Publications by authors named "Ivan G Costa"

66 Publications

The Expression of the Cancer-Associated lncRNA Is Modulated by EphrinA5-Induced Signaling.

Int J Mol Sci 2021 Jan 29;22(3). Epub 2021 Jan 29.

Division of Functional Epigenetics, Institute of Zoology (Biology 2), RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced expression. Computational analysis identified triple-helix-mediated DNA-binding sites of in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.
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http://dx.doi.org/10.3390/ijms22031332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866228PMC
January 2021

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation.

Cell Stem Cell 2021 Apr 8;28(4):653-670.e11. Epub 2021 Feb 8.

Department of Hematology, Erasmus MC Cancer Institute, Rotterdam 3015 CN, the Netherlands. Electronic address:

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.
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http://dx.doi.org/10.1016/j.stem.2021.01.006DOI Listing
April 2021

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

Nat Metab 2021 02 3;3(2):149-165. Epub 2021 Feb 3.

Department of Internal Medicine 1, Ulm University Hospital, Ulm, Germany.

Infection-related diabetes can arise as a result of virus-associated β-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.
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http://dx.doi.org/10.1038/s42255-021-00347-1DOI Listing
February 2021

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Cell Stem Cell 2021 Apr 9;28(4):637-652.e8. Epub 2020 Dec 9.

Department of Hematology, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands; Department of Cell Biology, Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany; Oncode Institute, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands. Electronic address:

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.
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http://dx.doi.org/10.1016/j.stem.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024900PMC
April 2021

Deconvolution of cellular subsets in human tissue based on targeted DNA methylation analysis at individual CpG sites.

BMC Biol 2020 11 24;18(1):178. Epub 2020 Nov 24.

Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, 52074, Aachen, Germany.

Background: The complex composition of different cell types within a tissue can be estimated by deconvolution of bulk gene expression profiles or with various single-cell sequencing approaches. Alternatively, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. DNAm is particularly suitable for deconvolution of cell types because each CG dinucleotide (CpG site) has only two states per DNA strand-methylated or non-methylated-and these epigenetic modifications are very consistent during cellular differentiation. So far, deconvolution of DNAm profiles implies complex signatures of many CpGs that are often measured by genome-wide analysis with Illumina BeadChip microarrays. In this study, we investigated if the characterization of cell types in tissue is also feasible with individual cell type-specific CpG sites, which can be addressed by targeted analysis, such as pyrosequencing.

Results: We compiled and curated 579 Illumina 450k BeadChip DNAm profiles of 14 different non-malignant human cell types. A training and validation strategy was applied to identify and test for cell type-specific CpGs. We initially focused on estimating the relative amount of fibroblasts using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. The combination of these two DNAm levels into a "FibroScore" correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts, and induced pluripotent stem cells. The accuracy of this eight CpG signature was tested in additional BeadChip datasets of defined cell mixtures and the results were comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types.

Conclusion: This proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings.
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http://dx.doi.org/10.1186/s12915-020-00910-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687708PMC
November 2020

Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia.

Leukemia 2020 10 20;34(10):2635-2647. Epub 2020 Jul 20.

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, University Hospital RWTH Aachen, Aachen, Germany.

Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34 cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.
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http://dx.doi.org/10.1038/s41375-020-0977-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515845PMC
October 2020

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Nat Commun 2020 04 27;11(1):2039. Epub 2020 Apr 27.

Institute for Cardiovascular Regeneration, Centre for Molecular Medicine, Goethe University Frankfurt am Main, Frankfurt am Main, Germany.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
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http://dx.doi.org/10.1038/s41467-020-15995-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184724PMC
April 2020

Deep learning-based clustering approaches for bioinformatics.

Brief Bioinform 2021 Jan;22(1):393-415

Fraunhofer Institute for Applied Information Technology FIT, Schloss Birlinghoven, Sankt Augustin, Germany.

