Differences In Transcriptional Activity Research Articles

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

1137-P: Liver Regulatory Variants and Genes at Metabolic Trait Loci

Genome-wide association studies (GWAS) have identified many loci that affect liver- and lipid-related metabolic traits, but the causal variants, target genes and mechanisms for most loci have not been characterized. GWAS loci are often enriched in tissue-specific transcriptional regulatory elements that drive physiological changes, and loci can be annotated with colocalized molecular quantitative traits for gene expression (eQTL) and accessible chromatin (caQTL) to identify candidate variants and genes. We previously profiled chromatin accessibility in 20 human liver samples, identified caQTL variants that colocalized with GWAS loci for metabolic traits, and predicted target genes based on promoter proximity, Hi-C chromatin contact with a promoter, and co-localization with liver eQTL. Here, we examined selected GWAS loci and tested variants for effects on gene regulation. We prioritized loci based on trait, gene function, and QTL characteristics and selected four variant-gene pairs to study. To evaluate effects of specific variants on gene expression, we performed transcriptional reporter assays in HepG2 cells. Three caQTL variants exhibited significant (P<0.05) allelic differences in transcriptional activity. rs13395911, near EFHD1 and associated with liver enzyme gamma glutamyl transferase, showed allelic differences in enhancer activity (P=0.008). In addition, rs9556404, near GPR180 and associated with triglycerides, and rs34003091, near ZNF329 and associated with LDL-cholesterol, also showed significant allelic effects on transcriptional activity (P<0.05). To study EFHD1 function in liver metabolism, we used gene-specific shRNA constructs in HepG2 cells and observed up to 90% reduction of EFHD1 mRNA expression compared to scramble shRNA. Subsequent metabolic assays will test role of EFHD1 on hepatic lipid and glucose metabolism. These data identified candidate regulatory variants that may be responsible for nearby GWAS loci and suggest genes as therapeutic targets for fatty liver disease. Disclosure G. Pandey: None. K. Currin: None. S. Vadlamudi: None. K. Broadaway: None. K. L. Mohlke: None.

Functional characterization and expression profile of microsomal FAD2 and FAD3 genes involved in linoleic and α-linolenic acid production in Leucas cephalotes.

The online version contains supplementary material available at 10.1007/s12298-021-01016-z.

Detection of genetic variation and activity analysis of the promoter region of the cattle tRNA-modified gene TRDMT1.

The tRNA modification gene in eukaryotes is relatively conservative. As an important modification gene, the TRDMT1 gene plays an important role in maintaining tRNA structural maintenance and reducing mistranslation of protein translation by methylation of specific tRNA subpopulations. Mouse and zebrafish TRDMT1 knockout experiments indicate that it may mediate growth and development through tRNA modification. However, there are no systematic reports on the function of tRNA-modified genes in livestock. In this study, Qinchuan cattle DNA pool sequencing technology was used. A GC mutation in the bp position upstream of the TRDMT1 translation initiator codon was found. At this locus, the dual-luciferase assay indicated that different genotypes cause differences in transcriptional activity (). Our experiment detected a natural genetic variation of a tRNA modification gene TRDMT1, which may provide potential natural molecular materials for the study of tRNA modification.

GATA2 regulates the CAD susceptibility gene ADTRP rs6903956 through preferential interaction with the G allele.

Myocardial infarction (MI) is a frequent outcome of coronary artery disease (CAD) and the key factor contributing to worldwide disability and death. Genetic factors contribute to the pathogenesis of CAD/MI, and SNP rs6903956 in the ADTRP gene was first found associated with CAD/MI in the Chinese Han population, which was successfully replicated in other cohorts. However, whether rs6903956 is a functional SNP and its risk mechanism to CAD/MI remains unknown. The ADTRP gene-encoded androgen-dependent TFPI regulating protein regulates vascular endothelial cell function, endothelial-monocyte adhesion, and thrombosis. The allele A of rs6903956, in particular, is associated with lower ADTRP mRNA levels in lymphocytes. In the current study, we found that SNP rs6903956 exhibits allelic differences in transcriptional activity by interacting with GATA2. Also, the A allele conferred a greater risk of CAD and MI, lowered transcriptional activity, and GATA2 binding ability as compared to the G allele. Our findings provide details on how rs6903956 regulates the expression of ADTRP and may provide novel insights into CAD pathology and susceptibility.

