN controlling cardiac mitochondrial/metabolic function and cell survival, also observed in fetal human cardiomyocytes. Overall, these multi-modal genomic studies enable the systematic discovery and validation of coordinated GRNs and transcriptional regulators controlling the development of distinct human cardiomyocyte populations.

A statistical simulation model to guide the choices of analytical methods in arrayed CRISPR screen experiments.

An arrayed CRISPR screen is a high-throughput functional genomic screening method, which typically uses 384 well plates and has different gene knockouts in different wells. Despite various computational workflows, there is currently no systematic way to find what is a good workflow for arrayed CRISPR screening data analysis. To guide this choice, we developed a statistical simulation model that mimics the data generating process of arrayed CRISPR screening experiments. Our model is flexible and can simulate effects on phenotypic readouts of various experimental factors, such as the effect size of gene editing, as well as biological and technical variations. With two examples, we showed that the simulation model can assist making principled choice of normalization and hit calling method for the arrayed CRISPR data analysis. This simulation model is implemented in an R package and can be downloaded from Github.

Combinatorial strategies targeting NEAT1 and AURKA as new potential therapeutic options for multiple myeloma.

Multiple myeloma (MM) is a dreadful disease, marked by the uncontrolled proliferation of clonal plasma cells (PCs) within the bone marrow (BM). MM is characterized by a highly heterogeneous clinical and molecular background, supported by severe genomic alterations. Important deregulation of long non-coding RNAs (lncRNAs) expression has been reported in MM patients, influencing progression and therapy resistance. NEAT1 is a lncRNA essential for nuclear paraspeckles and involved in gene expression regulation. We showed that NEAT1 supports MM proliferation making this lncRNA an attractive therapeutic candidate. Here, we used a combinatorial strategy integrating transcriptomic and computational approaches with functional high-throughput drug screening, to identify compounds that synergize with NEAT1 inhibition in restraining MM cells growth. AUKA inhibitors were identified as top-scoring drugs in these analyses. We showed that the combination of NEAT1 silencing and AURKA inhibitors in MM profoundly impairs microtubule organization and mitotic spindle assembly, finally leading to cell death. Analysis of the large publicly CoMMpass dataset showed that in MM patients AURKA expression is strongly associated with reduced progression-free (p < 0.0001) and overall survival probability (p < 0.0001) and patients displaying high expression levels of both NEAT1 and AURKA have a worse clinical outcome. Finally, using RNA-sequencing data from NEAT1 knockdown (KD) MM cells, we identified the AURKA allosteric regulator TPX2 as a new NEAT1 target in MM and as a mediator of the interplay between AURKA and NEAT1, therefore providing a possible explanation of the synergistic activity observed upon their combinatorial inhibition.

Identification and Functional Characterization of lncRNAs involved in Human Monocyte-to-Macrophage Differentiation.

Long noncoding RNAs (lncRNAs) make up the largest portion of RNA produced from the human genome, but only a small fraction have any ascribed functions. Although the role of protein-coding genes in macrophage biology has been studied extensively, our understanding of the role played by lncRNAs in this context is still in its early stages. There are over 20,000 lncRNAs in the human genome therefore, attempting to select a lncRNA to characterize functionally can be a challenge. Here we describe two approaches to identify and functionally characterize lncRNAs involved in monocyte-to-macrophage differentiation. The first involves the use of RNA-seq to infer possible functions and the second involves a high throughput functional screen. We examine the advantages and disadvantages of these methodologies and the pipelines for validation that assist in determining functional lncRNAs.

Abstract PO-038: Unbiased discovery of novel antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma

