Publications by authors named "Guglielmo Roma"

70 Publications

Mass Cytometry of CSF Identifies an MS-Associated B-cell Population.

Neurol Neuroimmunol Neuroinflamm 2021 03 15;8(2). Epub 2021 Feb 15.

From the Department of Biomedicine (D.J., J.R., E.G., I.C., M.D., J.K., T.D., N.S.R.S.), University Hospital Basel, University of Basel; Novartis Institutes for BioMedical Research (C. Rauld, C. Regairaz, L.R., A.W., R.C., G.R., J.M.C.); Swiss Institute of Bioinformatics (J.R.), Basel; Institute of Experimental Immunology (E.G., B.B.), University of Zurich; and Department of Medicine (E.G., M.D., J.K., T.D.), Neurologic Clinic and Policlinic, University Hospital and University of Basel, Switzerland.

Objective: To identify an MS-specific immune cell population by deep immune phenotyping and relate it to soluble signaling molecules in CSF.

Methods: We analyzed surface expression of 22 markers in paired blood/CSF samples from 39 patients using mass cytometry (cytometry by time of flight). We also measured the concentrations of 296 signaling molecules in CSF using proximity extension assay. Results were analyzed using highly automated unsupervised algorithmic informatics.

Results: Mass cytometry objectively identified a B-cell population characterized by the expression of CD49d, CD69, CD27, CXCR3, and human leukocyte antigen (HLA)-DR as clearly associated with MS. Concentrations of the B cell-related factors, notably FCRL2, were increased in MS CSF, especially in early stages of the disease. The B-cell trophic factor B cell activating factor (BAFF) was decreased in MS. Proteins involved in neural plasticity were also reduced in MS.

Conclusion: When analyzed without a priori assumptions, both the soluble and the cellular compartments of the CSF in MS were characterized by markers related to B cells, and the strongest candidate for an MS-specific cell type has a B-cell phenotype.
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http://dx.doi.org/10.1212/NXI.0000000000000943DOI Listing
March 2021

BET bromodomain inhibitors regulate keratinocyte plasticity.

Nat Chem Biol 2021 Mar 18;17(3):280-290. Epub 2021 Jan 18.

Novartis Institutes for BioMedical Research, Basel, Switzerland.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
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http://dx.doi.org/10.1038/s41589-020-00716-zDOI Listing
March 2021

A Simple and Efficient CRISPR Technique for Protein Tagging.

Cells 2020 Dec 5;9(12). Epub 2020 Dec 5.

Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland.

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.
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http://dx.doi.org/10.3390/cells9122618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762194PMC
December 2020

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Cell 2020 Sep;182(6):1623-1640.e34

Institute of Molecular and Clinical Ophthalmology Basel, 4031 Basel, Switzerland; Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; Department of Ophthalmology, University of Basel, 4031 Basel, Switzerland. Electronic address:

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.
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http://dx.doi.org/10.1016/j.cell.2020.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505495PMC
September 2020

The Genetic Architecture of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

Cell Mol Gastroenterol Hepatol 2021 28;11(1):199-220. Epub 2020 Aug 28.

Department of Medicine, Vatche and Tamar Manoukian Division of Digestive Diseases at UCLA and Pfleger Liver Institute, David Geffen School of Medicine at UCLA, Los Angeles, California. Electronic address:

Background & Aims: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics.

Methods: Chronic liver injury was induced by CCl injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen.

Results: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis.

Conclusions: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.
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http://dx.doi.org/10.1016/j.jcmgh.2020.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674618PMC
August 2020

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

PLoS One 2020 24;15(8):e0235551. Epub 2020 Aug 24.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446895PMC
September 2020

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis.

JCI Insight 2020 02 13;5(3). Epub 2020 Feb 13.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.
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http://dx.doi.org/10.1172/jci.insight.130651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098794PMC
February 2020

Farnesoid X Receptor Agonism, Acetyl-Coenzyme A Carboxylase Inhibition, and Back Translation of Clinically Observed Endpoints of Lipogenesis in a Murine NASH Model.

Hepatol Commun 2020 Jan 8;4(1):109-125. Epub 2019 Nov 8.

