Androgen receptor-mediated pharmacogenomic expression quantitative trait loci: implications for breast cancer response to AR-targeting therapy.

BACKGROUND: Endocrine therapy is the most important treatment modality of breast cancer patients whose tumors express the estrogen receptor α (ERα). The androgen receptor (AR) is also expressed in the vast majority (80-90%) of ERα-positive tumors. AR-targeting drugs are not used in clinical practice, but have been evaluated in multiple trials and preclinical studies. METHODS: We performed a genome-wide study to identify hormone/drug-induced single nucleotide polymorphism (SNP) genotype - dependent gene-expression, known as PGx-eQTL, mediated by either an AR agonist (dihydrotestosterone) or a partial antagonist (enzalutamide), utilizing a previously well characterized lymphoblastic cell line panel. The association of the identified SNPs-gene pairs with breast cancer phenotypes were then examined using three genome-wide association (GWAS) studies that we have published and other studies from the GWAS catalog. RESULTS: We identified 13 DHT-mediated PGx-eQTL loci and 23 Enz-mediated PGx-eQTL loci that were associated with breast cancer outcomes post ER antagonist or aromatase inhibitors (AI) treatment, or with pharmacodynamic (PD) effects of AIs. An additional 30 loci were found to be associated with cancer risk and sex-hormone binding globulin levels. The top loci involved the genes IDH2 and TMEM9, the expression of which were suppressed by DHT in a PGx-eQTL SNP genotype-dependent manner. Both of these genes were overexpressed in breast cancer and were associated with a poorer prognosis. Therefore, suppression of these genes by AR agonists may benefit patients with minor allele genotypes for these SNPs. CONCLUSIONS: We identified AR-related PGx-eQTL SNP-gene pairs that were associated with risks, outcomes and PD effects of endocrine therapy that may provide potential biomarkers for individualized treatment of breast cancer.

ACE2 and TMPRSS2 SARS-CoV-2 infectivity genes: deep mutational scanning and characterization of missense variants.

The human angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) proteins play key roles in the cellular internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus responsible for the coronavirus disease of 2019 (COVID-19) pandemic. We set out to functionally characterize the ACE2 and TMPRSS2 protein abundance for variant alleles encoding these proteins that contained non-synonymous single-nucleotide polymorphisms (nsSNPs) in their open reading frames (ORFs). Specifically, a high-throughput assay, deep mutational scanning (DMS), was employed to test the functional implications of nsSNPs, which are variants of uncertain significance in these two genes. Specifically, we used a 'landing pad' system designed to quantify the protein expression for 433 nsSNPs that have been observed in the ACE2 and TMPRSS2 ORFs and found that 8 of 127 ACE2, 19 of 157 TMPRSS2 isoform 1 and 13 of 149 TMPRSS2 isoform 2 variant proteins displayed less than ~25% of the wild-type protein expression, whereas 4 ACE2 variants displayed 25% or greater increases in protein expression. As a result, we concluded that nsSNPs in genes encoding ACE2 and TMPRSS2 might potentially influence SARS-CoV-2 infectivity. These results can now be applied to DNA sequence data for patients infected with SARS-CoV-2 to determine the possible impact of patient-based DNA sequence variation on the clinical course of SARS-CoV-2 infection.

IL17RB genetic variants are associated with acamprosate treatment response in patients with alcohol use disorder: A proteomics-informed genomics study.

Acamprosate is a Food and Drug Administration (FDA) approved medication for the treatment of alcohol use disorder (AUD). However, only a subset of patients achieves optimal treatment outcomes. Currently, no biological measures are utilized to predict response to acamprosate treatment. We applied our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Specifically, our previous open-label acamprosate clinical trial recruited 442 patients with AUD who were treated with acamprosate for three months. We first performed proteomics using baseline plasma samples to identify potential biomarkers associated with acamprosate treatment outcomes. Next, we applied our established "proteomics-informed genome-wide association study (GWAS)" research strategy, and identified 12 proteins, including interleukin-17 receptor B (IL17RB), associated with acamprosate treatment response.​ A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variantgenotypes for all of those IL17RB SNPs were protective for alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. In summary, this study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying acamprosate treatment response phenotypes. (The ClinicalTrials.gov Identifier: NCT00662571).

Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder.

