Drug Target Genes Research Articles

Multi-layered genetic approaches to identify approved drug targets

Drugs targeting genes linked to disease via evidence from human genetics have increased odds of approval. Approaches to prioritize such genes include genome-wide association studies (GWASs), rare variant burden tests in exome sequencing studies (Exome), or integration of a GWAS with expression/protein quantitative trait loci (eQTL/pQTL-GWAS). Here, we compare gene-prioritization approaches on 30 clinically relevant traits and benchmark their ability to recover drug targets. Across traits, prioritized genes were enriched for drug targets with odds ratios (ORs) of 2.17, 2.04, 1.81, and 1.31 for the GWAS, eQTL-GWAS, Exome, and pQTL-GWAS methods, respectively. Adjusting for differences in testable genes and sample sizes, GWAS outperforms e/pQTL-GWAS, but not the Exome approach. Furthermore, performance increased through gene network diffusion, although the node degree, being the best predictor (OR= 8.7), revealed strong bias in literature-curated networks. In conclusion, we systematically assessed strategies to prioritize drug target genes, highlighting the promises and pitfalls of current approaches.

Coregulation of extracellular vesicle production and fluconazole susceptibility in Cryptococcus neoformans.

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Genomic Innovation in Early Life Cardiovascular Disease Prevention and Treatment.

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality globally. Although CVD events do not typically manifest until older adulthood, CVD develops gradually across the life-course, beginning with the elevation of risk factors observed as early as childhood or adolescence and the emergence of subclinical disease that can occur in young adulthood or midlife. Genomic background, which is determined at zygote formation, is among the earliest risk factors for CVD. With major advances in molecular technology, including the emergence of gene-editing techniques, along with deep whole-genome sequencing and high-throughput array-based genotyping, scientists now have the opportunity to not only discover genomic mechanisms underlying CVD but use this knowledge for the life-course prevention and treatment of these conditions. The current review focuses on innovations in the field of genomics and their applications to monogenic and polygenic CVD prevention and treatment. With respect to monogenic CVD, we discuss how the emergence of whole-genome sequencing technology has accelerated the discovery of disease-causing variants, allowing comprehensive screening and early, aggressive CVD mitigation strategies in patients and their families. We further describe advances in gene editing technology, which might soon make possible cures for CVD conditions once thought untreatable. In relation to polygenic CVD, we focus on recent innovations that leverage findings of genome-wide association studies to identify druggable gene targets and develop predictive genomic models of disease, which are already facilitating breakthroughs in the life-course treatment and prevention of CVD. Gaps in current research and future directions of genomics studies are also discussed. In aggregate, we hope to underline the value of leveraging genomics and broader multiomics information for characterizing CVD conditions, work which promises to expand precision approaches for the life-course prevention and treatment of CVD.

150 risk variants for diverticular disease of intestine prioritize cell types and enable polygenic prediction of disease susceptibility

We conducted a genome-wide association study (GWAS) analysis of diverticular disease (DivD) of intestine within 724,372 individuals and identified 150 independent genome-wide significant DNA variants. Integration of the GWAS results with human gut single-cell RNA sequencing data implicated gut myocyte, mesothelial and stromal cells, and enteric neurons and glia in DivD development. Ninety-five genes were prioritized based on multiple lines of evidence, including SLC9A3, a drug target gene of tenapanor used for the treatment of the constipation subtype of irritable bowel syndrome. A DivD polygenic score (PGS) enables effective risk prediction (area under the curve [AUC], 0.688; 95% confidence interval [CI], 0.645-0.732) and the top 20% PGS was associated with ∼3.6-fold increased DivD risk relative to the remaining population. Our statistical and bioinformatic analyses suggest that the mechanism of DivD is through colon structure, gut motility, gastrointestinal mucus, and ionic homeostasis. Our analyses reinforce the link between gastrointestinal disorders and the enteric nervous system through genetics.