Tissue-specific cell-free DNA degradation quantifies circulating tumor DNA burden

Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we develop a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions is strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based on just 6 regulatory regions, could accurately predict ctDNA levels in colorectal cancer patients. Strikingly, a model restricted to blood-specific regulatory regions could predict ctDNA levels across both colorectal and breast cancer patients. Using compact targeted sequencing (<25 kb) of predictive regions, we demonstrate how the approach could enable quantitative low-cost tracking of ctDNA dynamics and disease progression.

Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe.

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Transcriptional variation of sensory-related genes in natural populations of Aedes albopictus

BackgroundThe Asian tiger mosquito, Aedes albopictus, is a highly dangerous invasive vector of numerous medically important arboviruses including dengue, chikungunya and Zika. In four decades it has spread from tropical Southeast Asia to many parts of the world in both tropical and temperate climes. The rapid invasion process of this mosquito is supported by its high ecological and genetic plasticity across different life history traits. Our aim was to investigate whether wild populations, both native and adventive, also display transcriptional genetic variability for functions that may impact their biology, behaviour and ability to transmit arboviruses, such as sensory perception.ResultsAntennal transcriptome data were derived from mosquitoes from a native population from Ban Rai, Thailand and from three adventive Mediterranean populations: Athens, Greece and Arco and Trento from Italy. Clear inter-population differential transcriptional activity was observed in different gene categories related to sound perception, olfaction and viral infection. The greatest differences were detected between the native Thai and the Mediterranean populations. The two Italian populations were the most similar.Nearly one million quality filtered SNP loci were identified.ConclusionThe ability to express this great inter-population transcriptional variability highlights, at the functional level, the remarkable genetic flexibility of this mosquito species. We can hypothesize that the differential expression of genes, including those involved in sensory perception, in different populations may enable Ae. albopictus to exploit different environments and hosts, thus contributing to its status as a global vector of arboviruses of public health importance.The large number of SNP loci present in these transcripts represents a useful addition to the arsenal of high-resolution molecular markers and a resource that can be used to detect selective pressure and adaptive changes that may have occurred during the colonization process.

Two coupled mutations abolished the binding of CEBPB to the promoter of CXCL14 that displayed an antiviral effect on PRRSV by activating IFN signaling.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease of pigs worldwide. Our previous study revealed that Tongcheng (TC) pigs display higher resistance to PRRS than Largewhite (LW) pigs, but the genetic mechanism remains unknown. Here, we first confirmed that CXCL14 was downregulated in lungs and porcine alveolar macrophages (PAMs) responding to PRRS virus (PRRSV) infection, but the decline in LW pigs was more obvious than that in TC pigs. Then, we found that the overexpression of CXCL14 activated type-I interferon (IFN-I) signaling by upregulating interferon beta (IFNB), which plays a major role in the antiviral effect. To further decipher the mechanism underlying its differential expression, we characterized the core promoter of CXCL14 as being located from -145 to 276bp of the transcription start site (TSS) and identified two main haplotypes that displayed significant differential transcriptional activities. We further identified two coupled point mutations that altered the binding status of CEBPB and were responsible for the differential expression in TC and LW pigs. The regulatory effect of CEBPB on CXCL14 was further confirmed by RNA interference (RNAi) and chromatin immunoprecipitation (ChIP), providing crucial clues for deciphering the mechanism of CXCL14 downregulation in unusual conditions. The present study revealed the potential antiviral effect of CXCL14, occurring via activation of interferon signaling, and suggested that CXCL14 contributes to the PRRS resistance of TC pigs.