Disease Area X Novartis Institutes for BioMedical Research Basel Switzerland.

A promising approach for the treatment of nonalcoholic steatohepatitis (NASH) is the inhibition of enhanced hepatic lipogenesis (DNL), which is the synthesis of fatty acids from nonlipid sources. This study assesses three approaches to DNL suppression in a newly developed dietary NASH mouse model: i) dietary intervention (switch from NASH-inducing diet to normal diet); ii) inhibition of acetyl-coenzyme A carboxylase (ACC), the enzyme catalyzing the rate-limiting step in DNL; and iii) activation of farnesoid X receptor (FXR), a major transcriptional regulator of DNL. C57BL/6J mice on a high-fat diet combined with consumption of a fructose-sucrose solution developed several of the liver histologic features seen in human disease, including steatosis, inflammation, and fibrosis, accompanied by elevated fibrosis biomarkers and liver injury enzymes. Obesity and metabolic impairments were associated with increased intestinal permeability and progression to adenoma and hepatocellular carcinoma. All three approaches led to resolution of established NASH with fibrosis in mice; however, some differences were noted, e.g., with respect to the degree of hepatic steatosis attenuation. While ACC inhibition resulted in elevated blood triglycerides and peripheral obesity, FXR activation prevented peripheral obesity in NASH mice. Comparative transcriptome analysis underlined the translatability of the mouse model to human NASH and revealed novel mechanistic insights into differential regulation of lipid, inflammatory, and extracellular matrix pathways by FXR agonism and ACC inhibition. Novel insights are provided on back translation of clinically observed endpoints of DNL inhibition by targeting ACC or FXR, which are promising therapeutic options for the treatment of NASH, in a newly developed diet-induced NASH mouse model.
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http://dx.doi.org/10.1002/hep4.1443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939503PMC
January 2020

AXIN2 Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.

Cell Stem Cell 2020 01 19;26(1):97-107.e6. Epub 2019 Dec 19.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

The existence of specialized liver stem cell populations, including AXIN2 pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2 pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS) hepatocytes into GS hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
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http://dx.doi.org/10.1016/j.stem.2019.10.011DOI Listing
January 2020

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

Nat Chem Biol 2020 01 9;16(1):50-59. Epub 2019 Dec 9.

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
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http://dx.doi.org/10.1038/s41589-019-0424-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116157PMC
January 2020

IRF2 is a master regulator of human keratinocyte stem cell fate.

Nat Commun 2019 10 14;10(1):4676. Epub 2019 Oct 14.

Chemical Biology & Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056, Basel, Switzerland.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.
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http://dx.doi.org/10.1038/s41467-019-12559-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791852PMC
October 2019

UTS2B Defines a Novel Enteroendocrine Cell Population and Regulates GLP-1 Secretion Through SSTR5 in Male Mice.

Endocrinology 2019 12;160(12):2849-2860

Novartis Institutes for BioMedical Research, Basel, Switzerland.

The gut-pancreas axis plays a key role in the regulation of glucose homeostasis and may be therapeutically exploited to treat not only type 2 diabetes but also hypoglycemia and hyperinsulinemia. We identify a novel enteroendocrine cell type expressing the peptide hormone urotensin 2B (UTS2B). UTS2B inhibits glucagon-like peptide-1 (GLP-1) secretion in mouse intestinal crypts and organoids, not by signaling through its cognate receptor UTS2R but through the activation of the somatostatin receptor (SSTR) 5. Circulating UTS2B concentrations in mice are physiologically regulated during starvation, further linking this peptide hormone to metabolism. Furthermore, administration of UTS2B to starved mice demonstrates that it is capable of regulating blood glucose and plasma concentrations of GLP-1 and insulin in vivo. Altogether, our results identify a novel cellular source of UTS2B in the gut, which acts in a paracrine manner to regulate GLP-1 secretion through SSTR5. These findings uncover a fine-tuning mechanism mediated by a ligand-receptor pair in the regulation of gut hormone secretion, which can potentially be exploited to correct metabolic unbalance caused by overactivation of the gut-pancreas axis.
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http://dx.doi.org/10.1210/en.2019-00549DOI Listing
December 2019

Age-Related Gene Expression Signature in Rats Demonstrate Early, Late, and Linear Transcriptional Changes from Multiple Tissues.