Abstract Monoclonal antibodies are among the most effective treatments for patients with blood cancers, and they primarily function by marking cells for destruction by the innate immune system. Macrophages are critical innate effectors of antibody therapies for lymphoma, but their anti-tumor capacity is limited by the CD47/SIRPa interaction. CD47 acts a “don’t eat me” signal that is highly expressed on the surface of many lymphomas, and it prevents phagocytosis by binding to the inhibitory receptor SIRPa expressed on macrophages. In multiple clinical trials, CD47-blocking therapies have demonstrated encouraging signs of efficacy for B-cell lymphomas, particularly when used in combination with rituximab, an opsonizing anti-CD20 antibody. However, the best antibodies and combination strategies to activate macrophages remains unknown. Here, we sought to define the repertoire of cell surface antigens that can be targeted to stimulate macrophage-mediated destruction of B-cell lymphomas. To achieve this goal, we developed a high-throughput functional screening platform to measure the ability of primary macrophages to attack B-cell lymphoma cells. We successfully applied this system to screen monoclonal antibody libraries targeting hundreds of distinct cell surface antigens across both mouse and human systems. We conducted screens using each antibody as a single agent, in combination with anti-CD20, or in combination with anti-CD47. From these efforts, we identified CD24, CD38, CXCR4, CD71, and multiple other novel and unique antigens that could be targeted alone or in combination to exert maximal macrophage-mediated destruction of B-cell lymphoma. Of note, some of the identified targets are predominantly expressed by the lymphoma cells, whereas others are expressed by the macrophages and act as unappreciated immune checkpoints. In validation studies, we defined a multitude of new antibody combinations that robustly stimulate macrophages to attack and eliminate lymphoma cells. Since some anti-CD47-antibodies have been limited by on-target hematologic toxicity, we also used the identified targets to engineer a collection of novel bispecific antibodies that induce macrophage phagocytosis without causing hematologic toxicity. We generated 156 bispecific antibodies, produced them recombinantly, and demonstrated they can maximize macrophage-mediated cytotoxicity of human B-cell lymphoma cells while minimizing binding to healthy blood cells. In mouse xenograft models, these bispecific antibodies exhibited significant single-agent activity in a model of aggressive B-cell lymphoma. Thus, our study has led to the development of a multitude of novel therapeutic candidates and combination strategies that can be developed further to maximize anti-tumor function and benefit patients with lymphoma. Furthermore, our approach can be rapidly applied to other hematologic malignancies to create innovative bispecific agents that maximize anti-tumor responses by macrophages or other innate immune cells. Citation Format: Juliano Ribeiro, Carlota Pages Geli, José Velarde, Anna Meglan, Jasmine Blandin, Kyle Vaccaro, Marta Crespo, Kipp Weiskopf. Unbiased discovery of novel antibody therapies that stimulate macrophage-mediated destruction of B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-038.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Abstract LB289: Single-cell CRISPR screens at scale to understand T cell biology

Abstract CRISPR screens have become the primary discovery engine in modern biology and are widely used to uncover novel targets in immuno-oncology. However, these genetic screens are usually coupled to rather simplistic read-outs such as cell fitness. In contrast, CROP-Seq (or “CRISPR Droplet” Sequencing) screens combine CRISPR perturbation with single-cell transcriptomics, enabling high-content phenotyping at single-cell resolution. Briefly, cells are perturbed with a pooled sgRNA library and transcriptomic profiles of each cell are recorded using conventional single-cell RNA sequencing platforms. Currently, the scale of these screens is limited to the perturbation of a couple of hundred genes, possibly up to 1.000 genes, because of increased costs for single-cell library preparation and next generation sequencing (NGS). At Myllia, we have built a platform allowing high-content CROP-Seq screens in both CRISPR-ready cancer cell lines as well as primary immune cells, e.g., human T cells. Using CRISPR KO, CRISPR interference or CRISPR activation workflows, we can help accelerate the identification of target genes and regulatory networks involved in immuno-oncology. Primary T cells are of great interest in the immunotherapy community as they are key players in autoimmune and inflammatory disease. Engineered T-lymphocytes such as CAR-T cells are currently developed as novel cellular medicines, yet many T cell-intrinsic features involved in CAR-T potency remain elusive. To enable the discovery of novel targets, we have performed multiple customized CROP-Seq CRISPR KO screens in primary human T cells investigating T cell stemness and effector phenotypes as well as CD4+ T helper cell differentiation towards Th1, Th2, or Th17 subsets. Here, we present screens for regulators of functional T cell plasticity and Th2 commitment in which we aimed to identify genes determining critical T cell fate decisions, potentially linking gene (dys)function to either autoimmune/inflammatory disease or functional persistence of cytotoxic T cells for cancer immunotherapy. Overall, the advanced CROP-Seq platform combining large-scale perturbations and targeted sequencing (TA) read-outs will catalyse a paradigm shift for CROP-Seq enabling high-throughput functional genomic screens that support the validation of drug targets in autoimmunity, inflammation and immuno-oncology. Citation Format: Nicole Untermoser, Johanna Irnstorfer, Anatoly Vasilyev, Nikola Vinko, Sumit Pawar, Anke Loregger, Adam Krejci, Henrik Schmidt, Tilmann Bürckstümmer. Single-cell CRISPR screens at scale to understand T cell biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB289.

RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics.

Aim: Our gut microbiome has its own functionalities which can be modulated by various xenobiotic and biotic components. The development and application of a high-throughput functional screening approach of individual gut microbiomes accelerates drug discovery and our understanding of microbiome-drug interactions. We previously developed the rapid assay of individual microbiome (RapidAIM), which combined an optimized culturing model with metaproteomics to study gut microbiome responses to xenobiotics. In this study, we aim to incorporate automation and multiplexing techniques into RapidAIM to develop a high-throughput protocol. Methods: To develop a 2.0 version of RapidAIM, we automated the protein analysis protocol, and introduced a tandem mass tag (TMT) multiplexing technique. To demonstrate the typical outcome of the protocol, we used RapidAIM 2.0 to evaluate the effect of prebiotic kestose on ex vivo individual human gut microbiomes biobanked with five different workflows. Results: We describe the protocol of RapidAIM 2.0 with extensive details on stool sample collection, biobanking, in vitro culturing and stimulation, sample processing, metaproteomics measurement, and data analysis. The analysis depth of 5,014 ± 142 protein groups per multiplexed sample was achieved. A test on five biobanking methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional responses to kestose. Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our internal ecology.

Abstract 2944: A high-throughput screening system for identifying therapeutic targets in immuno-oncology

Abstract INTRODUCTION: Over the last decade, cancer immunotherapy has revolutionized cancer treatment. However, more than half of patients don’t respond, and responders often lack lasting effects. Understanding how genetic variants in cancer cells influence their interaction with immune cells, especially T cells, is crucial to uncover underlying mechanisms. Genetic screens are powerful for uncovering responsible genes for various phenotypes, but challenges in obtaining ample samples to set up a proper immune and cancer co-culture system have limited their use for identifying immune modulators in cancer: primary mouse T cells expressing CD3 against a specific antigen (typically albumin) need to be extracted from transgenic mice and only syngeneic mouse cancer cell lines that match these T cells can be paired, restricting the utilization of human T cells and human cancer cells for this co-culture screening setup. METHODS: Here, we present a highly scalable co-culture system that enables genome-wide CRISPR screens on a pair of human primary T cells and any allogeneic human cancer cell lines. This system utilizes the expression of a membrane-targeted protein in mammalian cells, named as ImBridge, which directly engages and activates CD3+ immune cells. This mechanism bypasses the need for matched MHC on target cancer cells. RESULTS: We validated activation of CD3+ immune cells and selective cancer cell cytotoxicity by ImBridge. A genome-scale CRISPR screen utilizing the co-culture system unveiled known immune-oncology targets including PD-L1 and PTPN2 and revealed MEDiC_01 as a novel therapeutic target. Genetic loss of MEDiC_01 in cancer cells significantly sensitized them to cytotoxic cells, effectively eliminating cancers in the presence of human PBMCs. CONCLUSION: We demonstrated a scalable and efficient approach for conducting high-throughput functional screens to uncover genes that modulate interactions between effector T cells and target cancer cells. This can facilitate the identification of new immuno-oncology therapeutic targets, insights into underlying mechanisms, and genetic factors influencing immune susceptibility and evasion. Citation Format: Dippal Parikh, Soon Youn Choi, Ki Eun Pyo, Hong-Pyo Lee, Kyuho Han. A high-throughput screening system for identifying therapeutic targets in immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2944.

A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput Screening of hTRPV1 Agonists.

Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists. The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared. A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) < nonivamide (67 ± 3.05 nM) < piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists. The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.

Using Genomics to Identify Novel Therapeutic Targets for Aortic Disease.