Transcriptomic Heterogeneity of Gleason Grade Group 5 Prostate Cancer

Gleason grade group (GG) 5 prostate cancer has been associated with an aggressive natural history, and retrospective data support a role for treatment intensification. However, clinical outcomes remain heterogeneous in this cohort, and intensified treatments carry an increased risk of adverse events. We sought to explore the transcriptomic heterogeneity of GG 5 tumors by querying transcriptomic data from the tumors of 2138 patients with GG 5 disease who underwent prostatectomy. Four distinct consensus clusters were identified with respect to differential transcriptional activation of hallmark pathways, with distinct molecular subtyping profiles and different average genomic risks (AGRs). One cluster, accounting for 325 tumors (15.2% of the population), was enriched for genes related to the cell cycle/proliferation, metabolic pathways, androgen response pathways, and DNA repair, and had a higher AGR than the other clusters (p < 0.001). This clustering, with an identification of a high genomic risk cluster, was subsequently validated in a separate cohort of 1921 patients as well as a third cohort of 201 patients. The latter cohort had outcomes available, and it was found that patients in the high genomic risk cluster had significantly worse distant metastasis-free survival than the other clusters. Tumors in this high genomic risk cluster of GG 5 disease may be particularly likely to benefit from treatment intensification. Patient summaryIn this report, we examined differences in gene expression in tumors from men with Gleason grade group 5 prostate cancer. We identified significant diversity, with one specific subgroup of tumors associated with expression profiles that suggest a worse prognosis.

The In Vitro Functional Impairment of Thyroid Hormone Receptor Alpha 1 Isoform Mutants Is Mainly Dictated by Reduced Ligand Sensitivity.

Background: Thyroid hormone (TH) acts on TH receptors (TRs) and regulates gene transcription by binding of TRs to TH response elements (TREs) in target gene promoters. The transcriptional activity of TRs is modulated by interactions with TR-coregulatory proteins. Mutations in TRα cause resistance to thyroid hormone alpha (RTHα). In this study, we analyzed if, beyond reduced triiodothyronine (T3) affinity, altered interactions with cofactors or different TREs could account for the differential impaired transcriptional activity of different mutants. Methods: We evaluated four mutants derived from patients (D211G, M256T, A263S, and R384H) and three artificial mutants at equivalent positions in patients with RTHβ (T223A, L287V, and P398H). The in vitro transcriptional activity was evaluated on TRE-luciferase reporters (DR4, IR0, and ER6). The affinity for T3 and interaction with coregulatory proteins (nuclear receptor corepressor 1 [NCoR1] and steroid receptor coactivator 1 [SRC1]) were also determined. Results: We found that the affinity for T3 was significantly reduced for all mutants, except for TRα1-T223A. The reduction in the T3 sensitivity of the transcriptional activity on three TREs, the dissociation of the corepressor NCoR1, and the association of the coactivator SRC1 recruitment for each mutant correlated with the reduced affinity for T3. We did not observe mutation-specific alterations in interactions with cofactors or TREs. Conclusions: In summary, the degree of impaired transcriptional activity of mutants is mainly determined by their reduced affinity for T3.

Exploring the expression and preliminary function of chicken Gimap5 gene.

GTPase immune-associated protein 5 (Gimap5) plays a key role in maintaining T cell homeostasis, immunological tolerance and inflammatory processes. However, there are no reports on the chicken Gimap5 gene. In this study, the Gimap5 gene was first cloned from chicken and characterized its tissue expression characteristics in different developmental stages. The transcriptional activities of the Gimap5 gene in immune response were identified. The results showed that full-length cDNA sequence of Gimap5 contained 771 bp and encoded a 256-amino acid protein. The Gimap5 gene was transcribed in various tissues and different development stages. The transcriptional activities of Gimap5 gene in the most tissues increased with the development of chicken, but significantly up to peak in liver and large intestine of 10-month-old chicken. The Gimap5 gene exhibited differential transcriptional activities in immune-related tissues in immune responses, with down-regulated in liver (P < 0.01), spleen (P < 0.05) and bursa of Fabricius (P < 0.05), and up-regulated in thymus (P < 0.01). The results show that Gimap5 may be a multifunctional gene involved in tissue function, development and immune response in chicken. These data can provide the foundation for further study of Gimap5.

TA*p63 and GTAp63 achieve tighter transcriptional regulation in quality control by converting an inhibitory element into an additional transactivation domain

The p53 homolog p63 plays important roles in development of epithelial tissues and quality control in germ cells. These two functions are executed by two distinct isoforms of p63. They are created by different promotors resulting in isoforms having either an N-terminal transactivation domain (TAp63) or a truncated form (ΔNp63). In addition to these two N-terminal isoforms a third one with an even longer N-terminus, named TA*p63, has been found. A fourth N-terminal isoform, GTAp63, that closely resembles TA*p63 was discovered in male germ cells where it is involved in genetic quality control. Here, we characterize TA*p63α and GTAp63α and show that their N-terminal extensions stabilize the closed and only dimeric conformation adopted by the shorter TAp63α protein. Both proteins can be activated by the two kinases Chk2 and CK1 resulting in the open tetrameric state. In this conformation, the N-terminal extension acts as an additional transactivation domain enhancing transcriptional activity. Through this mechanism, the difference in transcriptional activity between the repressed and the active state of the protein gets enhanced relative to TAp63α. Finally, we show by mass spectrometry that TA*p63α is expressed in the breast cancer cell line Sum159 at the protein level together with mutant p53. Upon doxorubicin treatment, TA*p63α gets activated, providing a potential new tool to fight cancer.

