Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

Drug-induced cholestasis (DIC) is poorly understood and its preclinical prediction is mainly limited to assessing the compound's potential to inhibit the bile salt export pump (BSEP). Here, we evaluated two 3D spheroid models, one from primary human hepatocytes (PHH) and one from HepaRG cells, for the detection of compounds with cholestatic liability. By repeatedly co-exposing both models to a set of compounds with different mechanisms of hepatotoxicity and a non-toxic concentrated bile acid (BA) mixture for 8 days we observed a selective synergistic toxicity of compounds known to cause cholestatic or mixed cholestatic/hepatocellular toxicity and the BA mixture compared to exposure to the compounds alone, a phenomenon that was more pronounced after extending the exposure time to 14 days. In contrast, no such synergism was observed after both 8 and 14 days of exposure to the BA mixture for compounds that cause non-cholestatic hepatotoxicity. Mechanisms behind the toxicity of the cholestatic compound chlorpromazine were accurately detected in both spheroid models, including intracellular BA accumulation, inhibition of ABCB11 expression and disruption of the F-actin cytoskeleton. Furthermore, the observed synergistic toxicity of chlorpromazine and BA was associated with increased oxidative stress and modulation of death receptor signalling. Combined, our results demonstrate that the hepatic spheroid models presented here can be used to detect and study compounds with cholestatic liability.

Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved.

Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems-hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids-at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans.

Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health concern as reflected in its widespread distribution in the general population. Yet, treatment options are scarce which is at least in part due to lack of reliable human in vitro disease models. Here, we report a human hepatic 3D spheroid system cultured under defined chemical conditions that has the potential to mimic steatotic conditions in a reversible manner, useful for identification of novel drug treatment conditions. Primary human hepatocytes (PHH) from different donors were cultured as spheroid microtissues in physiological in vivo -like culture conditions. Hepatic steatosis was induced over the course of three weeks in culture by supplementing the culture medium with pathophysiological concentrations of free fatty acids, carbohydrates and insulin. Effects of steatosis in the 3D system were evaluated on transcriptional, metabolomic and lipidomic levels. Free fatty acids on one hand as well as a combination of insulin and monosaccharides, promoted lipid accumulation in hepatocytes and increased expression of lipogenic genes, such as fatty acid synthase. This milieu also promoted development of insulin resistance within 2 weeks as manifested by an increase in gluconeogenic and insulin resistance markers, which are observed in type 2 diabetes mellitus and metabolic syndrome. Induced steatosis was reversible after withdrawal of lipogenic substrates and a further reduction in cellular fat content was observed following treatment with different antisteatotic compounds, such as metformin, glucagon, olaparib and antioxidants. Taken together, these results demonstrate that the 3D hepatic spheroids can serve as a valuable, HTS compatible model for the study of liver steatosis and facilitate translational discovery of novel drug targets.

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a "positional information system" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The conserved FACT histone chaperone complex is required for this repression mechanism. Genome-wide Transcription Start Site (TSS) mapping reveals thousands of discrete intragenic TSS positions in fact mutants, including downstream promoters that initiate alternative transcript isoforms. We find that histone H3 lysine 4 mono-methylation (H3K4me1), an Arabidopsis RNAPII elongation signature, is enriched at FACT-repressed intragenic TSSs. Our analyses suggest that FACT is required to repress intragenic TSSs at positions that are in part characterized by elevated H3K4me1 levels. In sum, conserved and plant-specific chromatin features correlate with the co-transcriptional repression of intragenic TSSs. Our insights into TSS repression by RNAPII transcription promise to inform the regulation of alternative transcript isoforms and the characterization of gene regulation through the act of pervasive transcription across eukaryotic genomes.

Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.-Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Variability in genes implicated in drug pharmacokinetics or drug response can modulate treatment efficacy or predispose to adverse drug reactions. Besides common genetic polymorphisms, recent sequencing projects revealed a plethora of rare genetic variants in genes encoding proteins involved in drug metabolism, transport, and response.