The opioid epidemic represents a national crisis. Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. Given the extensive use of prescription opioids and the global opioid epidemic, it is essential to understand how opioids modulate brain cell type function at the single-cell level. We performed single nucleus RNA-seq (snRNA-seq) using iPSC-derived forebrain organoids from three male OUD subjects in response to oxycodone, buprenorphine, or vehicle for seven days. We utilized the snRNA-seq data to identify differentially expressed genes following drug treatment using the Seurat integrative analysis pipeline. We utilized iPSC-derived forebrain organoids and single-cell sequencing technology as an unbiased tool to study cell-type-specific and drug-specific transcriptional responses. After quality control filtering, we analyzed 25787 cells and identified sixteen clusters using unsupervised clustering analysis. Our results reveal distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. Specifically, buprenorphine displayed a significant influence on transcription regulation in glial cells. However, oxycodone induced type I interferon signaling in many cell types, including neural cells in brain organoids. Finally, we demonstrate that oxycodone, but not buprenorphine activated STAT1 and induced the type I interferon signaling in patients with OUD. These data suggest that elevation of STAT1 expression associated with OUD might play a role in transcriptional regulation in response to oxycodone. In summary, our results provide novel mechanistic insight into drug action at single-cell resolution.

TSPAN5 influences serotonin and kynurenine: pharmacogenomic mechanisms related to alcohol use disorder and acamprosate treatment response.

We previously reported that SNPs near TSPAN5 were associated with plasma serotonin (5-HT) concentrations which were themselves associated with selective serotonin reuptake inhibitor treatment outcomes in patients with major depressive disorder (MDD). TSPAN5 SNPs were also associated with alcohol consumption and alcohol use disorder (AUD) risk. The present study was designed to explore the biological function of TSPAN5 with a focus on 5-HT and kynurenine concentrations in the tryptophan pathway. Ethanol treatment resulted in decreased 5-HT concentrations in human induced pluripotent stem cell (iPSC)-derived neuron culture media, and the downregulation of gene expression of TSPAN5, DDC, MAOA, MAOB, TPH1, and TPH2 in those cells. Strikingly, similar observations were made when the cells were treated with acamprosate-an FDA approved drug for AUD therapy. These results were replicated in iPSC-derived astrocytes. Furthermore, TSPAN5 interacted physically with proteins related to clathrin and other vesicle-related proteins, raising the possibility that TSPAN5 might play a role in vesicular function in addition to regulating expression of genes associated with 5-HT biosynthesis and metabolism. Downregulation of TSPAN5 expression by ethanol or acamprosate treatment was also associated with decreased concentrations of kynurenine, a major metabolite of tryptophan that plays a role in neuroinflammation. Knockdown of TSPAN5 also influenced the expression of genes associated with interferon signaling pathways. Finally, we determined that TSPAN5 SNPs were associated with acamprosate treatment outcomes in AUD patients. In conclusion, TSPAN5 can modulate the concentrations of 5-HT and kynurenine. Our data also highlight a potentially novel pharmacogenomic mechanism related to response to acamprosate.

SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.

SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19.

CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes; however, clinically actionable genetic variability in drug metabolism due to these genes has been limited to a few common alleles. The identification and functional characterization of less-common open reading frame sequence variation might help to individualize therapy with drugs that are substrates for the enzymes encoded by these genes. The present study identified seven uncharacterized variants each in CYP2C9 and CYP2C19 using next-generation sequence data for 1013 subjects, and functionally characterized the encoded proteins. Constructs were created and transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and liquid chromatography- tandem mass spectrometry with tolbutamide (CYP2C9) and (S)-mephenytoin (CYP2C19) as prototypic substrates. The results were compared with the SIFT, Polyphen, and Provean functional prediction software programs. Cytochrome P450 oxidoreductase (CPR) activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants of CYP2C9 (P < 0.05) and CYP2C19 (P < 0.0005). However, CYP2C9 709G>C and CYP2C19 65A>G activities were much lower than predicted based on protein content. Substrate intrinsic clearance values for CYP2C9 218C>T, 343A>C, and CYP2C19 337G>A, 518C>T, 556C>T, and 557G>A were less than 25% of wild-type allozymes. CPR activity levels were similar for all variants. In summary, sequencing of CYP2C9 and CYP2C19 in 1013 subjects identified low-frequency variants that had not previously been functionally characterized. In silico predictions were not always consistent with functional assay results. These observations emphasize the need for high-throughput methods for pharmacogene variant mutagenesis and functional characterization.

TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor κB p65: Single Nucleotide Polymorphism and Estrogen Dependence.

T-cell leukemia 1A (TCL1A) single-nucleotide polymorphisms (SNPs) have been associated with aromatase inhibitor-induced musculoskeletal adverse events. We previously demonstrated that TCL1A is inducible by estradiol (E2) and plays a critical role in the regulation of cytokines, chemokines, and Toll-like receptors in a TCL1A SNP genotype and estrogen-dependent fashion. Furthermore, TCLIA SNP-dependent expression phenotypes can be "reversed" by exposure to selective estrogen receptor modulators such as 4-hydroxytamoxifen (4OH-TAM). The present study was designed to comprehensively characterize the role of TCL1A in transcriptional regulation across the genome by performing RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays with lymphoblastoid cell lines. RNA-seq identified 357 genes that were regulated in a TCL1A SNP- and E2-dependent fashion with expression patterns that were 4OH-TAM reversible. ChIP-seq for the same cells identified 57 TCL1A binding sites that could be regulated by E2 in a SNP-dependent fashion. Even more striking, nuclear factor-κB (NF-κB) p65 bound to those same DNA regions. In summary, TCL1A is a novel transcription factor with expression that is regulated in a SNP- and E2-dependent fashion-a pattern of expression that can be reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq results suggest that TCL1A also acts as a transcriptional coregulator with NF-κB p65, an important immune system transcription factor.

TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88-Dependent Nuclear Factor-κB Activation: Single-Nucleotide Polymorphism- and Selective Estrogen Receptor Modulator-Dependent Modification of Inflammation and Immune Response.

In a previous genome-wide association study (GWAS) for musculoskeletal adverse events during aromatase inhibitor therapy for breast cancer, we reported that single nucleotide polymorphisms (SNPs) near the TCL1A gene were associated with this adverse drug reaction. Functional genomic studies showed that TCL1A expression was induced by estradiol, but only in cells with the variant sequence for the top GWAS SNP (rs11849538), a SNP that created a functional estrogen response element. In addition, TCL1A genotype influenced the downstream expression of a series of cytokines and chemokines and had a striking effect on nuclear factor κB (NF-κB) transcriptional activity. Furthermore, this SNP-dependent regulation could be reversed by selective ER modulators (SERMs). The present study was designed to pursue mechanisms underlying TCL1A SNP-mediated, estrogen-dependent NF-κB activation. Functional genomic studies were performed using a panel of 300 lymphoblastoid cell lines for which we had generated genome-wide SNP and gene expression data. It is known that toll-like receptors (TLRs) can regulate NF-κB signaling by a process that requires the adaptor protein MYD88. We found that TLR2, TLR7, TLR9, and TLR10 expression, as well as that of MYD88, could be modulated by TCL1A in a SNP and estrogen-dependent fashion and that these effects were reversed in the presence of SERMs. Furthermore, MYD88 inhibition blocked the TCL1A SNP and estrogen-dependent NF-κB activation, as well as protein-protein interaction between TCL1A and MYD88. These observations greatly expand the range of pathways influenced by TCL1A genotype and raise the possibility that this estrogen- and SNP-dependent regulation might be altered pharmacologically by SERMs.

Molecular mechanisms involved in alcohol craving, IRF3, and endoplasmic reticulum stress: a multi-omics study.

Alcohol use disorder (AUD) is the most prevalent substance use disorder worldwide. Acamprosate and naltrexone are anti-craving drugs used in AUD pharmacotherapy. However, molecular mechanisms underlying their anti-craving effect remain unclear. This study utilized a patient-derived induced pluripotent stem cell (iPSC)-based model system and anti-craving drugs that are used to treat AUD as "molecular probes" to identify possible mechanisms associated with alcohol craving. We examined the pathophysiology of craving and anti-craving drugs by performing functional genomics studies using iPSC-derived astrocytes and next-generation sequencing. Specifically, RNA sequencing performed using peripheral blood mononuclear cells from AUD patients with extreme values for alcohol craving intensity prior to treatment showed that inflammation-related pathways were highly associated with alcohol cravings. We then performed a genome-wide assessment of chromatin accessibility and gene expression profiles of induced iPSC-derived astrocytes in response to ethanol or anti-craving drugs. Those experiments identified drug-dependent epigenomic signatures, with IRF3 as the most significantly enriched motif in chromatin accessible regions. Furthermore, the activation of IRF3 was associated with ethanol-induced endoplasmic reticulum (ER) stress which could be attenuated by anti-craving drugs, suggesting that ER stress attenuation might be a target for anti-craving agents. In conclusion, we found that craving intensity was associated with alcohol consumption and treatment outcomes. Our functional genomic studies suggest possible relationships among craving, ER stress, IRF3 and the actions of anti-craving drugs.

Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) analogs have been tested as potential therapeutics for substance use disorders. Prior research suggests that FGF21 administration might affect alcohol consumption and reward behaviors. Our recent report showed that plasma FGF21 levels were positively correlated with alcohol use in patients with alcohol use disorder (AUD). FGF21 has a short half-life (0.5-2 h) and crosses the blood-brain barrier. Therefore, we set out to identify molecular mechanisms for both the naïve form of FGF21 and a long-acting FGF21 molecule (PF-05231023) in induced pluripotent stem cell (iPSC)-derived forebrain neurons. METHODS: We performed RNA-seq in iPSC-derived forebrain neurons treated with naïve FGF21 or PF-05231023 at physiologically relevant concentrations. We obtained plasma levels of FGF21 and GABA from our previous AUD clinical trial (n = 442). We performed ELISA for FGF21 in both iPSC-derived forebrain neurons and forebrain organoids. We determined protein interactions using co-immunoprecipitation. Finally, we applied ChIP assays to confirm the occupancy of REST, EZH2 and H3K27me3 by FGF21 using iPSC-derived forebrain neurons with and without drug exposure. RESULTS: We identified 4701 and 1956 differentially expressed genes in response to naïve FGF21 or PF-05231023, respectively (FDR < 0.05). Notably, 974 differentially expressed genes overlapped between treatment with naïve FGF21 and PF-05231023. REST was the most important upstream regulator of differentially expressed genes. The GABAergic synapse pathway was the most significant pathway identified using the overlapping genes. We also observed a significant positive correlation between plasma FGF21 and GABA concentrations in AUD patients. In parallel, FGF21 and PF-05231023 significantly induced GABA levels in iPSC-derived neurons. Finally, functional genomics studies showed a drug-dependent occupancy of REST, EZH2, and H3K27me3 in the promoter regions of genes involved in GABA catabolism which resulted in transcriptional repression. CONCLUSIONS: Our results highlight a significant role in the epigenetic regulation of genes involved in GABA catabolism related to FGF21 action. (The ClinicalTrials.gov Identifier: NCT00662571).

Genetic predisposition to major depressive disorder differentially impacts alcohol consumption and high-risk drinking situations in men and women with alcohol use disorder.

Lifetime history of major depressive disorder (MDD) has a sex-specific association with pretreatment alcohol consumption in patients with alcohol dependence. Here, we investigated the association of genetic load for MDD estimated using a polygenic risk score (PRS) with pretreatment alcohol consumption assessed with Timeline Follow Back in a sample of 287 men and 156 women meeting DSM-IV-TR criteria for alcohol dependence. Preferred drinking situations were assessed using the Inventory of Drug Taking Situations (IDTS). Linear models were used to test for association of normalized alcohol consumption measures with the MDD-PRS, adjusting for ancestry, age, sex, and number of days sober at baseline. We fit models both with and without adjustment for MDD history and alcohol-use-related PRSs as covariates. Higher MDD-PRS was associated with lower 90-day total alcohol consumption in men (ß = -0.16, p = 0.0012) but not in women (ß = 0.11, p = 0.18). The association of MDD-PRS with IDTS measures was also sex-specific: higher MDD-PRS was associated with higher propensity to drink in temptation-related situations in women, while the opposite (negative association)was found in men. MDD-PRS was not associated with lifetime MDD history in our sample, and adjustment for lifetime MDD and alcohol-related PRSs did not impact the results. Our results suggest that genetic load for MDD impacts pretreatment alcohol consumption in a sex-specific manner, which is similar to, but independent from, the effect of history of MDD. The clinical implications of these findings and contributing biological and psychological factors should be investigated in future studies.