ScSLAM-seq reveals core features of transcription dynamics in single cells.

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.

Epigenetic regulation of BMP2 gene in osteoporosis: a DNA methylation study.

Osteoporosis is a multifactorial disease in which genetic factors and epigenetic modifications play a major role. DNA methylation is known for gene silencing and its effect on BMP2 promoter has been studied here to understand its regulatory activity in osteoporosis pathogenicity. CpG methylation in the BMP2 promoter was analyzed by performing bisulfite specific PCR on the gDNA samples extracted from whole blood of osteoporotic (n = 24) and healthy (n = 24) individuals. Disproportionate allele frequency of CpG sites was calculated statistically. Differential BMP2 expression was estimated using quantitative RT-PCR technique. Luciferase reporter assay was performed to determine and confirm differential transcriptional activity of BMP2 promoter due to methylation. Total of 14 CpG sites were reporter in the BMP2 promoter of which, CpG site at - 267th position upstream to TSS was found to have disproportionate allele frequency among osteoporotic and healthy individuals and was found to be significantly associated with osteoporosis condition. Functional and gene expression analysis of this methylated site using luciferase reporter vector and Real Time PCR approach, suggested reduced transcriptional activity of BMP2 promoter as well as decreased gene expression in disease condition. BMP2 is being a central signaling molecule, aberrant methylation in the promoter region may result into down regulation of osteoblast markers involved in bone formation.

Differential involvement of the three nuclear estrogen receptors during oogenesis in European sea bass (Dicentrarchus labrax)†.

Estrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.

Design of PPAR-γ agonist based on algal metabolites and the endogenous ligand 15-deoxy-Δ12, 14-prostaglandin J2

In a previous study, we synthesized endocyclic enone jasmonate derivatives that function as anti-inflammatory and PPAR-γ-activating entities by using key functional moieties of anti-inflammatory algal metabolites. Herein, we designed additional derivatives containing an exocyclic enone moiety that resembles the key structure of the natural PPAR-γ ligand, 15-deoxy-Δ12, 14-prostaglandin J2 (15 d-PGJ2). The exocyclic enone moiety of 15 d-PGJ2 is essential for covalent bonding with the Cys285 residue in the PPAR-γ ligand-binding domain (LBD). In silico analysis of the designed compounds indicated that they may form hydrogen bonds with key amino acid residues in the PPAR-γ LBD, and thus, secure a position in the bioactive cavity in a similar fashion as does rosiglitazone and 15 d-PGJ2. By a luciferase reporter assay on rat liver Ac2F cells, the synthesized compounds were evaluated for PPAR-γ transcriptional activity. The differential PPAR-γ transcriptional activities of the geometric and enantiomeric isomers of the selected analog were also evaluated; based on our results, the enantiopure compound (+)-(R,E)-6a1 was suggested as a potential PPAR-γ ligand.

Abstract 3485: Transcriptomic and metabolomic profiles in renal cell carcinoma (RCC) tumors reflect ontogeny of RCC subtypes