Most DNA in the genomes of higher organisms does not code for proteins. RNA Polymerase II (Pol II) transcribes non-coding DNA into long non-coding RNAs (lncRNAs), but biological roles of lncRNA are unclear. We find that mutations in the yeast lncRNA CUT60 result in poor growth. Defective termination of CUT60 transcription causes read-through transcription across the ATP16 gene promoter. Read-through transcription localizes chromatin signatures associated with Pol II elongation to the ATP16 promoter. The act of Pol II elongation across this promoter represses functional ATP16 expression by a Transcriptional Interference (TI) mechanism. Atp16p function in the mitochondrial ATP-synthase complex promotes mitochondrial DNA stability. ATP16 repression by TI through inefficient termination of CUT60 therefore triggers mitochondrial genome loss. Our results expand the functional and mechanistic implications of non-coding DNA in eukaryotes by highlighting termination of nuclear lncRNA transcription as mechanism to stabilize an organellar genome.

As the use of next-generation sequencing (NGS) for the Mendelian diseases diagnosis is expanding, the performance of this method has to be improved in order to achieve higher quality. Typically, performance measures are considered to be designed in the context of each application and, therefore, account for a spectrum of clinically relevant variants. We present EphaGen, a new computational methodology for bioinformatics quality control (QC). Given a single NGS dataset in BAM format and a pre-compiled VCF-file of targeted clinically relevant variants it associates this dataset with a single arbiter parameter. Intrinsically, EphaGen estimates the probability to miss any variant from the defined spectrum within a particular NGS dataset. Such performance measure virtually resembles the diagnostic sensitivity of given NGS dataset. Here we present case studies of the use of EphaGen in context of BRCA1/2 and CFTR sequencing in a series of 14 runs across 43 blood samples and 504 publically available NGS datasets. EphaGen is superior to conventional bioinformatics metrics such as coverage depth and coverage uniformity. We recommend using this software as a QC step in NGS studies in the clinical context. Availability: https://github.com/m4merg/EphaGen or https://hub.docker.com/r/m4merg/ephagen.

The liver is a highly regenerative organ. While mature hepatocytes under homeostatic conditions are largely quiescent, upon injury, they rapidly enter the cell cycle to recover the damaged tissue. In rodents, a variety of injury models have provided important insights into the molecular underpinnings that govern the proliferative activation of quiescent hepatocytes. However, little is known about the molecular mechanisms of human hepatocyte regeneration and experimental methods to expand primary human hepatocytes (PHH). Here, a 3D spheroid model of PHH is established to study hepatocyte regeneration and integrative time-lapse multi-omics analyses show that upon isolation from the native liver PHH acquire a regenerative phenotype, as seen in vivo upon partial hepatectomy. However, proliferation is limited. By analyzing global promoter motif activities, it is predicted that activation of Wnt/β-catenin and inhibition of p53 signaling are critical factors required for human hepatocyte proliferation. Functional validations reveal that activation of Wnt signaling through external cues alone is sufficient to inhibit p53 and its proliferative senescence-inducing target PAI1 (SERPINE1) and drive proliferation of >50% of all PHH. A scalable 3D culture model is established to study the molecular and cellular biology of human hepatocyte regeneration. By using this model, an essential role of Wnt/β-catenin signaling during human hepatocyte regeneration is identified.

Bone injuries represent a major social and financial impairment, commonly requiring surgical intervention due to a limited healing capacity of the tissue, particularly regarding critical-sized defects and non-union fractures. Regenerative medicine with the application of bone implants has been developing in the past decades towards the manufacturing of appropriate devices. This work intended to evaluate medical 316L stainless steel (SS)-based devices covered by a polymer poly (L-lactic acid) (PLLA) coating for bone lesion mechanical and functional support. SS316L devices were subjected to a previously described silanization process, following a three-layer PLLA film coating. Devices were further characterized and evaluated towards their cytocompatibility and osteogenic potential using human dental pulp stem cells, and biocompatibility via subcutaneous implantation in a rat animal model. Results demonstrated PLLA-SS316L devices to present superior in vitro and in vivo outcomes and suggested the PLLA coating to provide osteo-inductive properties to the device. Overall, this work represents a preliminary study on PLLA-SS316L devices' potential towards bone tissue regenerative techniques, showing promising outcomes for bone lesion support.

The quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data.