Transcription‐based drug repurposing in Alzheimer disease psychosis

AbstractBackgroundThere are at least 50 million people with Alzheimer’s Disease (AD) or other dementias worldwide. About 50% of people with AD may develop psychotic symptoms (delusions and hallucinations). However, there is no effective pharmacological therapy for psychosis in AD and all current treatments are based on antipsychotics originally designed for other disorders like schizophrenia. In this study, we identify the molecular signature of psychosis in AD in post‐mortem brain samples and then use the connectivity map (CMAP) and library of network‐based cellular signatures (LINC) to predict candidate drugs based on the disease transcriptional signature.MethodWe generated RNA‐Seq data (Illumina Stranded mRNA Prep) and compared the gene expression levels across 4 groups: Control, AD with no psychosis (AD), AD with Delusions (ADD), and AD with both Delusions and Hallucinations (ADDH). The final identified differentially expressed up and down‐regulated genes (DEGs) were used as query signatures in the CMAP and LINC databases to identify drugs that could potentially reverse the gene expression signatures in disease samples. To investigate the disease mechanisms and the effect of repurposed drugs on it, a drug‐target interaction network was reconstructed and combined with the human interactome. Enrichment analysis of this network identifies AD and psychosis‐related pathways and the potential effect the nominated drugs have on them.ResultAfter filtering out the common DEGs, 357, 171, and 66 unique genes remained in the AD, ADD, and ADDH signature sets, respectively. The signatureSearch package was used to search transcriptome signatures against CMAP and LINC. The top ranked drugs, based on negative connectivity scores, were selected as final results. Gene set enrichment analysis of the drug target genes presented some key pathways and activities related to psychiatric disorders. For example, serotonin receptor signalling pathway is one of the most over‐represented pathway of target genes in both ADD and ADDH groups.ConclusionThis study can improve our understanding of the molecular mechanisms underlying AD psychosis. The candidate drugs we have identified could be repurposed for clinical trials of psychosis in AD and provide valuable knowledge for future pharmacological research.

Analysis of immunogenic cell death in atherosclerosis based on scRNA-seq and bulk RNA-seq data.

Regulated cell death plays a very important role in atherosclerosis (AS). Despite a large number of studies, there is a lack of literature on immunogenic cell death (ICD) in AS. Carotid atherosclerotic plaque single-cell RNA (scRNA) sequencing data were analyzed to define involved cells and determine their transcriptomic characteristics. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, CIBERSORT, ESTIMATE and ssGSEA (Gene Set Enrichment Analysis), consensus clustering analysis, random forest (RF), Decision Curve Analysis (DCA), and the Drug-Gene Interaction and DrugBank databases were applied for bulk sequencing data. All data were downloaded from Gene Expression Omnibus (GEO). mDCs and CTLs correlated obviously with AS occurrence and development (k2(mDCs)=48.333, P<0.001; k2(CTL)=130.56, P<0.001). In total, 21 differentially expressed genes were obtained for the bulk transcriptome; KEGG enrichment analysis results were similar to those for differentially expressed genes in endothelial cells. Eleven genes with a gene importance score>1.5 were obtained in the training set and validated in the test set, resulting in 8 differentially expressed genes for ICD. A model to predict occurrence of AS and 56 drugs that may be used to treat AS were obtained with these 8 genes. Immunogenic cell death occurs mainly in endothelial cells in AS. ICD maintains chronic inflammation in AS and plays a crucial role in its occurrence and development. ICD related genes may become drug-targeted genes for AS treatment.

Isolation, Identification and Molecular Mechanism Analysis of the Nematicidal Compound Spectinabilin from Newly Isolated Streptomyces sp. DT10.

Plant parasitic nematodes (PPNs) are highly destructive and difficult to control, while conventional chemical nematicides are highly toxic and cause serious environmental pollution. Additionally, resistance to existing pesticides is becoming increasingly common. Biological control is the most promising method for the controlling of PPNs. Therefore, the screening of nematicidal microbial resources and the identification of natural products are of great significance and urgency for the environmentally friendly control of PPNs. In this study, the DT10 strain was isolated from wild moss samples and identified as Streptomyces sp. by morphological and molecular analysis. Using Caenorhabditis elegans as a model, the extract of DT10 was screened for nematicidal activity, which elicited 100% lethality. The active compound was isolated from the extracts of strain DT10 using silica gel column chromatography and semipreparative high-performance liquid chromatography (HPLC). The compound was identified as spectinabilin (chemical formula C28H31O6N) using liquid chromatography mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Spectinabilin exhibited a good nematicidal activity on C. elegans L1 worms, with a half-maximal inhibitory concentration (IC50) of 2.948 μg/mL at 24 h. The locomotive ability of C. elegans L4 worms was significantly reduced when treated with 40 μg/mL spectinabilin. Further analysis of spectinabilin against known nematicidal drug target genes in C. elegans showed that it acts via target(s) different from those of some currently used nematicidal drugs such as avermectin and phosphine thiazole. This is the first report on the nematicidal activity of spectinabilin on C. elegans and the southern root-knot nematode Meloidogyne incognita. These findings may pave the way for further research and application of spectinabilin as a potential biological nematicide.