Cell Rep 2019 Sep;28(12):3263-3273.e3

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, 181 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

To understand the changes in gene expression that occur as a result of age, which might create a permissive or causal environment for age-related diseases, we produce a multi-time point age-related gene expression signature (AGES) from liver, kidney, skeletal muscle, and hippocampus of rats, comparing 6-, 9-, 12-, 18-, 21-, 24-, and 27-month-old animals. We focus on genes that changed in one direction throughout the lifespan of the animal, either early in life (early logistic changes), at mid-age (mid-logistic), late in life (late-logistic), or linearly, throughout the lifespan of the animal. The pathways perturbed because of chronological age demonstrate organ-specific and more-global effects of aging and point to mechanisms that could potentially be counter-regulated pharmacologically to treat age-associated diseases. A small number of genes are regulated by aging in the same manner in every tissue, suggesting they may be more-universal markers of aging.
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http://dx.doi.org/10.1016/j.celrep.2019.08.043DOI Listing
September 2019

Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.

PLoS One 2019 29;14(8):e0221762. Epub 2019 Aug 29.

Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland.

Background & Aims: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known.

Methods: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles.

Results: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues.

Conclusions: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715171PMC
March 2020

PAX8 activates metabolic genes via enhancer elements in Renal Cell Carcinoma.

Nat Commun 2019 08 20;10(1):3739. Epub 2019 Aug 20.

Disease Area Oncology, Novartis Institute for Biomedical Research, Basel, Switzerland.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.
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http://dx.doi.org/10.1038/s41467-019-11672-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702156PMC
August 2019

CellSIUS provides sensitive and specific detection of rare cell populations from complex single-cell RNA-seq data.

Genome Biol 2019 07 17;20(1):142. Epub 2019 Jul 17.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.
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http://dx.doi.org/10.1186/s13059-019-1739-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637521PMC
July 2019

PipeIT: A Singularity Container for Molecular Diagnostic Somatic Variant Calling on the Ion Torrent Next-Generation Sequencing Platform.

J Mol Diagn 2019 09 21;21(5):884-894. Epub 2019 Jun 21.

Institute of Pathology, University Hospital Basel, Basel, Switzerland; Department for Biomedical Research, University of Bern, Bern, Switzerland. Electronic address:

The accurate identification of somatic mutations has become a pivotal component of tumor profiling and precision medicine. In molecular diagnostics laboratories, somatic mutation analyses on the Ion Torrent sequencing platform are typically performed on the Ion Reporter platform, which requires extensive manual review of the results and lacks optimized analysis workflows for custom targeted sequencing panels. Alternative solutions that involve custom bioinformatics pipelines involve the sequential execution of software tools with numerous parameters, leading to poor reproducibility and portability. We describe PipeIT, a stand-alone Singularity container of a somatic mutation calling and filtering pipeline for matched tumor-normal Ion Torrent sequencing data. PipeIT is able to identify pathogenic variants in BRAF, KRAS, PIK3CA, CTNNB1, TP53, and other cancer genes that the clinical-grade Oncomine workflow identified. In addition, PipeIT analysis of tumor-normal paired data generated on a custom targeted sequencing panel achieved 100% positive predictive value and 99% sensitivity compared with the 68% to 80% positive predictive value and 92% to 96% sensitivity using the default tumor-normal paired Ion Reporter workflow, substantially reducing the need for manual curation of the results. PipeIT can be rapidly deployed to and ensures reproducible results in any laboratory and can be executed with a single command with minimal input files from the users.
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http://dx.doi.org/10.1016/j.jmoldx.2019.05.001DOI Listing
September 2019

The liver-specific protein 2 (LISP2) is an early marker of liver stage development.

Elife 2019 05 16;8. Epub 2019 May 16.

