High-throughput Functional Assays Research Articles

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes.

ABSTRACTIt has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea.IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

Clinical likelihood ratios and balanced accuracy for 44 in silico tools against multiple large-scale functional assays of cancer susceptibility genes

PurposeWhere multiple in silico tools are concordant, the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework affords supporting evidence toward pathogenicity or benignity, equivalent to a likelihood ratio of ~2. However, limited availability of “clinical truth sets” and prior use in tool training limits their utility for evaluation of tool performance. MethodsWe created a truth set of 9,436 missense variants classified as deleterious or tolerated in clinically validated high-throughput functional assays for BRCA1, BRCA2, MSH2, PTEN, and TP53 to evaluate predictive performance for 44 recommended/commonly used in silico tools. ResultsOver two-thirds of the tool–threshold combinations examined had specificity of <50%, thus substantially overcalling deleteriousness. REVEL scores of 0.8–1.0 had a Positive Likelihood Ratio (PLR) of 6.74 (5.24–8.82) compared to scores <0.7 and scores of 0–0.4 had a Negative Likelihood Ratio (NLR) of 34.3 (31.5–37.3) compared to scores of >0.7. For Meta-SNP, the equivalent PLR = 42.9 (14.4–406) and NLR = 19.4 (15.6–24.9). ConclusionAgainst these clinically validated “functional truth sets," there was wide variation in the predictive performance of commonly used in silico tools. Overall, REVEL and Meta-SNP had best balanced accuracy and might potentially be used at stronger evidence weighting than current ACMG/AMP prescription, in particular for predictions of benignity.

24.1 HIGH-THROUGHPUT FUNCTIONAL ANALYSIS OF RISK GENES OF AUTISM SPECTRUM DISORDER IN ZEBRAFISH

Advances in whole-exome sequencing have led to the identification of a growing number of high-confidence risk genes in autism spectrum disorder (ASD). However, our understanding of the mechanisms by which gene disruption leads to alterations in specific cell types and neural circuits predisposing to behavioral dysfunction, remains limited. The objective of this research is to investigate the extent to which ASD risk genes might converge on common pathways in the developing vertebrate brain as a path toward identifying potential pharmacological targets. We leveraged the advantages of zebrafish to perform high-throughput functional assays of multiple ASD risk genes. Using CRISPR/Cas9, we generated zebrafish mutants lacking the function of 10 high-confidence ASD risk genes. We performed large-scale pharmaco-behavioral profiling assays and targeted drug screens. To identify how gene disruption leads to alterations in brain signaling, we performed whole-brain activity mapping, which allows for the identification of regional differences in baseline brain activity. Using a quantitative behavioral profiling approach, we characterized the behavioral “fingerprints” of 10 zebrafish ASD risk gene mutants. We identified points of convergence and divergence across mutant behavioral profiles of the following risk genes: CHD8, CNTNAP2, CUL3, DYRK1A, GRIN2B, KATNAL2, KDM5B, POGZ, SCN2A, and TBR1. In addition, we screened 775 FDA-approved drugs in wild-type fish and used these behavioral profiles to predict potential suppressors of the mutant behavioral phenotypes. Using pharmaco-behavioral profiling, we previously showed that estrogenic compounds selectively rescue the behavioral phenotype of zebrafish mutants of the ASD and epilepsy-associated gene, CNTNAP2. In addition, using whole-brain activity mapping, we identified differences in baseline brain activity in zebrafish ASD risk gene mutants. These studies highlight the potential of high-throughput functional screens in zebrafish to identify potential points of convergence across ASD risk genes.

Comprehensive functional evaluation of variants of fibroblast growth factor receptor genes in cancer

Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

Better understanding of roles of complement in pathology has fuelled an explosion of interest in complement-targeted therapeutics. The C5-blocking monoclonal antibody (mAb) eculizumab, the first of the new wave of complement blocking drugs, was FDA approved for treatment of Paroxysmal Nocturnal Hemoglobinuria in 2007; its expansion into other diseases has been slow and remains restricted to rare and ultra-rare diseases such as atypical hemolytic uremic syndrome. The success of eculizumab has provoked other Pharma to follow this well-trodden track and made C5 blockade the busiest area of complement drug development. C5 blockade inhibits generation of C5a and C5b, the former an anaphylatoxin, the latter the nidus for formation of the pro-inflammatory membrane attack complex. In order to use anti-complement drugs in common complement-driven diseases, more affordable and equally effective therapeutics are needed. To address this, we explored complement inhibition downstream of C5. Novel blocking mAbs targeting C7 and/or the C5b-7 complex were generated, identified using high throughput functional assays and specificity confirmed by immunochemical assays and surface plasmon resonance (SPR). Selected mAbs were tested in rodents to characterize pharmacokinetics, and therapeutic capacity. Administration of a mouse C7-selective mAb to wildtype mice, or a human C7 specific mAb to C7-deficient mice reconstituted with human C7, completely inhibited serum lytic activity for >48 h. The C5b-7 complex selective mAb 2H2, most active in rat serum, efficiently inhibited serum lytic activity in vivo for over a week from a single low dose (10 mg/kg); this mAb effectively blocked disease and protected muscle endplates from destruction in a rat myasthenia model. Targeting C7 and C7-containing terminal pathway intermediates is an innovative therapeutic approach, allowing lower drug dose and lower product cost, that will facilitate the expansion of complement therapeutics to common diseases.

Abstract 818: Organoid based functional test to predict personalized treatment in cholangiocarcinoma

Abstract Introduction: It is difficult to find targeted therapies that work across the genetically diverse Cholangiocarcinoma (CCA) patient population; genomic sequencing alone has not resulted in substantial changes in treatment choices for this rare cancer. We reasoned that CCA patients would benefit from a highly personalized diagnostic test, where patient-derived tumor organoids are tested with a library of targeted drugs in a high-throughput functional assay. Methods: Seventeen patients with CCA were enrolled in an IRB-approved ongoing study from 2017 to 2019; biopsies were collected from surgical resection and shipped on ice overnight to a CLIA-certified laboratory (SEngine Precision Medicine) for organoid derivation and high throughput drug testing. Tumor organoids were challenged with up to 120 FDA-approved and investigational drugs to obtain 6-dose, 3-log response curves. Drug responses were benchmarked against an in-house database of pan-cancer patients to concurrently assess both sensitivity and the uniqueness of each patient's response. Clinical history and genomic information were collected from each patient for retrospective and prospective analysis. Results: 14/19 (74%) samples were successfully derived from 17 patients and screened. Patient age ranged from 36 to 73 (mean=51). The 120-drug library was adapted based on total viable tumor cells per sample, resulting in average of 71 drugs per screen. Top scoring drugs were Ceritinib, MK2206, Belinostat, Taselisib, AZD4547, Everolimus and Poziotinib. In general, the drug sensitivity highlighted the following pathways: AKT, ALK, FGFR, PI3K and EGFR. Drugs were selected per patient and classified as either good or exceptional response and correlated with mutations in known genomic biomarkers in 7 of 12 drug/target pairs. Retrospective analysis demonstrated 100% concordance (n=3) between ex vivo drug resistance and prior treatments. We observed multiple mutation/drug pairs which showed resistance in our functional screen. We achieved a high success rate and clinically relevant turnaround times, from biopsy to final report generation average 17 days (range 9 to 35). Conclusion: This study shows the feasibility of functional testing of organoids derived from CCA patients in a CLIA-certified diagnostic test. While these results correlate well with genomically predicted drug sensitivities, all patients showed additional drug sensitivities beyond those predicted by genomics offering patients additional potential treatment options. This study highlights the importance of functional data in a genetically heterogenous tumor type such as CCA. Each patient represents a mosaic of drug sensitivities, reflecting unique combinations of genetic and epigenetic alterations, and this test can identify the personalized drug combination for them. Citation Format: Astrid Margossian, Franz X. Schaub, Annie Richardson, Michael Churchill, Rachele Rosati, Alexandra Dullea, G Adam Whitney, Hallie Swan, Robert Diaz, Lauren Appleyard, Vijaykrishna Gadi, Ali Zarrinpar, Milind Javle, Christopher Kemp, Shalini Pereira, Carla Grandori. Organoid based functional test to predict personalized treatment in cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 818.