Abstract Introduction The renal cell carcinoma (RCC) subtypes clear cell RCC (ccRCC), papillary RCC (papRCC) and chromophobe RCC (chRCC) are proposed to arise from distinct cell types of the nephron. The subtypes differ not only in their incidence, but also in terms of metastasis risk and prognosis. The most common form ccRCC, accounting for ~75% of the cases, is characterized by reprogramming of tumor metabolism and is therefore assumed to be a metabolic disease. In contrast, little is known about the metabolic alterations in non-ccRCC subtypes. In this study, we applied transcriptomics and metabolomics profiling to identify key pathways altered in specific RCC subtypes and in ccRCC-derived metastases. In addition, the potential of metabolites to improve subtype classification and to identify promising drug targets for new therapeutic interventions was elucidated. Experimental Procedures Genome-wide expression analyses were performed in ccRCC (n=58), chRCC (n=19), papRCC (n=14), matching non-tumor tissues, and ccRCC-derived metastases (n=9) using HTA 2.0 gene expression arrays. The transcriptomic profiles were compared to expression data of RCC cohorts of TCGA (KIRC, n=452; KIRP, n=260; KICH, n=59). In addition, a targeted metabolomics approach using LC/MS technology was applied in these tissues. Results Pathway enrichment analyses based on metabolic gene expression signatures revealed metabolic alterations in proximal vs. distal cells of the nephron. Accordingly, the respective RCC subtypes arising from proximal (ccRCC, papRCC) or distal (chRCC) tubular cells showed major differences in transcriptional activity of metabolic pathways, reflecting their cell type of origin. Based on metabolite levels, hierarchical cluster analyses separated the samples into three groups, differentiating non-tumor tissues, chRCC, as well as ccRCC, ccRCC-derived metastases and papRCC, indicating differences depending on RCC ontogeny also for metabolic profiles. ccRCC-derived metastases clustered with primary ccRCC tumors, irrespective of affected organs. When comparing ccRCC and chRCC subtypes, major differences were found, e.g. for certain biogenic amines of the polyamine pathway. Differential regulation of this pathway was also reflected on transcriptome level. In vitro experiments revealed that interfering with the polyamine pathway by inhibiting the ornithine-decarboxylase with difluoromethylornithine, reduced cell viability and mitochondrial activity in RCC cells. Conclusion In this study, we elucidated the transcriptomic and metabolomic profiles of RCC tumors, which confirm the distinct ontogeny of RCC subtypes from different parts of the nephron. Furthermore, we found that identification and quantification of differentially regulated metabolites can improve RCC classification and represent an attractive opportunity to reveal subtype-specific therapeutic targets. Citation Format: Pascale Fisel, Florian Büttner, Anna Reustle, Verena Klumpp, Stefan Winter, Steffen Rausch, Jörg Hennenlotter, Stephan Kruck, Arnulf Stenzl, Judith Wahrheit, Denise Sonntag, Marcus Scharpf, Falko Fend, Abbas Agaimy, Arndt Hartmann, Jens Bedke, Matthias Schwab, Elke Schaeffeler. Transcriptomic and metabolomic profiles in renal cell carcinoma (RCC) tumors reflect ontogeny of RCC subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3485.

Abstract 3343: Identifying kinases and phosphatases that regulate STAT3 activity

Abstract The STAT3 transcription factor is hyperactivated in many cancers where it induces cancer progression by driving drug resistance, metastasis and pro-cancer inflammation. We therefore sought new strategies to target the activation of STAT3. Many kinases and signalling pathways can cause STAT3 activation, and it is not well understood how this is regulated in cancer cells. In some rare solid and hematological cancers, such as NK-/T-cell large granular lymphocytic leukemia and inflammatory hepatocellular adenomas, STAT3 has also been found to be mutated, resulting in a constitutively active STAT3 protein. In testing mock transfected vs. STAT3(Y640F)-transfected cells, as a means to introduce activated STAT3 in already transformed cells, in a screen against 525 anticancer agents, we identified that activated STAT3 protects the cells from cytotoxic effects of several classes of anticancer compounds. Therefore, we performed RNAi screens to identify kinases and phosphatases that regulate STAT3 activity and therefore may act as drug-sensitizing targets. Using STAT3 transcription-driven luciferase reporter cell lines expressing active wild type (WT) or mutant (Y640F) STAT3, we identified several candidate genes (CDC7, CDK8, CSK, CSNK2A1, PI4KII, PTPRH, DDR2) whose downregulation led to differential STAT3 transcriptional activity. Strikingly, none of the hits caused highly selective effect on either mutant or wild-type STAT3 expressing cells, suggesting that the phosphoregulation of hyperactive mutant and IL6 induced wild-type STAT3 are mechanistically similar. Using small molecule inhibitors targeting CDK8, CSNK2A1, DDR2 and CDC7, we confirmed that STAT3 transcriptional activity was inhibited in a dose and time dependent fashion without severely affecting cell viability. Small molecules targeting of CSNK2A1, DDR2 and CDC7 kinase activity caused a slow inhibition of STAT3 transcriptional activity where the strongest effects were only seen after 72 h. CDK8 kinase inhibition, on the other hand, reduced STAT3 activity effectively already after 4 h. This suggests that CDK8 regulates STAT3 activity more directly than CSNK2A1, DDR2 and CDC7. Inhibition of CDK8 and CSNK2A1 resulted in decrease of STAT3 phosphorylation in the STAT3(Y640F)-transfected cells, and knockdown of CSNK2A1 inhibited the nuclear localization of STAT3. In conclusion, we have found that hyperactive STAT3(Y640F) can protect cancer cells from several drug classes, mimicking the case when hyperactivated wild type STAT3 is acting as malignancy promoting protein in cancers. In a functional genetics screen, we found six kinases and a phosphatase that regulate transcriptional activity of STAT3. Using small molecule inhibitors, we could confirm that targeting the hit kinases regulate STAT3 transcriptional activity. Together, our data suggest that there are multiple kinases that may be targeted to counteract STAT3 mediated drug resistance in cancers. Citation Format: Elina Parri, Heikki Kuusanmäki, Arjan van Adrichem, Meri Kaustio, Laura Turunen, Krister Wennerberg. Identifying kinases and phosphatases that regulate STAT3 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3343.