Clustering is central to many data-driven bioinformatics research and serves a powerful computational method. In particular, clustering helps at analyzing unstructured and high-dimensional data in the form of sequences, expressions, texts and images. Further, clustering is used to gain insights into biological processes in the genomics level, e.g. clustering of gene expressions provides insights on the natural structure inherent in the data, understanding gene functions, cellular processes, subtypes of cells and understanding gene regulations. Subsequently, clustering approaches, including hierarchical, centroid-based, distribution-based, density-based and self-organizing maps, have long been studied and used in classical machine learning settings. In contrast, deep learning (DL)-based representation and feature learning for clustering have not been reviewed and employed extensively. Since the quality of clustering is not only dependent on the distribution of data points but also on the learned representation, deep neural networks can be effective means to transform mappings from a high-dimensional data space into a lower-dimensional feature space, leading to improved clustering results. In this paper, we review state-of-the-art DL-based approaches for cluster analysis that are based on representation learning, which we hope to be useful, particularly for bioinformatics research. Further, we explore in detail the training procedures of DL-based clustering algorithms, point out different clustering quality metrics and evaluate several DL-based approaches on three bioinformatics use cases, including bioimaging, cancer genomics and biomedical text mining. We believe this review and the evaluation results will provide valuable insights and serve a starting point for researchers wanting to apply DL-based unsupervised methods to solve emerging bioinformatics research problems.
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http://dx.doi.org/10.1093/bib/bbz170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820885PMC
January 2021

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Leukemia 2020 04 14;34(4):1062-1074. Epub 2019 Nov 14.

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.
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http://dx.doi.org/10.1038/s41375-019-0629-zDOI Listing
April 2020

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data.

Nucleic Acids Res 2019 11;47(20):10580-10596

Department of Genetics, Saarland University, 66123 Saarbrücken, Germany.

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-seq, ATAC-seq and NOMe-seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However, we also find a high proportion of assay specific NDRs that are often 'called' by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-seq and DNase I-seq provide a superb high NDR calling rate for relatively low sequencing costs in comparison to NOMe-seq, NOMe-seq singles out for its genome-wide coverage allowing to not only detect NDRs but also endogenous DNA methylation and as we show here genome wide segmentation into heterochromatic B domains and local phasing of nucleosomes outside of NDRs. In summary, our comparisons strongly suggest to consider assay specific differences for the experimental design and for generalized and comparative functional interpretations.
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http://dx.doi.org/10.1093/nar/gkz799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6847574PMC
November 2019

Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia.

BMC Cancer 2019 Jul 4;19(1):658. Epub 2019 Jul 4.

Department of Hematology Oncology Hemostaseology and Stem Cell Transplantation Faculty of Medicine, University Hospital RWTH Aachen, Pauwelsstr 30, 52074, Aachen, Germany.

Background: Expression of Bcr-Abl in hematopoietic stem cells is sufficient to cause chronic myeloid leukemia (CML) and tyrosine kinase inhibitors (TKI) induce molecular remission in the majority of CML patients. However, the disease driving stem cell population is not fully targeted by TKI therapy, and leukemic stem cells (LSC) capable of re-inducing the disease can persist. Single-cell RNA-sequencing technology recently identified an enriched inflammatory gene signature with TNFα and TGFβ being activated in TKI persisting quiescent LSC. Here, we studied the effects of human TNFα antibody infliximab (IFX), which has been shown to induce anti-inflammatory effects in mice, combined with TKI treatment on LSC function.

Methods: We first performed GSEA-pathway analysis using our microarray data of murine LSK cells (lin; Sca-1; c-kit) from the SCLtTA/Bcr-Abl CML transgenic mouse model. Bcr-Abl positive cell lines were generated by retroviral transduction. Clonogenic potential was assessed by CFU (colony forming unit). CML mice were treated with nilotinib or nilotinib plus infliximab, and serial transplantation experiments were performed.

Results: Likewise to human CML, TNFα signaling was specifically active in murine CML stem cells, and ectopic expression of Bcr-Abl in murine and human progenitor cell lines induced TNFα expression. In vitro exposure to human (IFX) or murine (MP6-XT22) TNFα antibody reduced clonogenic growth of CML cells. Interestingly, TNFα antibody treatment enhanced TKI-induced effects on immature cells in vitro. Additionally, in transplant and serial transplant experiments, using our transgenic CML mouse model, we could subsequently show that IFX therapy boosted TKI-induced effects and further reduced the proportion of malignant stem cells in vivo.

Conclusion: TNFα signaling is induced in CML stem cells, and anti-inflammatory therapy enhances TKI-induced decline of LSC, confirming that successful targeting of persisting CML stem cells can be enhanced by addressing their malignant microenvironment simultaneously.
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http://dx.doi.org/10.1186/s12885-019-5871-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610865PMC
July 2019

Hematopoietic stem and progenitor cell proliferation and differentiation requires the trithorax protein Ash2l.

Sci Rep 2019 06 4;9(1):8262. Epub 2019 Jun 4.

Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.

Post-translational modifications of core histones participate in controlling the expression of genes. Methylation of lysine 4 of histone H3 (H3K4), together with acetylation of H3K27, is closely associated with open chromatin and gene transcription. H3K4 methylation is catalyzed by KMT2 lysine methyltransferases that include the mixed-lineage leukemia 1-4 (MLL1-4) and SET1A and B enzymes. For efficient catalysis, all six require a core complex of four proteins, WDR5, RBBP5, ASH2L, and DPY30. We report that targeted disruption of Ash2l in the murine hematopoietic system results in the death of the mice due to a rapid loss of mature hematopoietic cells. However, linSca1Kit (LSK) cells, which are highly enriched in hematopoietic stem and multi-potent progenitor cells, accumulated in the bone marrow. The loss of Ash2l resulted in global reduction of H3K4 methylation and deregulated gene expression, including down-regulation of many mitosis-associated genes. As a consequence, LSK cells accumulated in the G2-phase of the cell cycle and were unable to proliferate and differentiate. In conclusion, Ash2l is essential for balanced gene expression and for hematopoietic stem and multi-potent progenitor cell physiology.
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http://dx.doi.org/10.1038/s41598-019-44720-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547667PMC
June 2019

Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.

Gut 2020 03 10;69(3):551-563. Epub 2019 May 10.

Department of Medicine III, RWTH-University Hospital Aachen, Aachen, Germany.

Objective: Bone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in the liver and bone marrow. We therefore aimed at characterising in depth the functional adaptations of myeloid cells in fatty liver.

Design: We employed single-cell RNA sequencing to comprehensively assess the heterogeneity of myeloid cells in the liver and bone marrow during NAFLD, by analysing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol 'Western diet' for 16 weeks. We also characterised NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo.

Results: Single-cell RNA sequencing identified distinct myeloid cell clusters in the liver and bone marrow. In both compartments, monocyte-derived populations were largely expanded in NASH-affected mice. Importantly, the liver myeloid compartment adapted a unique inflammatory phenotype during NAFLD progression, exemplarily characterised by downregulated inflammatory calprotectin (S100A8/A9) in macrophage and dendritic cell subsets. This distinctive gene signature was also found in their bone marrow precursors. The NASH myeloid phenotype was principally recapitulated by in vitro exposure of bone marrow-derived macrophages with fatty acids, depended on toll-like receptor 4 signalling and defined a characteristic response pattern to lipopolysaccharide stimulation. This imprinted and stable NASH myeloid immune phenotype functionally determined inflammatory responses following acute liver injury (acetaminophen poisoning) in vivo.

Conclusion: Liver myeloid leucocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.
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http://dx.doi.org/10.1136/gutjnl-2019-318382DOI Listing
March 2020

Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha.

J Hematol Oncol 2019 04 2;12(1):36. Epub 2019 Apr 2.

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr 30, 52074, Aachen, Germany.

Background: Interferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells.

Methods: Gene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression.

Results: Microarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells.

Conclusions: JAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.
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http://dx.doi.org/10.1186/s13045-019-0722-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444528PMC
April 2019

Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling.

Leukemia 2019 08 6;33(8):1964-1977. Epub 2019 Mar 6.

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.

Tyrosine kinase inhibitor (TKI) therapy effectively blocks oncogenic Bcr-Abl signaling and induces molecular remission in the majority of CML patients. However, the disease-driving stem cell population is not fully targeted by TKI therapy in the majority of patients, and leukemic stem cells (LSCs) capable of re-inducing the disease can persist. In TKI-resistant CML, STAT3 inhibition was previously shown to reduce malignant cell survival. Here, we show therapy-resistant cell-extrinsic STAT3 activation in TKI-sensitive CML cells, using cell lines, HoxB8-immortalized murine BM cells, and primary human stem cells. Moreover, we identified JAK1 but not JAK2 as the STAT3-activating kinase by applying JAK1/2 selective inhibitors and genetic inactivation. Employing an IL-6-blocking peptide, we identified IL-6 as a mediator of STAT3 activation. Combined inhibition of Bcr-Abl and JAK1 further reduced CFUs from murine CML BM, human CML MNCs, as well as CD34 CML cells, and similarly decreased LT-HSCs in a transgenic CML mouse model. In line with these observations, proliferation of human CML CD34 cells was strongly reduced upon combined Bcr-Abl and JAK1 inhibition. Remarkably, the combinatory therapy significantly induced apoptosis even in quiescent LSCs. Our findings suggest JAK1 as a potential therapeutic target for curative CML therapies.
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http://dx.doi.org/10.1038/s41375-019-0427-7DOI Listing
August 2019

Identification of transcription factor binding sites using ATAC-seq.