Genome-wide multi-trait analysis of irritable bowel syndrome and related mental conditions identifies 38 new independent variants

BackgroundIrritable bowel syndrome (IBS) is a chronic disorder of gut-brain interaction frequently accompanied by mental conditions, including depression and anxiety. Despite showing substantial heritability and being partly determined by a genetic component, the genetic underpinnings explaining the high rates of comorbidity remain largely unclear and there are no conclusive data on the temporal relationship between them. Exploring the overlapping genetic architecture between IBS and mental conditions may help to identify novel genetic loci and biological mechanisms underlying IBS and causal relationships between them.MethodsWe quantified the genetic overlap between IBS, neuroticism, depression and anxiety, conducted a multi-trait genome-wide association study (GWAS) considering these traits and investigated causal relationships between them by using the largest GWAS to date.ResultsIBS showed to be a highly polygenic disorder with extensive genetic sharing with mental conditions. Multi-trait analysis of IBS and neuroticism, depression and anxiety identified 42 genome-wide significant variants for IBS, of which 38 are novel. Fine-mapping risk loci highlighted 289 genes enriched in genes upregulated during early embryonic brain development and gene-sets related with psychiatric, digestive and autoimmune disorders. IBS-associated genes were enriched for target genes of anti-inflammatory and antirheumatic drugs, anesthetics and opioid dependence pharmacological treatment. Mendelian-randomization analysis accounting for correlated pleiotropy identified bidirectional causal effects between IBS and neuroticism and depression and causal effects of the genetic liability of IBS on anxiety.ConclusionsThese findings provide evidence of the polygenic architecture of IBS, identify novel genome-wide significant variants for IBS and extend previous knowledge on the genetic overlap and relationship between gastrointestinal and mental disorders.

In vitro and in vivo evaluation of efficacy of berberine chloride: Phyto-alternative approach against Trypanosoma evansi infection

Current chemotherapy against the Surra organism, Trypanosoma evansi has several limitations in terms of efficacy, toxicity, availability and emerging resistance. These reasons make the search of new chemo-preventive and chemo-therapeutic agent with high potency and low toxicity. Alkaloid phyto-molecules, berberine has shown promising anti-kinetoplastid activity against T. cruzi, T. congolense, T. brucei, Leishmania donovani and L. tropica. However, till date, there is no investigation of therapeutic efficacy of berberine chloride (BC) against T. evansi. The IC50 value of BC for growth inhibition of T. evansi at 24 h of culture was calculated as 12.15 µM. The specific selectivity index (SSI) of BC was calculated as 19.01 and 10.43 against Vero cell line and Equine PBMC’s, respectively. Thirteen drug target genes affecting various metabolic pathways were studied to investigate the mode of trypanocidal action of BC. In transcript analysis, the mRNA expression of arginine kinase 1 remained refractory to exposure with BC, which provides metabolic plasticity in adverse environmental conditions. In contrary, rest all the drug target gene were down-regulated, which indicates that drug severely affect DNA replication, cell proliferation, energy homeostasis, redox homeostasis and calcium homeostasis of T. evansi, leading to the death of parasite in low concentrations. It is the first attempt to investigate in vitro anti-trypanosomal activity of BC against T. evansi. These data imply that phytochemicals as alternative strategies can be explored in the future as an alternative treatment for Surra in animal.