Aortic disease, including dissection, aneurysm, and rupture, carries significant morbidity and mortality and is a notable cause of sudden cardiac death. Much of our knowledge regarding the genetic basis of aortic disease has relied on the study of individuals with Mendelian aortopathies and, until recently, the genetic determinants of population-level variance in aortic phenotypes remained unclear. However, the application of machine learning methodologies to large imaging datasets has enabled researchers to rapidly define aortic traits and mine dozens of novel genetic associations for phenotypes such as aortic diameter and distensibility. In this review, we highlight the emerging potential of genomics for identifying causal genes and candidate drug targets for aortic disease. We describe how deep learning technologies have accelerated the pace of genetic discovery in this field. We then provide a blueprint for translating genetic associations to biological insights, reviewing techniques for locus and cell type prioritization, high-throughput functional screening, and disease modeling using cellular and animal models of aortic disease.

An improved method for large scale generation and high-throughput functional characterization of human iPSC-derived microglia

Neuroscience drug discovery has faced significant challenges due to restricted access to relevant human cell models and limited translatability of existing preclinical findings to human pathophysiology. Induced pluripotent stem cells (iPSCs) have emerged as a promising solution, offering the potential to generate patient-specific cell types, including in the recent years, iPSC-derived microglia (iMGL). Current methods rely on complex and time-consuming differentiation procedures, leading to considerable batch-to-batch variability consequently hindering the establishment of standardized and reproducible high-throughput functional screening approaches. Addressing these challenges is critical in ensuring the generation of homogenous iMGL populations with consistent functional properties. In this study we describe an improved high-yield protocol for generating iMGL, which allows for increased reproducibility and flexibility in the execution of high-throughput functional screens. We introduce a two-step process in embryoid bodie (EB) maintenance and a stop point allowing for cryopreservation at the hematopoietic progenitor cell (iHPC) stages. Furthermore, we demonstrate inter-operator robustness of this modified protocol in a range of high-throughput functional assays including phagocytosis, lysosomal acidification, chemotaxis, and cytokine release. Our study underscores the importance of quality control checks at various stages of iPSC-differentiation and functional assay set up, highlighting novel workarounds to the existing challenges such as limited yield, flexibility, and reproducibility, all critical in drug discovery.

MiR-4270 acts as a tumor suppressor by directly targeting Bcl-xL in human osteosarcoma cells.

Chondrosarcomas and osteosarcomas are malignant bone tumors with a poor prognosis when unresectable or metastasized. Moreover, radiotherapy and chemotherapy could be ineffective. MiRNAs represent an alternative therapeutic approach. Based on high-throughput functional screening, we identified four miRNAs with a potential antiproliferative effect on SW1353 chondrosarcoma cells. Individual functional validations were then performed in SW1353 cells, as well as in three osteosarcoma cell lines. The antiproliferative and cytotoxic effects of miRNAs were evaluated in comparison with a positive control, miR-342-5p. The cytotoxic effect of four selected miRNAs was not confirmed on SW1353 cells, but we unambiguously revealed that miR-4270 had a potent cytotoxic effect on HOS and MG-63 osteosarcoma cell lines, but not on SaOS-2 cell line. Furthermore, like miR-342-5p, miR-4270 induced apoptosis in these two cell lines. In addition, we provided the first report of Bcl-xL as a direct target of miR-4270. MiR-4270 also decreased the expression of the anti-apoptotic protein Mcl-1, and increased the expression of the pro-apoptotic protein Bak. Our findings demonstrated that miR-4270 has tumor suppressive activity in osteosarcoma cells, particularly through Bcl-xL downregulation.

Abstract P1034: Functional Modelling Of Arrhythmias With Human Cardiac Organoids

Engineered heart tissues (EHTs) have become a well-established model of human cardiac muscle. Metabolic maturity, improved sarcoplasmic reticulum (SR) Ca2+ handling, and the incorporation of critical non-myocyte cell populations are advantages that EHTs provide over immature 2D cardiomyocyte models. Our human cardiac organoid-EHT hybrid (hCO) platform was developed to be compatible with standardized 96-well tissue culture formats to enable high-throughput functional screening, while retaining the physiological advantages of larger EHTs. Here, we explored the applicability of hCOs as a model of various arrhythmic mechanisms. QT-prolongation could be mimicked with LTCC agonist BayK8644 or hERG blocker dofetilide, which increased the relaxation time of the hCOs and induced arrhythmia at high concentrations. Ectopy was induced by perturbing SR Ca2+ cycling with ryanodine or caffeine. HCN4 blocker ivabradine slowed rate, and at high concentrations produced a complex pause-burst phenotype. Increasing the cardiomyocyte-to-fibroblast ratio with growth factors, or the addition of extra fibroblasts prolonged contraction duration and induced ectopy. Inflammatory cytokines also increased relaxation time and provoked ectopy. From this data we propose that hCOs are a sensitive and reliable platform for studying arrhythmias and we are currently exploring the impact of CPVT-linked RYR2 and CASQ2 mutations on hCO arrhythmogenesis.