Genome-wide association study for circulating FGF21 in patients with alcohol use disorder: Molecular links between the SNHG16 locus and catecholamine metabolism.

OBJECTIVE: Alcohol consumption can increase circulating levels of fibroblast growth factor 21 (FGF21). The effects of FGF21 in the central nervous system are associated with the regulation of catecholamines, neurotransmitters that play a crucial role in reward pathways. This study aims to identify genetic variants associated with FGF21 levels and evaluate their functional role in alcohol use disorder (AUD). METHODS: We performed a genome-wide association study (GWAS) using DNA samples from 442 AUD subjects recruited from the Mayo Clinic Center for the Individualized Treatment of Alcoholism Study. Plasma FGF21 levels were measured using Olink proximity extension immunoassays. Alcohol consumption at time of entry into the study was measured using the self-reported timeline followback method. Functional genomic studies were performed using HepG2 cells and induced pluripotent stem cell (iPSC)-derived brain organoids. RESULTS: Plasma FGF21 levels were positively correlated with recent alcohol consumption and gamma-glutamyl transferase levels, a commonly used marker for heavy alcohol use. One variant, rs9914222, located 5' of SNHG16 on chromosome 17 was associated with plasma FGF21 levels (p = 4.60E-09). This variant was also associated with AUD risk (ß: -3.23; p:0.0004). The rs9914222 SNP is an eQTL for SNHG16 in several brain regions, i.e., the variant genotype was associated with decreased expression of SNHG16. The variant genotype for the rs9914222 SNP was also associated with higher plasma FGF21 levels. Knockdown of SNHG16 in HepG2 cells resulted in increased FGF21 concentrations and decreased expression and enzyme activity for COMT, an enzyme that plays a key role in catecholamine metabolism. Finally, we demonstrated that ethanol significantly induced FGF21, dopamine, norepinephrine, and epinephrine concentrations in iPSC-derived brain organoids. CONCLUSIONS: GWAS for FGF21 revealed a SNHG16 genetic variant associated with FGF21 levels which are associated with recent alcohol consumption. Our data suggest that SNHG16 can regulate FGF21 concentrations and decrease COMT expression and enzyme activity which, in turn, have implications for the regulation of catecholamines. (The ClinicalTrials.gov Identifier: NCT00662571).

Genetic variants associated with acamprosate treatment response in alcohol use disorder patients: A multiple omics study.

BACKGROUND AND PURPOSE: Acamprosate is an anti-craving drug used for the pharmacotherapy of alcohol use disorder (AUD). However, only some patients achieve optimal therapeutic outcomes. This study was designed to explore differences in metabolomic profiles between patients who maintained sobriety and those who relapsed, to determine whether those differences provide insight into variation in acamprosate treatment response phenotypes. EXPERIMENTAL APPROACH: We previously conducted an acamprosate trial involving 442 AUD patients, and 267 of these subjects presented themselves for a 3-month follow-up. The primary outcome was abstinence. Clinical information, genomic data and metabolomics data were collected. Baseline plasma samples were assayed using targeted metabolomics. KEY RESULTS: Baseline plasma arginine, threonine, α-aminoadipic acid and ethanolamine concentrations were associated with acamprosate treatment outcomes and baseline craving intensity, a measure that has been associated with acamprosate treatment response. We next applied a pharmacometabolomics-informed genome-wide association study (GWAS) strategy to identify genetic variants that might contribute to variations in plasma metabolomic profiles that were associated with craving and/or acamprosate treatment outcome. Gene expression data for induced pluripotent stem cell-derived forebrain astrocytes showed that a series of genes identified during the metabolomics-informed GWAS were ethanol responsive. Furthermore, a large number of those genes could be regulated by acamprosate. Finally, we identified a series of single nucleotide polymorphisms that were associated with acamprosate treatment outcomes. CONCLUSION AND IMPLICATIONS: These results serve as an important step towards advancing our understanding of disease pathophysiology and drug action responsible for variation in acamprosate response and alcohol craving in AUD patients.

Plasma TNFSF10 levels associated with acamprosate treatment response in patients with alcohol use disorder.