Repurposing antidiabetic drugs for rheumatoid arthritis: results from a two-sample Mendelian randomization study

Despite increasing therapeutic options to treat rheumatoid arthritis (RA), many patients fail to reach treatment targets. The use of antidiabetic drugs like thiazolidinediones has been associated with lower RA risk. We aimed to explore the repurposing potential of antidiabetic drugs in RA prevention by assessing associations between genetic variation in antidiabetic drug target genes and RA using Mendelian randomization (MR). A two-sample MR design was used to estimate the association between the antidiabetic drug and RA risk using summary statistics from genome-wide association studies (GWAS). We selected independent genetic variants from the gene(s) that encode the target protein(s) of the investigated antidiabetic drug as instruments. We extracted the associations of instruments with blood glucose concentration and RA from the UK Biobank and a GWAS meta-analysis of clinically diagnosed RA, respectively. The effect of genetic variation in the drug target(s) on RA risk was estimated by the Wald ratio test or inverse-variance weighted method. Insulin and its analogues, thiazolidinediones, and sulfonylureas had valid genetic instruments (n = 1, 1, and 2, respectively). Genetic variation in thiazolidinedione target (gene: PPARG) was inversely associated with RA risk (odds ratio [OR] 0.38 per 0.1mmol/L glucose lowering, 95% confidence interval [CI] 0.20–0.73). Corresponding ORs (95%CIs) were 0.83 (0.44–1.55) for genetic variation in the targets of insulin and its analogues (gene: INSR), and 1.12 (0.83, 1.49) 1.25 (0.78-2.00) for genetic variation in the sulfonylurea targets (gene: ABCC8 and KCNJ11). In conclusion, genetic variation in the thiazolidinedione target is associated with a lower RA risk. The underlying mechanisms warrant further exploration.

Association between antihypertensive drugs and hepatocellular carcinoma: A trans-ancestry and drug-target Mendelian randomization study.

Antihypertensive drugs were recently reported to have an oncogenic role in common cancer, however, whether these drugs would affect the risk of hepatocellular carcinoma (HCC) remains unclear. A drug-target Mendelian randomization method was adopted to examine the long-term effect of 12 antihypertensive drugs classes on the risk of HCC in Europeans and East Asians. To proxy antihypertensive drugs, we leveraged genetic variants located near or within drug target genes that were associated with systolic blood pressure (SBP). Genetically proxied drugs associated with reduced risk of coronary artery disease were included in primary analysis. Genetic summary statistics of SBP and HCC were derived from publicly available large-scale genome-wide association studies in Europeans and East Asians respectively. Expression quantitative trait loci (eQTLs) of drugs target genes were used to proxy drugs in a sensitivity analysis. Genetically proxied thiazides and related diuretics were associated with decreased risk of HCC in both Europeans (OR [95% CI]: 0.79 [0.73, 0.86] per 1 mmHg reduction in SBP; p < 0.001) and East Asians (0.60 [0.45, 0.82]; p = 0.001). Genetically proxied beta-adrenoceptor blockers (BBs) were strongly associated with increased risk of HCC in Europeans (1.46 [1.12, 1.91]; p = 0.004). These findings were replicated in deCODE genetics study and remained consistent when using eQTLs to proxy antihypertensive drugs. Our findings suggested that thiazides diuretics may lower the risk of HCC in both Europeans and East Asians, while BBs may increase the risk of HCC specifically in Europeans. Further studies are warranted to explore the potential of repurposing or retargeting antihypertensive drugs for HCC prevention.