Genome Biol 2019 02 26;20(1):45. Epub 2019 Feb 26.

Institute for Computational Genomics, Joint Research Center for Computational Biomedicine, RWTH Aachen University Medical School, Aachen, 52074, Germany.

Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) is a simple protocol for detection of open chromatin. Computational footprinting, the search for regions with depletion of cleavage events due to transcription factor binding, is poorly understood for ATAC-seq. We propose the first footprinting method considering ATAC-seq protocol artifacts. HINT-ATAC uses a position dependency model to learn the cleavage preferences of the transposase. We observe strand-specific cleavage patterns around transcription factor binding sites, which are determined by local nucleosome architecture. By incorporating all these biases, HINT-ATAC is able to significantly outperform competing methods in the prediction of transcription factor binding sites with footprints.
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http://dx.doi.org/10.1186/s13059-019-1642-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391789PMC
February 2019

Detection of RNA-DNA binding sites in long noncoding RNAs.

Nucleic Acids Res 2019 04;47(6):e32

Institute for Computational Genomics, Joint Research Center for Computational Biomedicine, RWTH Aachen Medical Faculty, Aachen 52074, Germany.

Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes.
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http://dx.doi.org/10.1093/nar/gkz037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451187PMC
April 2019

Isolation and genome-wide characterization of cellular DNA:RNA triplex structures.

Nucleic Acids Res 2019 03;47(5):2306-2321

Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Heidelberg, Germany.

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.
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http://dx.doi.org/10.1093/nar/gky1305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411930PMC
March 2019

Variants of cause transcript-specific DNA methylation patterns and affect hematopoiesis.

Life Sci Alliance 2018 Dec 13;1(6):e201800153. Epub 2018 Dec 13.

Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Aachen, Germany.

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing of has characteristic epigenetic and functional sequels. Specific transcripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating that variants also affect malignancies. Our results demonstrate that specific variants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.
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http://dx.doi.org/10.26508/lsa.201800153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293073PMC
December 2018

yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.

Cell Stem Cell 2019 02 13;24(2):318-327.e8. Epub 2018 Dec 13.

Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; Institute for Neurophysiology, University of Cologne, 50931 Cologne, Germany; CECAD, Cologne Cluster of Excellence in Cellular Stress Responses in Ageing-Associated Diseases, University of Cologne, 50931 Cologne, Germany. Electronic address:

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.
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http://dx.doi.org/10.1016/j.stem.2018.11.005DOI Listing
February 2019

A Drug-Target Network-Based Supervised Machine Learning Repurposing Method Allowing the Use of Multiple Heterogeneous Information Sources.

Methods Mol Biol 2019 ;1903:281-289

Institute for Computational Genomics, Centre of Medical Technology (MTZ), RWTH Aachen University Medical School, Aachen, Germany.

Drug-target networks have an important role in pharmaceutical innovation, drug lead discovery, and recent drug repositioning tasks. Many different in silico approaches for the identification of new drug-target interactions have been proposed, many of them based on a particular class of machine learning algorithms called kernel methods. These pattern classification algorithms are able to incorporate previous knowledge in the form of similarity functions, i.e., a kernel, and they have been successful in a wide range of supervised learning problems. The selection of the right kernel function and its respective parameters can have a large influence on the performance of the classifier. Recently, multiple kernel learning algorithms have been introduced to address this problem, enabling one to combine multiple kernels into large drug-target interaction spaces in order to integrate multiple sources of biological information simultaneously. The Kronecker regularized least squares with multiple kernel learning (KronRLS-MKL) is a machine learning algorithm that aims at integrating heterogeneous information sources into a single chemogenomic space to predict new drug-target interactions. This chapter describes how to obtain data from heterogeneous sources and how to implement and use KronRLS-MKL to predict new interactions.
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http://dx.doi.org/10.1007/978-1-4939-8955-3_17DOI Listing
June 2019

CXCR6 protects from inflammation and fibrosis in NEMO mice.

Biochim Biophys Acta Mol Basis Dis 2019 02 24;1865(2):391-402. Epub 2018 Nov 24.