Virtual Screening for Ligand Discovery at the σ1 Receptor

The σ1 receptor is a transmembrane protein implicated in several pathophysiological conditions, including neurodegenerative disease (J. Pharmacol. Sci.2015127 (1), 1729), drug addiction (Behav. Pharmacol.201627 (2-3 Spec Issue), 10015), cancer (Handb. Exp. Pharmacol.2017244237308), and pain (Neural Regener. Res.201813 (5), 775778). However, there are no high-throughput functional assays for σ1 receptor drug discovery. Here, we assessed high-throughput structure-based computational docking for discovery of novel ligands of the σ1 receptor. We screened a library of over 6 million compounds using the Schrödinger Glide package, followed by experimental characterization of top-scoring candidates. 77% of tested candidates bound σ1 with high affinity (KD < 1 μM). These include compounds with high selectivity for the σ1 receptor compared to the genetically unrelated but pharmacologically similar σ2 receptor, as well as compounds with substantial crossreactivity between the two receptors. These results establish structure-based virtual screening as a highly effective platform for σ1 receptor ligand discovery and provide compounds to prioritize in studies of σ1 biology.

Genetics of mitochondrial diseases: Identifying mutations to help diagnosis.

Mitochondrial diseases are amongst the most genetically and phenotypically diverse groups of inherited diseases. The vast phenotypic overlap with other disease entities together with the absence of reliable biomarkers act as driving forces for the integration of unbiased methodologies early in the diagnostic algorithm, such as whole exome sequencing (WES) and whole genome sequencing (WGS). Such approaches are used in variant discovery and in combination with high-throughput functional assays such as transcriptomics in simultaneous variant discovery and validation. By capturing all genes, they not only increase the diagnostic rate in heterogenous mitochondrial disease patients, but accelerate novel disease gene discovery, and are valuable in side-stepping the risk of overlooking unexpected or even treatable genetic disease diagnoses.

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer.

The clinical utility of BRCA1/2 genotyping was recently extended from the selection of subjects at high risk for hereditary breast and ovary cancer to the identification of candidates for poly (ADP-ribose) polymerase (PARP) inhibitor treatment. This underscores the importance of accurate interpretation of BRCA1/2 genetic variants and of reducing the number of variants of uncertain significance (VUSs). Two recent studies by Findlay et al. and Starita et al. introduced high-throughput functional assays, and proactively analyzed variants in specific regions regardless of whether they had been previously observed. We retrospectively reviewed all BRCA1 and BRCA2 germline genetic test reports from patients with breast or ovarian cancer examined at Asan Medical Center (Seoul, Korea) between September 2011 and December 2018. Variants were assigned pathogenic or benign strong evidence codes according to the functional classification and were reclassified according to the ACMG/AMP 2015 guidelines. Among 3684 patients with available BRCA1 and BRCA2 germline genetic test reports, 429 unique variants (181 from BRCA1) were identified. Of 34 BRCA1 variants intersecting with the data reported by Findlay et al., three missense single-nucleotide variants from four patients (0.11%, 4/3684) were reclassified from VUSs to likely pathogenic variants. Four variants scored as functional were reclassified into benign or likely benign variants. Three variants that overlapped with the data reported by Starita et al. could not be reclassified. In conclusion, proactive high-throughput functional study data are useful for the reclassification of clinically observed VUSs. Integrating additional evidence, including functional assay results, may help reduce the number of VUSs.

Tumor-Derived Organoid Culture For Functional Personalized Oncology In Cholangiocarcinoma

Presenter: Ali Zarrinpar MD, PhD | University of Florida Background: Given the low incidence of tumors such as cholangiocarcinoma (CCA), it is difficult to establish the efficacy of standard treatment protocols with any certainty. This compels clinicians to select therapeutic approaches based on anecdotal evidence or small case series. The advent of tumor genetic sequencing has not resulted in substantial changes in treatment choices, as these fail to identify targets in the vast majority of cases. We have developed a high-throughput functional assay to test a library of targeted drugs against individual patient-derived tumor organoids from CCA. We seek to show the feasibility of this approach to yield experimentally-validated therapeutic options for patients. Methods: Four consecutive patients with CCA who were undergoing a planned standard of care biopsy or resection were enrolled in an IRB-approved prospective pilot study. Approximately 200 mg of tumor was shipped on ice overnight to SEngine Precision Medicine for growth of tumor-derived organoid culture and analysis of drug sensitivities. Organoids were evaluated using a multi-dose response to each drug in a library of 120 FDA-approved and investigational drugs. These Results were compared to all previous patients. This established both functional sensitivity and the uniqueness of each patient’s response. Results: Patient characteristics such as demographics, tumor pathology, Next Generation Sequencing (NGS) recommendations, and organoid-derived drug recommendations are shown in the Table. We optimized operational logistics, assessed tumor growth rates, and measured chemosensitivity reporting timelines. All four specimens resulted in adequate growth to characterize drug sensitivities. No NGS studies performed on these tumors resulted in targeted chemotherapy recommendations. Conversely, tumor organoid assays rapidly identified selective, personalized drugs in all four cases. Conclusion: We have shown the feasibility of this approach in CCA. This pilot study has facilitated the expansion process, quantified the turn-around time, and identified technical and logistic barriers for future clinical trials. Our preliminary Results will inform the design of a future prospective clinical trial to establish and validate this method to select personalized cancer treatments for rare malignancies.