Abstract 3118: Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks

Abstract Introduction: PARP inhibitors (PARPi) are known to have a synthetic lethal (SL) relationship with BRCA1/2 mutated cancers (ie: BRCA loss confers sensitivity to PARPi), but heterogeneity in response exists. PARPi have also found utility in BRCA-non mutated cancers, suggesting other factors may contribute to response. Recently, PARPi trials in pediatric solid tumors have had mixed results. SL interactions have been shown to exist in a network relationship, as such the aim of this study was to identify a clinically relevant SL signature (including synthetic dosage lethality and synthetic rescue) that would predict response to PARPi in pediatric cancers. Methods: Pre-treatment genomic copy number alteration, RNA sequencing, and survival data from 519 pediatric cancer patients (INFORM Consortium, 91% solid tumor) were obtained. SL partner genes for PARPi were selected if co-alteration of the drug target (PARP1/2/3) and candidate gene was 1) found to be under-represented in the patient transcriptome, 2) associated with improved survival, and 3) the genes were phylogenetically similar to PARP1/2/3. The 100 gene signature was validated on two independent pediatric cancer drug screens: in-vivo orthotopic patient-derived xenograft (OPDX) mouse models (n=24) and in-vitro PRISM cell lines (n=54). It was also validated on a cisplatin (PARP surrogate) treated pediatric osteosarcoma clinical trial (n=37). Comparisons were made to 1) a signature generated with the same pipeline using adult TCGA data, 2) a validated adult synthetic lethality pipeline (SELECT), and 3) a published 60-gene PARPi response signature (BRCAness score). Results: The pediatric signature predicted response in all drug screens evaluated. Four cell line screens: Talazoparib (AUC 0.72, p = 0.003), Rucaparib (AUC 0.69, p = 0.01), Niraparib (AUC 0.67, p = 0.03), Olaparib (AUC 0.64, p = 0.06), and two OPDX screens: Talazoparib (AUC 0.74, p = 0.03), Olaparib (AUC 0.74, p = 0.03). The pediatric signature outperformed both adult signatures (AUCs 0.34-0.61, all p’s &amp;gt;0.05), and the BRCAness score (AUCs 0.41-0.53, p’s &amp;gt;0.05) except in the Olaparib cell line (BRCAness: AUC 0.68, p = 0.01). In the pediatric osteosarcoma dataset, the pediatric signature predicted death (AUC 0.68) and two-year recurrence (AUC 0.72), outperforming the other measures (death: AUC 0.49-0.66; two-year recurrence: AUCs 0.36-0.44). Discussion: We developed a pediatric derived, clinically relevant, synthetic lethal signature that predicts response to PARPi in pan-pediatric cancer drug screens and in a cisplatin treated osteosarcoma clinical trial. Our findings support the goal of identifying precision oncology frameworks to improve the care of children with cancer. Citation Format: Matthew Nagy, Fiorella Schischlik, Kun Wang, Nishanth Ulhas Nair, E. Michael Gertz, Lipika R. Pal, Natalie Jaeger, Christopher Previti, Dina ElHarouni, Stefan M. Pfister, Eytan Ruppin. Predicting response to PARP inhibitors in pediatric cancer via synthetic lethal networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3118.

Abstract 2690: In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC)

Abstract Purpose: To identify druggable gene targets that would sensitize NSCLCs to available chemotherapy agents. Experimental Procedures: We developed a CRISPR-Cas9 lentivirus library of 12,474 sgRNAs targeting 660 FDA proved ‘druggable’ putative proteins (18 guides/gene) and investigated their potential to chemosensitize (“drop out” in the presence of taxane) the NSCLC lung adenocarcinoma (LUAD) line NCI-H2009 (TP53 and KRAS mutant) in parallel studies in vitro (tissue culture) &amp; in vivo (xenografts). Key elements of our experiments were: the use low doses of paclitaxel (IC10 values for in vitro and in vivo doses that barely affected tumor growth) compared to control treatment; treatment schedules in vitro and in vivo that mirrored those used in patients; multiple biologic replicates for each transfection and drug selection; and large representation (2,000 cells/sgRNA) for each guide. At the end time point (1 month) we harvested multiple replicates of the taxane and control treatments and sequenced each to identify each guide that “remained” or “dropped out.” Data Summary: We were looking for guides that selectively dropped out in the setting of low dose taxane exposure comparing taxane to control treatment and in vitro to in vivo results. Of importance, we found little overlap between guides that dropped out during taxane exposure in tissue culture vs those that dropped out in xenografts. From the top 10 gene “sensitizers” that selectively dropped out only with taxane treatment and selectively dropped out in xenografts compared to 2D mass tissue culture, we focused on SOAT1 (Sterol O-Acyltransferase 1). SOAT1 is a key enzyme for lipid metabolism which mediates conversion of intracellular free cholesterol to cholesteryl esters which are then stored as lipid droplets. SOAT1 is a potential cancer therapeutic target with a clinically available inhibitor, avasimibe. H2009 cells with SOAT1 hemizygously removed (CRISPR) grew well in vitro and in vivo in the absence of chemotherapy treatment, but were dramatically sensitized to taxanes compared to parental H2009 cells. While avasimibe treatment, at concentrations achievable in patients, sensitized NSCLC to taxanes, this sensitization was not as dramatic as hemizygous removal by CRISPR. Of equal importance, SOAT1 H2009 hemizygous knockout (KO) cells were also dramatically sensitized to etoposide, pemetrexed, and gemcitabine, other chemotherapy agents used in NSCLC treatment. Mechanistically, RNAseq analysis identified G2/M cell cycle related gene sets as enriched in the H2009 SOAT1 KO xenografts compared to SOAT1 wildtype xenografts with paclitaxel treatment. Conclusions: Our results using CRISPR screening in vivo, identified SOAT1 as a critical therapeutic chemosensitizing target requiring only 50% inhibition of activity for sensitizing NSCLC to several chemotherapy agents routinely used in the clinic. Citation Format: Long Shan Li, Kenneth Huffman, Huiyu Li, Michael Peyton, Hyunsil Park, Kimberley Avila, Luc Girard, Mathew Augustine, Joshua T. Mendell, John D. Minna. In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2690.