Novartis Institute for Tropical Diseases, Emeryville, United States.

hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro () and in vivo (), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.
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http://dx.doi.org/10.7554/eLife.43362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542585PMC
May 2019

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Cell Stem Cell 2019 07 9;25(1):39-53.e10. Epub 2019 May 9.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
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http://dx.doi.org/10.1016/j.stem.2019.04.005DOI Listing
July 2019

Self-organization and symmetry breaking in intestinal organoid development.

Nature 2019 05 24;569(7754):66-72. Epub 2019 Apr 24.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.
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http://dx.doi.org/10.1038/s41586-019-1146-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544541PMC
May 2019

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

Nat Chem Biol 2019 02 14;15(2):179-188. Epub 2019 Jan 14.

Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
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http://dx.doi.org/10.1038/s41589-018-0200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251565PMC
February 2019

Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite during progression into dormancy.

Elife 2018 12 27;7. Epub 2018 Dec 27.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Europe.

Relapses of dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.
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http://dx.doi.org/10.7554/eLife.41081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344078PMC
December 2018

mTORC1 signaling suppresses Wnt/β-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Proc Natl Acad Sci U S A 2018 10 8;115(44):E10362-E10369. Epub 2018 Oct 8.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA 02139;

Wnt/β-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/β-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/β-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/β-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/β-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/β-catenin pathway to influence stem cell maintenance.
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http://dx.doi.org/10.1073/pnas.1808575115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217415PMC
October 2018

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.

Nature 2018 08 1;560(7718):377-381. Epub 2018 Aug 1.

Chemical Biology & Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury-repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the 'pulmonary ionocyte', which co-expresses FOXI1, multiple subunits of the vacuolar-type H-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.
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http://dx.doi.org/10.1038/s41586-018-0394-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108322PMC
August 2018

TORC1 inhibition enhances immune function and reduces infections in the elderly.

Sci Transl Med 2018 07;10(449)

Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

Inhibition of the mechanistic target of rapamycin (mTOR) protein kinase extends life span and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this phase 2a randomized, placebo-controlled clinical trial was to determine whether low-dose mTOR inhibitor therapy enhanced immune function and decreased infection rates in 264 elderly subjects given the study drugs for 6 weeks. A low-dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor that selectively inhibits target of rapamycin complex 1 (TORC1) downstream of mTOR was safe and was associated with a significant ( = 0.001) decrease in the rate of infections reported by elderly subjects for a year after study drug initiation. In addition, we observed an up-regulation of antiviral gene expression and an improvement in the response to influenza vaccination in this treatment group. Thus, selective TORC1 inhibition has the potential to improve immune function and reduce infections in the elderly.
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http://dx.doi.org/10.1126/scitranslmed.aaq1564DOI Listing
July 2018

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

ChemMedChem 2018 09 16;13(18):1997-2007. Epub 2018 Aug 16.

Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, 4002, Basel, Switzerland.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.
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http://dx.doi.org/10.1002/cmdc.201800344DOI Listing
September 2018

TRRAP is a central regulator of human multiciliated cell formation.

J Cell Biol 2018 06 27;217(6):1941-1955. Epub 2018 Mar 27.

Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, MA

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.
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http://dx.doi.org/10.1083/jcb.201706106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987713PMC
June 2018

Genomic Analysis Revealed New Oncogenic Signatures in -Mutant Hepatocellular Carcinoma.

Front Genet 2018 2;9. Epub 2018 Feb 2.

Institute of Pathology, University Hospital Basel, Basel, Switzerland.

The gene is the most commonly mutated gene in human cancers and mutations in have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in -mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct -mutant subsets, three of which were defined by mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type or with missense mutations, but not in HCCs with deleterious mutations. Finally, whereas patients with HCCs harboring deleterious mutations had worse overall and disease-free survival than patients with -wild-type HCCs, patients with HCCs harboring missense mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among -mutant HCCs in studies of biomarkers and molecular characterization of HCCs.
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http://dx.doi.org/10.3389/fgene.2018.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801302PMC
February 2018

Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy.

Proc Natl Acad Sci U S A 2018 01 21;115(2):E180-E189. Epub 2017 Dec 21.

Novartis Institutes for BioMedical Research, Basel CH 4002, Switzerland;

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.
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http://dx.doi.org/10.1073/pnas.1711023115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777035PMC
January 2018