The genetic background for interindividual variability of the polymorphic CYP2D6 enzyme activity remains incompletely understood and the role of NFIB genetic polymorphism for this variability was evaluated in this translational study. We investigated the effect of NFIB expression in vitro using 3D liver spheroids, Huh7 cells, and the influence of the NFIB polymorphism on metabolism of risperidone in patients in vivo. We found that NFIB regulates several important pharmacogenes, including CYP2D6. NFIB inhibited CYP2D6 gene expression in Huh7 cells and NFIB expression in livers was predominantly nuclear and reduced at the mRNA and protein level in carriers of the NFIB rs28379954 T>C allele. Based on 604 risperidone treated patients genotyped for CYP2D6 and NFIB, we found that the rate of risperidone hydroxylation was elevated in NFIB rs28379954 T>C carriers among CYP2D6 normal metabolizers, resulting in a similar rate of drug metabolism to what is observed in CYP2D6 ultrarapid metabolizers, with no such effect observed in CYP2D6 poor metabolizers lacking functional enzyme. The results indicate that NFIB constitutes a novel nuclear factor in the regulation of cytochrome P450 genes, and that its polymorphism is a predictor for the rate of CYP2D6 dependent drug metabolism in vivo.

The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in response to toxicity and that more focus should be placed on alterations of ECM-derived protein expression as biomarkers of liver disease and chronic drug-induced hepatotoxicity, changes that can be studied in stable in vivo-like hepatic cell systems, such as the spheroids.

Genetic variation in genes encoding cytochrome P450 enzymes influences the metabolism of drugs and endogenous compounds. The locus containing the cytochrome genes CYP2C8 and CYP2C9 on chromosome 10 exhibits linkage disequilibrium between the CYP2C8*3 and CYP2C9*2 alleles, forming a haplotype of ~300 kilobases. This haplotype is associated with altered metabolism of several drugs, most notably reduced metabolism of warfarin and phenytoin, leading to toxicity at otherwise therapeutic doses. Here we show that this haplotype is inherited from Neandertals.

Cytochrome P4502C19 (CYP2C19) is an important enzyme involved in the metabolism of antiulcer drugs and antidepressants. However, despite the well documented drug-dependent variability of CYP2C19 expression, the mechanisms underlying the regulation of the enzyme remain unknown. In this study we investigated whether the transcription factor family GATA is involved in the regulation of CYP2C19 gene expression.

The liver plays a central role in the maintenance of homeostasis and health in general. However, there is substantial inter-individual variation in hepatic gene expression, and although numerous genetic factors have been identified, less is known about the epigenetic factors.

Genotyping of cell-free DNA (cfDNA) in plasma samples has the potential to allow for a noninvasive assessment of tumor biology, avoiding the inherent shortcomings of tissue biopsy. Next generation sequencing (NGS), a leading technology for liquid biopsy analysis, continues to be hurdled with several major issues with cfDNA samples, including low cfDNA concentration and high fragmentation. In this study, by employing Ion Torrent PGM semiconductor technology, we performed a comparison between two multi-biomarker amplicon-based NGS panels characterized by a substantial difference in average amplicon length. In course of the analysis of the peripheral blood from 13 diagnostic non-small cell lung cancer patients, equivalence of two panels, in terms of overall diagnostic sensitivity and specificity was shown. A pairwise comparison of the allele frequencies for the same somatic variants obtained from the pairs of panel-specific amplicons, demonstrated an identical analytical sensitivity in range of 140 to 170 bp amplicons in size. Further regression analysis between amplicon length and its coverage, illustrated that NGS sequencing of plasma cfDNA equally tolerates amplicons with lengths in the range of 120 to 170 bp. To increase the sensitivity of mutation detection in cfDNA, we performed a computational analysis of the features associated with genome-wide nucleosome maps, evident from the data on the prevalence of cfDNA fragments of certain sizes and their fragmentation patterns. By leveraging the support vector machine-based machine learning approach, we showed that a combination of nucleosome map associated features with GC content, results in the increased accuracy of prediction of high inter-sample sequencing coverage variation (areas under the receiver operating curve: 0.75, 95% CI: 0.750-0.752 vs. 0.65, 95% CI: 0.63-0.67). Thus, nucleosome-guided fragmentation should be utilized as a guide to design amplicon-based NGS panels for the genotyping of cfDNA samples.