Abstract 5320: The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets

Abstract In recent years, an increasing number of therapeutic antibodies have shown to be effective for the treatment of cancer and other diseases. However, limitations in the traditional discovery process, including immune tolerance of highly homologous genes, challenges with antibody sequence humanization, clone selection, and model selection for drug efficacy and safety evaluation, often hinder the process of identifying new therapeutic antibodies. The RenMice™ HiTS (Hyperimmune Target Specific) Platform is a library of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific drug target gene knocked out. These mice are designed to establish robust immune responses and generate antibodies that bind to more epitopes of the target protein, including conserved domains. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins (e.g. GPCRs/ion channels). Here, we show that the platform can be used to generate antibodies that cross-react with multiple species, like human, monkey, dog, and mouse targets, by immunizing with both human and mouse or dog antigen. We provide examples for newer campaigns, including species cross-reactivity and internalization of novel antibodies targeting NECTIN-4, and high-throughput in vivo efficacy screening of novel anti-PD-1 antibodies in wild-type mice. In the future, we will evaluate the preliminary toxicity of these cross-reactive antibodies in preclinical animal models. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of developable antibodies that recognize novel epitopes and challenging targets. Citation Format: Xiaoqian Zhang, Shufang Fu, Shujin Zhang, Xin Ji, Li Hui, James Jin, Jing Zhang. The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5320.

Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in non-tuberculous mycobacteria isolates from Kenya.

Non-tuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing Mycobacteria (RGM), and rifampicin for Slow-Growing Mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene. NTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in the rrl gene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058 (A2058G, A2058C, A2058T) of the rrl gene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included for rrs characterization, 11.1% (6/54) exhibited mutations at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in the rpoB gene at positions S531W, S531L, S531Y, F506L, E509H with M.gastri having multiple mutations at positions D516V, H526D and, S531F. We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.

Genetic association of lipids and lipid-lowering drug target genes with non-alcoholic fatty liver disease.

Some observational studies found that dyslipidaemia is a risk factor for non-alcoholic fatty liver disease (NAFLD), and lipid-lowering drugs may lower NAFLD risk. However, it remains unclear whether dyslipidaemia is causative for NAFLD. This Mendelian randomisation (MR) study aimed to explore the causal role of lipid traits in NAFLD and evaluate the potential effect of lipid-lowering drug targets on NAFLD. Genetic variants associated with lipid traits and variants of genes encoding lipid-lowering drug targets were extracted from the Global Lipids Genetics Consortium genome-wide association study (GWAS). Summary statistics for NAFLD were obtained from two independent GWAS datasets. Lipid-lowering drug targets that reached significance were further tested using expression quantitative trait loci data in relevant tissues. Colocalisation and mediation analyses were performed to validate the robustness of the results and explore potential mediators. No significant effect of lipid traits and eight lipid-lowering drug targets on NAFLD risk was found. Genetic mimicry of lipoprotein lipase (LPL) enhancement was associated with lower NAFLD risks in two independent datasets (OR1=0.60 [95% CI 0.50-0.72], p1=2.07×10-8; OR2=0.57 [95% CI 0.39-0.82], p2=3.00×10-3). A significant MR association (OR=0.71 [95% CI, 0.58-0.87], p=1.20×10-3) and strong colocalisation association (PP.H4=0.85) with NAFLD were observed for LPL expression in subcutaneous adipose tissue. Fasting insulin and type 2 diabetes mediated 7.40% and 9.15%, respectively, of the total effect of LPL on NAFLD risk. Our findings do not support dyslipidaemia as a causal factor for NAFLD. Among nine lipid-lowering drug targets, LPL is a promising candidate drug target in NAFLD. The mechanism of action of LPL in NAFLD may be independent of its lipid-lowering effects. Capital's Funds for Health Improvement and Research (2022-4-4037). CAMS Innovation Fund for Medical Sciences (CIFMS, grant number: 2021-I2M-C&T-A-010).