Acamprosate is an anti-craving drug used in alcohol use disorder (AUD) pharmacotherapy. However, only a subset of patients achieves optimal treatment outcomes. The identification of predictive biomarkers of acamprosate treatment response in patients with AUD would be a substantial advance in addiction medicine. We designed this study to use proteomics data as a quantitative biological trait as a step toward identifying inflammatory modulators that might be associated with acamprosate treatment outcomes. The NIAAA-funded Mayo Clinic Center for the Individualized Treatment of Alcoholism study had previously recruited 442 AUD patients who received 3 months of acamprosate treatment. However, only 267 subjects returned for the 3-month follow-up visit and, as a result, had treatment outcome information available. Baseline alcohol craving intensity was the most significant predictor of acamprosate treatment outcomes. We performed plasma proteomics using the Olink target 96 inflammation panel and identified that baseline plasma TNF superfamily member 10 (TNFSF10) concentration was associated with alcohol craving intensity and variation in acamprosate treatment outcomes among AUD patients. We also performed RNA sequencing using baseline peripheral blood mononuclear cells from AUD patients with known acamprosate treatment outcomes which revealed that inflammation-related pathways were highly associated with relapse to alcohol use during the 3 months of acamprosate treatment. These observations represent an important step toward advancing our understanding of the pathophysiology of AUD and molecular mechanisms associated with acamprosate treatment response. In conclusion, applying omics-based approaches may be a practical approach for identifying biologic markers that could potentially predict alcohol craving intensity and acamprosate treatment response.

Genetic contributions to alcohol use disorder treatment outcomes: a genome-wide pharmacogenomics study.

Naltrexone can aid in reducing alcohol consumption, while acamprosate supports abstinence; however, not all patients with alcohol use disorder (AUD) benefit from these treatments. Here we present the first genome-wide association study of AUD treatment outcomes based on data from the COMBINE and PREDICT studies of acamprosate and naltrexone, and the Mayo Clinic CITA study of acamprosate. Primary analyses focused on treatment outcomes regardless of pharmacological intervention and were followed by drug-stratified analyses to identify treatment-specific pharmacogenomic predictors of acamprosate and naltrexone response. Treatment outcomes were defined as: (1) time until relapse to any drinking (TR) and (2) time until relapse to heavy drinking (THR; ≥ 5 drinks for men, ≥4 drinks for women in a day), during the first 3 months of treatment. Analyses were performed within each dataset, followed by meta-analysis across the studies (N = 1083 European ancestry participants). Single nucleotide polymorphisms (SNPs) in the BRE gene were associated with THR (min p = 1.6E-8) in the entire sample, while two intergenic SNPs were associated with medication-specific outcomes (naltrexone THR: rs12749274, p = 3.9E-8; acamprosate TR: rs77583603, p = 3.1E-9). The top association signal for TR (p = 7.7E-8) and second strongest signal in the THR (p = 6.1E-8) analysis of naltrexone-treated patients maps to PTPRD, a gene previously implicated in addiction phenotypes in human and animal studies. Leave-one-out polygenic risk score analyses showed significant associations with TR (p = 3.7E-4) and THR (p = 2.6E-4). This study provides the first evidence of a polygenic effect on AUD treatment response, and identifies genetic variants associated with potentially medication-specific effects on AUD treatment response.

Selective Serotonin Reuptake Inhibitor Pharmaco-Omics: Mechanisms and Prediction.

Selective serotonin reuptake inhibitors (SSRIs) are a standard of care for the pharmacotherapy of patients suffering from Major Depressive Disorder (MDD). However, only one-half to two-thirds of MDD patients respond to SSRI therapy. Recently, a "multiple omics" research strategy was applied to identify genetic differences between patients who did and did not respond to SSRI therapy. As a first step, plasma metabolites were assayed using samples from the 803 patients in the PGRN-AMPS SSRI MDD trial. The metabolomics data were then used to "inform" genomics by performing a genome-wide association study (GWAS) for plasma concentrations of the metabolite most highly associated with clinical response, serotonin (5-HT). Two genome-wide or near genome-wide significant single nucleotide polymorphism (SNP) signals were identified, one that mapped near the TSPAN5 gene and another across the ERICH3 gene, both genes that are highly expressed in the brain. Knocking down TSPAN5 and ERICH3 resulted in decreased 5-HT concentrations in neuroblastoma cell culture media and decreased expression of enzymes involved in 5-HT biosynthesis and metabolism. Functional genomic studies demonstrated that ERICH3 was involved in clathrin-mediated vesicle formation and TSPAN5 was an ethanol-responsive gene that may be a marker for response to acamprosate pharmacotherapy of alcohol use disorder (AUD), a neuropsychiatric disorder highly co-morbid with MDD. In parallel studies, kynurenine was the plasma metabolite most highly associated with MDD symptom severity and application of a metabolomics-informed pharmacogenomics approach identified DEFB1 and AHR as genes associated with variation in plasma kynurenine levels. Both genes also contributed to kynurenine-related inflammatory pathways. Finally, a multiply replicated predictive algorithm for SSRI clinical response with a balanced predictive accuracy of 76% (compared with 56% for clinical data alone) was developed by including the SNPs in TSPAN5, ERICH3, DEFB1 and AHR. In summary, application of a multiple omics research strategy that used metabolomics to inform genomics, followed by functional genomic studies, identified novel genes that influenced monoamine biology and made it possible to develop a predictive algorithm for SSRI clinical outcomes in MDD. A similar pharmaco-omic research strategy might be broadly applicable for the study of other neuropsychiatric diseases and their drug therapy.