Department of Medicine III, RWTH-University Hospital Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. Electronic address:

Chronic inflammation in the liver provokes fibrosis and, on long-term, carcinogenesis. This sequence is prototypically recapitulated in mice with hepatocyte-specific knock-out of the NF-κB essential modulator (NEMO), termed NEMO mice, in which increased hepatocyte apoptosis and compensatory regeneration cause steatosis, inflammation and fibrosis. Natural killer T (NKT) cells carrying the chemokine receptor CXCR6 participate in liver inflammation and injury responses. Here, we investigated the role of CXCR6 in the NEMO mouse model. Unexpectedly, genetic deletion of CXCR6 enhanced hepatocyte death, inflammation and fibrosis in NEMO mice. Although CXCR6 expression is restricted to immune cells in the liver, the adoptive transfer of CXCR6 cells did not protect NEMOCxcr6 mice from hepatic injury. Gene array analyses revealed up-regulated stress response and metabolism pathways in hepatocytes from NEMOCxcr6 mice, functionally corresponding to an increased susceptibility of these hepatocytes to TNFα-induced cell death in vitro. These data revealed a novel CXCR6-dependent mechanism of suppressing inflammatory hepatocytic responses to cellular stress.
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http://dx.doi.org/10.1016/j.bbadis.2018.11.020DOI Listing
February 2019

JAK2V617F but not CALR mutations confer increased molecular responses to interferon-α via JAK1/STAT1 activation.

Leukemia 2019 04 23;33(4):995-1010. Epub 2018 Nov 23.

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30-40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR- as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.
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http://dx.doi.org/10.1038/s41375-018-0295-6DOI Listing
April 2019

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2018 09 27;115(37):9282-9287. Epub 2018 Aug 27.

Department of Internal Medicine III, University Hospital Rheinisch-Westfälische Technische Hochschule (RWTH), 52074 Aachen, Germany;

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.
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http://dx.doi.org/10.1073/pnas.1807155115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140539PMC
September 2018

Transcription factor motif enrichment in whole transcriptome analysis identifies STAT4 and BCL6 as the most prominent binding motif in systemic juvenile idiopathic arthritis.

Arthritis Res Ther 2018 05 30;20(1):98. Epub 2018 May 30.

Department of Pediatrics, Universitätsklinikum Aachen, Aachen, Germany.

Background: The term systemic juvenile idiopathic arthritis (sJIA) describes an autoinflammatory condition characterized by arthritis and severe systemic inflammation, which in later stages can transform into interleukin (IL)-17-driven autoimmune arthritis. IL-1 antagonists have been used with good efficacy in the early stages of sJIA.

Methods: A whole transcriptome analysis of peripheral blood RNA samples was performed in six patients with sJIA and active systemic disease, before initiating treatment with the IL-1β receptor antagonist anakinra, and after induction of inactive disease, compared with a single-sample control cohort of 21 patients in several clinical stages of sJIA activity. Whole transcriptomes were compared longitudinally and interindividually including gene ontology and motif enrichment analysis of differentially expressed genes.

Results: There were 741 transcripts were identified using a threshold with a p value <0.01 and a fold change > 2. HLADRB1 and CD74 were identified as the most strongly upregulated genes in inactive compared to active disease; CD177 expression was significantly enhanced in active disease compared to inactive disease. Motif enrichment analysis revealed STAT4, BCL6, and STAT3 as the most prominent transcription factors that were present during active disease. In addition, strong upregulation of the major histocompatability complex II (MHCII) ligand CD74 was found in both active and inactive sJIA compared to healthy controls.

Conclusion: Using transcription factor motif enrichment, this study identifies novel putative pathways in sJIA (STAT4, BCL6) implicating B cell activation at an earlier stage than predicted in refractory disease. The implication of BCL-6 dependent pathways argues for occurrence of autoimmunity early within the process of sJIA chronification. Transcriptional regulation of HLA-DRB1, a recently described independent genetic risk factor, in combination with its cooperating partner CD74 in patients where sJIA is confirmed, supports pathogenic involvement in alterations in antigen presentation during sJIA.
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http://dx.doi.org/10.1186/s13075-018-1603-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977738PMC
May 2018

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Mol Cell 2018 05 26;70(4):730-744.e6. Epub 2018 Apr 26.

Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany. Electronic address:

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.
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http://dx.doi.org/10.1016/j.molcel.2018.03.030DOI Listing
May 2018

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice.

PLoS One 2017 31;12(10):e0187030. Epub 2017 Oct 31.