Non-steroidal anti-inflammatory drug target gene associations with major depressive disorders: a Mendelian randomisation study integrating GWAS, eQTL and mQTL Data

Previous observational studies reported associations between non-steroidal anti-inflammatory drugs (NSAIDs) and major depressive disorder (MDD), however, these associations are often inconsistent and underlying biological mechanisms are still poorly understood. We conducted a two-sample Mendelian randomisation (MR) study to examine relationships between genetic variants and NSAID target gene expression or DNA methylation (DNAm) using publicly available expression, methylation quantitative trait loci (eQTL or mQTL) data and genetic variant-disease associations from genome-wide association studies (GWAS of MDD). We also assessed drug exposure using gene expression and DNAm levels of NSAID targets as proxies. Genetic variants were robustly adjusted for multiple comparisons related to gene expression, DNAm was used as MR instrumental variables and GWAS statistics of MDD as the outcome. A 1-standard deviation (SD) lower expression of NEU1 in blood was related to lower C-reactive protein (CRP) levels of −0.215 mg/L (95% confidence interval (CI): 0.128–0.426) and a decreased risk of MDD (odds ratio [OR] = 0.806; 95% CI: 0.735–0.885; p = 5.36 × 10−6). A concordant direction of association was also observed for NEU1 DNAm levels in blood and a risk of MDD (OR = 0.886; 95% CI: 0.836–0.939; p = 4.71 × 10−5). Further, the genetic variants associated with MDD were mediated by NEU1 expression via DNAm (β = −0.519; 95% CI: −0.717 to −0.320256; p = 3.16 × 10−7). We did not observe causal relationships between inflammatory genetic marker estimations and MDD risk. Yet, we identified a concordant association of NEU1 messenger RNA and an adverse direction of association of higher NEU1 DNAm with MDD risk. These results warrant increased pharmacovigilance and further in vivo or in vitro studies to investigate NEU1 inhibitors or supplements for MDD.

PDE3A and GSK3B as Atrial Fibrillation Susceptibility Genes in the Chinese Population via Bioinformatics and Genome-Wide Association Analysis

Atrial fibrillation (AF) is the most common cardiac arrhythmia, with uncovered genetic etiology and pathogenesis. We aimed to screen out AF susceptibility genes with potential pathogenesis significance in the Chinese population. Differentially expressed genes (DEGs) were screened by the Limma package in three GEO data sets of atrial tissue. AF-related genes were identified by combination of DEGs and public GWAS susceptibility genes. Potential drug target genes were selected using the DrugBank, STITCH and TCMSP databases. Pathway enrichment analyses of AF-related genes were performed using the databases GO and KEGG databases. The pathway gene network was visualized by Cytoscape software to identify gene-gene interactions and hub genes. GWAS analysis of 110 cases of AF and 1201 controls was carried out through a genome-wide efficient mixed model in the Fangshan population to verify the results of bioinformatic analysis. A total of 3173 DEGs were identified, 57 of which were found to be significantly associated with of AF in public GWAS results. A total of 75 AF-related genes were found to be potential therapeutic targets. Pathway enrichment analysis selected 79 significant pathways and classified them into 7 major pathway networks. A total of 35 hub genes were selected from the pathway networks. GWAS analysis identified 126 AF-associated loci. PDE3A and GSK3B were found to be overlapping genes between bioinformatic analysis and GWAS analysis. We screened out several pivotal genes and pathways involved in AF pathogenesis. Among them, PDE3A and GSK3B were significantly associated with the risk of AF in the Chinese population. Our study provided new insights into the mechanisms of action of AF.

Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya

Background: Nontuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing mycobacteria (RGM), and rifampicin for Slow-Growing mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene. Results: Twenty-eight (23%) of the NTM harbored mutations associated with resistance to at least one of the macrolide-based therapy antibiotics. Twelve (10.4%) NTM comprising 7(58.3%) of RGM and 5(41.7%) of SGM had mutations in the rrl gene. For ten (83.3%) NTM, mutations were found at position 2058, while for two (16.6%) NTM, mutations were found at position 2059. Six (11.1%) of the 54 RGM exhibited mutations in the aminoglycoside target gene rrs at location 1408. Ten (14.7%) of the 68 SGM harbored mutations in the rpoB gene with 40 percent having mutations at codon 531. Conclusion: We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.

Clinical application of circulating tumor DNA (ctDNA) in patients with metastatic gastric cancer.

441 Background: Circulating tumor DNA (ctDNA) is a minimally invasive approach, effective for the real-time follow-up of cancer. However, the clinical significance in gastric cancer is not yet determined. Therefore, we investigated both ctDNA and tissue biopsy NGS at diagnosis of stage IV gastric cancer patients to evaluate the relationship of ctDNA with clinical parameters and concordance of ctDNA and tissue NGS. Methods: All tissue samples were sequenced with the targeted panel sequencing – CancerMaster Panel V2 (in-house 522 gene panel, Kwon et al., ASCO GI 2021) or TruSight Oncology (TSO) 500 (illumina, Inc. USA). 20mL of whole blood in DxTube(Dxome, Korea) was used for library preparation using DxSeq ctDNA TMB500 for Illumina Platform Kit (Dxome, Korea) according to the manufacturer’s instructions. For each sample, PBMC was sequenced as germline paired controls using the identical panel, and library kits targeting an average depth of &gt;3,000x. ctDNA analysis using TMB500 panel reports total ctDNA amount, genomic alterations including copy number variations, tumor mutational burden (ctTMB) and MSI status (Lee et al, Clinical Chemistry accepted). Her-2, EBV, dMMR status in the tissues were performed in standard manner. Results: ctDNA were analysed since 2 samples were not qualified. 27 chromosomal instable (CIN) type and 46 Genome stable (GS) type based on pathologic evaluation were identified. Higher ctDNA were detected in CIN group than GS group (17.8 vs. 6.8, p&lt;0.01) and ctTMB was higher in EBV or CIN than GS group (12.8 vs. 9.1, p=0.02). Patients with high CEA, LDH or systemic metastases including liver, bone, lung, or distant lymph node showed higher ctTMB than those without such factors (14.7 vs. 8.5, p&lt;0.01, 12 vs. 8.8, p&lt;0.01, and 12.5 vs. 7.8, p&lt;0.01, respectively). ctDNA amount was higher in LDH high and systemic metastases patients (31.3 vs. 6.9, p&lt;0.01 and 12.8 vs 6.3, p&lt;0.01, respectively). Among 53 patients with paired ctDNA and tissue NGS, 49 (92.4%) and 47 (88.7%) had detectable ctDNA alterations and tissue alterations respectively, with concordance of 95.9%. Tissue TMB was higher than ctTMB (14.8 vs. 8.0, p&lt;0.01), but the two showed positive correlation (Pearson c. 0.34, p=0.01). Druggable targets (TP53 mutation, KRAS, ERBB2, FGFR, MET or RICTOR amplification) were more frequently detected by ctDNA than tissue NGS. In 25 patients with TP53 mutation in both tissue and ctDNA, TP53 genetic alterations were identical except 1 patient. When compared with ERBB2 IHC, specificity was 93.6% in ctDNA, 97.9% in tissue NGS and sensitivity of both were 50%. Patients with false positive of ERBB2 in ctDNA showed lesser copy number than those with true positive in ctDNA. Conclusions: Our study showed a potential role of ctDNA in advanced gastric cancer, suggesting ctDNA’s sensitivity in detecting various druggable target genes. Investigation to reveal relationship between ctDNA and clinical outcomes is ongoing.