Abstract 3753: Indoximod modulates AhR-driven transcription of genes that control immune function

Abstract The IDO pathway mediates immunosuppressive effects by metabolizing tryptophan (Trp) into kynurenine (Kyn). The depletion of Trp stimulates downstream signaling through nutrient sensors GCN2 and mTOR, while the production of Kyn stimulates signaling through the aryl-hydrocarbon receptor (AHR) transcription factor. The activation of these signaling pathways has pleiotropic effects on immune cells, including influencing the differentiation of dendritic cells (DCs), helper T cells, and regulatory T cells (Treg) as well as enhancing the proliferation of effector T cells and Treg. Indoximod has been demonstrated to relieve IDO-mediated immunosuppression in vitro and in vivo, by creating an artificial Trp-sufficiency signal that bypasses activation of GCN2 and inhibition of mTOR in conditions of Trp deprivation. We hypothesized that indoximod's activity could also include the disruption of AHR activation by Kyn and other Trp catabolites, causing differential AHR signaling and transcriptional activity, resulting in an augmented antitumor immune response. We observed that indoximod activates AHR-dependent transcriptional activity in HepG2 cells as evidenced by increased AHR-driven luciferase and endogenous CYP1A1 activity. AHR activation by indoximod was also observed in primary human T cells, as measured by the induction of CYP1A1 mRNA. In a Kyn-driven Treg differentiation assay, indoximod altered transcription of genes associated with T helper and Treg phenotypes. Indoximod induced upregulation of Rorc expression, a TH17-associated transcription factor, while concurrently downregulating transcription of Foxp3, the master transcription factor of Treg cells. These effects were reverted by an AHR inhibitor. Consistent with the gene expression profiles, indoximod shifted the cellular phenotype from Foxp3+ Treg toward Th17-producing CD4 helper T cells. Transcription of Ido1 is controlled by IFNγ and AHR-response elements in its promoter. Consistent with the role of indoximod in blocking the IDO pathway, we observed that indoximod downregulated the expression and function of IDO in in vitro derived plasmacytoid DC that normally would express IDO under the same differentiation conditions. When these in vitro differentiated human pDCs were used in an MLR culture, the indoximod-induced downregulation of IDO resulted in decreased Kyn production and increased T cell proliferation. Moreover, indoximod treatment of tumor-bearing mice decreased expression of IDO in the pDC in tumor-draining LN. Together, these data suggest that indoximod modulates AHR signaling to exert multiple immunomodulatory effects, including a shift from suppressive Foxp3+ Treg toward TH17 helper T cells as well as the downregulation of IDO expression in pDC, contributing to enhanced antitumor immunity. Citation Format: Erik L. Brincks, James Adams, Michael Essmann, Benjamin A. Turner, Lifu Wang, Jiyuan Ke, Agnieszka Marcinowicz, Nicholas Vahanian, Charles J. Link, Mario R. Mautino. Indoximod modulates AhR-driven transcription of genes that control immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3753.