Biointerface Laboratory, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany.

Fetuin-A / α2-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663435PMC
November 2017

Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis.

Hepatology 2018 04 19;67(4):1270-1283. Epub 2018 Feb 19.

Department of Medicine III, University Hospital Aachen, Aachen, Germany.

Macrophages are key regulators of liver fibrosis progression and regression in nonalcoholic steatohepatitis (NASH). Liver macrophages comprise resident phagocytes, Kupffer cells, and monocyte-derived cells, which are recruited through the chemokine receptor C-C motif chemokine receptor 2 (CCR2). We aimed at elucidating the therapeutic effects of inhibiting monocyte infiltration in NASH models by using cenicriviroc (CVC), an oral dual chemokine receptor CCR2/CCR5 antagonist that is under clinical evaluation. Human liver tissues from NASH patients were analyzed for CCR2 macrophages, and administration of CVC was tested in mouse models of steatohepatitis, liver fibrosis progression, and fibrosis regression. In human livers from 17 patients and 4 controls, CCR2 macrophages increased parallel to NASH severity and fibrosis stage, with a concomitant inflammatory polarization of these cluster of differentiation 68 , portal monocyte-derived macrophages (MoMF). Similar to human disease, we observed a massive increase of hepatic MoMF in experimental models of steatohepatitis and liver fibrosis. Therapeutic treatment with CVC significantly reduced the recruitment of hepatic Ly-6C MoMF in all models. In experimental steatohepatitis with obesity, therapeutic CVC application significantly improved insulin resistance and hepatic triglyceride levels. In fibrotic steatohepatitis, CVC treatment ameliorated histological NASH activity and hepatic fibrosis. CVC inhibited the infiltration of Ly-6C monocytes, without direct effects on macrophage polarization, hepatocyte fatty acid metabolism, or stellate cell activation. Importantly, CVC did not delay fibrosis resolution after injury cessation. RNA sequencing analysis revealed that MoMF, but not Kupffer cells, specifically up-regulate multiple growth factors and cytokines associated with fibrosis progression, while Kupffer cells activated pathways related to inflammation initiation and lipid metabolism.

Conclusion: Pharmacological inhibition of CCR2 monocyte recruitment efficiently ameliorates insulin resistance, hepatic inflammation, and fibrosis, corroborating the therapeutic potential of CVC in patients with NASH. (Hepatology 2018;67:1270-1283).
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http://dx.doi.org/10.1002/hep.29544DOI Listing
April 2018

Clustering of RNA-Seq samples: Comparison study on cancer data.

Methods 2018 01 2;132:42-49. Epub 2017 Aug 2.

College of Science and Engineering, James Cook University, Townsville, Queensland, Australia; Institute of Mathematics and Computer Sciences, University of São Paulo, São Carlos, São Paulo, Brazil. Electronic address:

RNA-Seq is becoming the standard technology for large-scale gene expression level measurements, as it offers a number of advantages over microarrays. Standards for RNA-Seq data analysis are, however, in its infancy when compared to those of microarrays. Clustering, which is essential for understanding gene expression data, has been widely investigated w.r.t. microarrays. In what concerns the clustering of RNA-Seq data, however, a number of questions remain open, resulting in a lack of guidelines to practitioners. Here we evaluate computational steps relevant for clustering cancer samples via an empirical analysis of 15mRNA-seq datasets. Our evaluation considers strategies regarding expression estimates, number of genes after non-specific filtering and data transformations. We evaluate the performance of four clustering algorithms and twelve distance measures, which are commonly used for gene expression analysis. Results support that clustering cancer samples based on a gene quantification should be preferred. The use of non-specific filtering leading to a small number of features (1,000) presents, in general, superior results. Data should be log-transformed previously to cluster analysis. Regarding the choice of clustering algorithms, Average-Linkage and k-medoids provide, in general, superior recoveries. Although specific cases can benefit from a careful selection of a distance measure, Symmetric Rank-Magnitude correlation provides consistent and sound results in different scenarios.
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http://dx.doi.org/10.1016/j.ymeth.2017.07.023DOI Listing
January 2018

Surface Topography Guides Morphology and Spatial Patterning of Induced Pluripotent Stem Cell Colonies.

Stem Cell Reports 2017 08 27;9(2):654-666. Epub 2017 Jul 27.

Helmholtz Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074 Aachen, Germany; Institute of Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Medical School, 52074 Aachen, Germany. Electronic address:

The relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2017.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550028PMC
August 2017