Identification of novel compounds against Tat-mediated human immunodeficiency virus-1 transcription by high-throughput functional screening assay

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2′-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase.Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants

Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.

Abstract GMM-051: A CLIA-CERTIFIED HIGH-THROUGHPUT DRUG SCREENING PLATFORM FOR OVARIAN CANCER TO INFORM PERSONALIZED CANCER CARE AND DISCOVER NOVEL COMBINATIONS

Abstract BACKGROUND: Metastatic disease in ovarian cancer is difficult to treat and patients often exhaust standard-of-care regimens. To gain a better understanding of potential treatments, genomic data can be used. However, the interpretation of the genomic data for ovarian cancer is challenging given complex mutation patterns and the absence of recurrent druggable alterations beyond DNA repair deficiency. Here we present the first CLIA certified high-throughput functional assay employing organoid cultures derived from primary patient specimens to directly aid oncologists for personalized treatment selection in combination with genomic data (P.A.R.I.S® Assay, SEngine Precision Medicine, Seattle, WA). EXPERIMENTAL PROCEDURES: Organoids are exposed to a library of 123 clinically relevant drugs. The library was developed to include chemotherapies and FDA approved drugs currently used in the clinic, as well as promising drugs undergoing clinical trials. Compounds are evaluated at a multi-dose response curve and ranked by SPM™ score, which weighs both the sensitivity (degree of cell death) and specificity, which compares the response of the patient's tumor cells to the drug relative to all prior patients. In addition to single agent screens, optimal combinations can also be tested. The results are further integrated with genomic data when available, and drug-responses are reported to the clinician. SEngine has performed &amp;gt;200 drug screens across &amp;gt;20 different tumor types and established high reproducibility of the high-throughput platform. Ovarian samples were derived from either ascites or biopsies. RESULTS: Of the 7 samples directly derived from patients, 1 was from ascites, 4 were from biopsies or surgical excisions, and 2 had both. In addition, 13 cell lines were screened with the SEngine drug library. While every patient exhibited a unique pattern of response consistent with the heterogeneity of the complex genetic landscape of ovarian cancer, sensitivities for certain drugs such as HDAC, PI3K and tyrosine-kinase inhibitors were frequently found. Several n=1 cases will be presented to highlight correlations with retrospective clinical responses as well as with genomic alterations. These results highlight how genomic data and functional testing can be combined to optimize personalized cancer care. To identify optimal drug combinations with PARP inhibitors, we have performed an unbiased screen where patient derived ovarian cancer cell lines were challenged with SEngine drug library in the presence of rucaparib. The screen indicated multiple drugs, such as bromodomain, BCL2 and cyclin dependent kinase (CDK) inhibitors as well as dasatinib as potential sensitizers to rucaparib. Confirmation of these results in additional patient derived organoids and PDX models are in progress. IMPACT: We developed a robust ex vivo screening platform to objectively quantify patient specific sensitivity to a panel of more than 123 oncology drugs and potential novel combinations. SEngine is compiling a registry capturing clinical data, outcome following the P.A.R.I.S® test, and genomic data. Combining the power of high-throughput technology and organoid isolation with genomic data will enable the rapid selection of optimal individualized therapies as single agents or in combination and guide design of future clinical trials. Citation Format: Franz X Schaub, Sabrina Maisel, Reid Shaw, Rachele Rosati, Caroline Bridgwater, Michael Churchill, Robert Diaz, Stephanie Tatem Murphy, Shalini Pereira, Christopher Kemp, VK Gadi, and Carla Grandori. A CLIA-CERTIFIED HIGH-THROUGHPUT DRUG SCREENING PLATFORM FOR OVARIAN CANCER TO INFORM PERSONALIZED CANCER CARE AND DISCOVER NOVEL COMBINATIONS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-051.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein.

Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2 O2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays.

Functional personalized oncology to determine drug sensitivity in cholangiocarcinoma.

e15672 Background: Rare tumors, such as cholangiocarcinoma (CC), have no established treatment protocols, forcing clinicians to select therapies based on educated guesses or small case series. Genomic tumor analyses fail to identify options for most patients. Here, we show the feasibility of using a high-throughput functional assay to test targeted drug libraries on individual patient-derived tumor organoids from CC to yield experimentally-validated therapeutic options. Methods: Three consecutive patients with CC who were undergoing a standard of care biopsy or resection were enrolled in an IRB-approved study. Patient characteristics, demographics, tumor pathology, including Next Generation Sequencing (NGS) data and organoid-derived drug recommendations are shown in the Table. Specimen (~ 200 mg of tumor) was shipped on ice overnight to SEngine Precision Medicine for tumor-derived organoid culture and sensitivity analysis. Organoids were evaluated using a multi-dose response to each drug in a library of 120 FDA-approved and investigational drugs and compared those responses to all prior patients. This established both functional sensitivity and the uniqueness of each patient’s response. Results: We tested operational logistics, tumor growth rates, and chemosensitivity reporting timelines. This has allowed us to: 1) operationalize the expansion process, 2) quantify turn-around time, and 3) identify any technical or logistic barriers. All specimens resulted in adequate growth for sensitivity characterization. None of the NGS studies resulted in recommendations for sensitivity to targeted chemotherapy. Conversely, tumor organoid assays rapidly identified selective, personalized drugs. Conclusions: We have shown the feasibility of this approach in CC. Our preliminary results will inform the design of a future prospective clinical trial to establish and validate this method to select personalized cancer treatments for rare malignancies.[Table: see text]

The evolution of Great Apes has shaped the functional enhancers' landscape in human embryonic stem cells

High-throughput functional assays of enhancer activity have recently enabled the genome-scale definition of molecular, structural, and biochemical features of these genomic regulatory regions. To infer the evolutionary origin of DNA sequences operating as functional enhancers in human embryonic stem cells (hESC), we examined the patterns of evolutionary conservation and divergence in the genome-wide functional enhancers' landscape of hESC. We show that a prominent majority (up to 94%) of DNA sequences identified in hESC as functional enhancers are conserved in humans and our closest evolutionary relatives, Chimpanzee and Bonobo. More than 91% of functional enhancers that are highly conserved in both Chimpanzee and Bonobo, are conserved among other Great Apes and >75% are conserved in the Rhesus genome. In striking contrast, <5% of DNA sequences operating in hESC as functional enhancers are conserved in rodents. Conserved in primates enhancers' sequences are complemented by 1619 sequences of enhancers that are specific to humans. Enhancers that harbor human-specific sequences appear enriched among the invariant enhancer module maintaining activity in different pluripotent states and these regions are associated with pluripotency- and embryonic-lineage-related genes. However, functional enhancers make up only a minority of all conserved in primates or human-specific transcription factor binding sites. Our analyses revealed that sequences that are conserved during ~8 million years of primate evolution dominate the genomic landscape of functional enhancers in both primed and naïve hESC. Collectively, these observations revealed thousands of evolutionarily conserved sequences that function as a core regulatory network in human embryonic stem cells which has recently undergone further extension after divergence of modern humans from our closest relatives, Chimpanzee and Bonobo.

Functional assays for transcription mechanisms in high-throughput.

Dramatic increases in the scale of programmed synthesis of nucleic acid libraries coupled with deep sequencing have powered advances in understanding nucleic acid and protein biology. Biological systems centering on nucleic acids or encoded proteins greatly benefit from such high-throughput studies, given that large DNA variant pools can be synthesized and DNA, or RNA products of transcription, can be easily analyzed by deep sequencing. Here we review the scope of various high-throughput functional assays for studies of nucleic acids and proteins in general, followed by discussion of how these types of study have yielded insights into the RNA Polymerase II (Pol II) active site as an example. We discuss methodological considerations in the design and execution of these experiments that should be valuable to studies in any system.