High-throughput functional screen identifies YWHAZ as a key regulator of pancreatic cancer metastasis

Pancreatic cancer is a leading cause of cancer death due to its early metastasis and limited response to the current therapies. Metastasis is a complicated multistep process, which is determined by complex genetic alterations. Despite the identification of many metastasis-related genes, distinguishing the drivers from numerous passengers and establishing the causality in cancer pathophysiology remains challenging. Here, we established a high-throughput and piggyBac transposon-based genetic screening platform, which enables either reduced or increased expression of chromosomal genes near the incorporation site of the gene search vector cassette that contains a doxycycline-regulated promoter. Using this strategy, we identified YWHAZ as a key regulator of pancreatic cancer metastasis. We demonstrated that functional activation of Ywhaz by the gene search vector led to enhanced metastatic capability in mouse pancreatic cancer cells. The metastasis-promoting role of YWHAZ was further validated in human pancreatic cancer cells. Overexpression of YWHAZ resulted in more aggressive metastatic phenotypes in vitro and a shorter survival rate in vivo by modulating epithelial-to-mesenchymal transition. Hence, our study established a high-throughput screening method to investigate the functional relevance of novel genes and validated YWHAZ as a key regulator of pancreatic cancer metastasis.

A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

Deep Mutational Scanning and High-throughput Functional Testing to Resolve Variants of Unknown Significance in Sarcoglycanopathies (N4.001)

<h3>Objective:</h3> The goal of this study was to accurately predict the pathogenicity of all possible protein-coding genetic variation in genes causing sarcoglycanopathies. <h3>Background:</h3> Limb-girdle muscular dystrophies R3/R4/R5 are due to recessive mutations in <i>SGCA</i>, <i>SGCB</i> and <i>SGCG</i>. Genetic variation identified within genetic test reports leads to diagnostic uncertainty. Only 14% of missense variants in the sarcoglycan genes have been asserted as either benign or pathogenic; with the vast majority being “variants of unknown significance (VUS).” VUS in LGMD genes leads to an under recognition of these diseases that have treatments within the pre-clinical pipeline. <h3>Design/Methods:</h3> To help resolve VUSs, we performed Deep Mutational Scanning (DMS) to create lentiviral libraries of all possible coding variations in <i>SGCA</i>, <i>SGCB</i> and <i>SGCG</i>. Using these libraries we performed high throughput and orthogonal functional screens in cells followed by sequencing to determine a functional score for each unique variant. <h3>Results:</h3> We tested the effect of all possible coding variants in <i>SGCB</i> to obtain a functional score that predicts the pathogenicity of each genetic variant. The measured scores were perfectly predictive of known pathogenic and benign variants and out-performed all tested bioinformatic prediction algorithms. Validation studies of questionably pathogenic vs benign variants identified in patients support the robustness of our results. Additionally, patient acquired clinical data demonstrated that our DMS functional score was quantitatively correlated with with disease onset and severity. <h3>Conclusions:</h3> In summary, we have generated a valuable resource or “look up table” of functionally defined pathogenic variants in sarcoglycan genes. This type of approach can be utilized for other LGMD genes. In the future these mutational libraries can be used to perform high throughput analysis in the presence and absence of small molecules that may elucidate variant specific therapies. <b>Disclosure:</b> Dr. Haller has nothing to disclose. Dr. Li has nothing to disclose. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Casma therapeutics. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Abata. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Acceleron. Dr. Weihl has received personal compensation in the range of $5,000-$9,999 for serving as an Expert Witness for Wilson Elser Moskowitz Edelman &amp; Dicker LLP.

MicroRNA-216a is essential for cardiac angiogenesis

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.