Ketamine and Active Ketamine Metabolites Regulate STAT3 and the Type I Interferon Pathway in Human Microglia: Molecular Mechanisms Linked to the Antidepressant Effects of Ketamine.

Inflammation is an important biological process which contributes to risk for depression, in part as a result of the production of proinflammatory cytokines and of alterations in glutamatergic neurotransmission. Ketamine has anti-inflammatory properties which might contribute to its antidepressant effects. This study was designed to clarify mechanisms of action for ketamine and its active metabolites, (2R,6R;2S,6S)-hydroxynorketamine (HNK), which also appear to play a major role in ketamine's rapid antidepressant effects. An HMC3 human microglial cell line was used as a model system to test a possible role for ketamine in immune response regulation that might contribute to its antidepressant effects. Our results highlight the fact that ketamine and its two active metabolites can regulate the type I interferon pathway mediated, at least partially, through signal transducer and activation of transcription 3 (STAT3) which plays a major role in the immune response. Specifically, STAT3 downstream genes that were modulated by either ketamine or its active metabolites were enriched in the "response to type I interferon" pathway. Our data also suggest that STAT3 might play a role in ketamine's antidepressant effects, mediated, at least in part, through eukaryotic elongation factor 2 (EEF2), resulting in the augmentation of brain-derived neurotropic factor (BDNF) expression and promoting the synthesis of synaptic proteins postsynaptic density protein 95 (PSD95) and synapsin I (SYN1).

Ketamine and ketamine metabolites as novel estrogen receptor ligands: Induction of cytochrome P450 and AMPA glutamate receptor gene expression.

Major depressive disorder (MDD) is the most common psychiatric illness worldwide, and it displays a striking sex-dependent difference in incidence, with two thirds of MDD patients being women. Ketamine treatment can produce rapid antidepressant effects in MDD patients, effects that are mediated-at least partially-through glutamatergic neurotransmission. Two active metabolites of ketamine, (2R,6R)-hydroxynorketamine (HNK) and (2S,6S)-HNK, also appear to play a key role in ketamine's rapid antidepressant effects through the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors. In the present study, we demonstrated that estrogen plus ketamine or estrogen plus active ketamine metabolites displayed additive effects on the induction of the expression of AMPA receptor subunits. In parallel, the expression of estrogen receptor alpha (ERα) was also significantly upregulated. Even more striking, radioligand binding assays demonstrated that [3H]-ketamine can directly bind to ERα (KD: 344.5 ±â€¯13 nM). Furthermore, ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites displayed similar affinity for ERα (IC50: 2.31 ±â€¯0.1, 3.40 ±â€¯0.2, and 3.53 ±â€¯0.2 µM, respectively) as determined by [3H]-ketamine displacement assays. Finally, induction of AMPA receptors by either estrogens or ketamine and its metabolites was lost when ERα was knocked down or silenced pharmacologically. These results suggest a positive feedback loop by which estrogens can augment the effects of ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites on the ERα-induced transcription of CYP2A6 and CYP2B6, estrogen inducible enzymes that catalyze ketamine's biotransformation to form the two active metabolites. These observations provide novel insight into ketamine's molecular mechanism(s) of action and have potential implications for the treatment of MDD.