Publications by authors named "Walter Carbone"

21 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

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

A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite .

Elife 2017 12 7;6. Epub 2017 Dec 7.

Novartis Institute for Tropical Diseases, Singapore, Singapore.

liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite . Hypnozoites express only 34% of physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.
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http://dx.doi.org/10.7554/eLife.29605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758109PMC
December 2017

A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples.

BMC Genomics 2017 06 5;18(1):442. Epub 2017 Jun 5.

Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.

Background: RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A) + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives.

Methods: We compared for the first time three commercial Illumina library preparation kits (TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access) as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels (intact, degraded, and highly degraded).

Results: We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results (R  > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input.

Conclusions: We found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. We also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples.
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http://dx.doi.org/10.1186/s12864-017-3827-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460543PMC
June 2017

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology.

SLAS Discov 2017 06 6;22(5):571-582. Epub 2017 Jan 6.

1 Novartis Institutes for BioMedical Research (NIBR), Developmental and Molecular Pathways (DMP), Basel, Switzerland.

Oral and intestinal mucositis is a debilitating side effect of radiation treatment. A mouse model of radiation-induced mucositis leads to weight loss and tissue damage, reflecting the human ailment as it responds to keratinocyte growth factor (KGF), the standard-of-care treatment. Cultured intestinal crypt organoids allowed the development of an assay monitoring the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay resembles the mouse model as KGF and roof plate-specific spondin-1 (RSPO1) enhanced crypt organoid recovery following radiation. Screening identified compounds that increased the survival of organoids postradiation. Testing of these compounds revealed that the organoids changed their responses over time. Unbiased transcriptome analysis was performed on crypt organoid cultures at various time points in culture to investigate this adaptive behavior. A number of genes and pathways were found to be modulated over time, providing a rationale for the altered sensitivity of the organoid cultures. This report describes an in vitro assay that reflects aspects of human disease. The assay was used to identify bioactive compounds, which served as probes to interrogate the biology of crypt organoids over prolonged culture. The pathways that are changing over time may offer potential targets for treatment of mucositis.
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http://dx.doi.org/10.1177/2472555216683651DOI Listing
June 2017

Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells.

Sci Rep 2017 02 16;7:42728. Epub 2017 Feb 16.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland.

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.
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http://dx.doi.org/10.1038/srep42728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311948PMC
February 2017

Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening.

BMC Genomics 2016 09 9;17(1):723. Epub 2016 Sep 9.

Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.

Background: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.

Results: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.

Conclusions: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.
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http://dx.doi.org/10.1186/s12864-016-3042-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016932PMC
September 2016

Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.

Elife 2016 06 28;5. Epub 2016 Jun 28.

Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland.

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.
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http://dx.doi.org/10.7554/eLife.17290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924995PMC
June 2016

The RSPO-LGR4/5-ZNRF3/RNF43 module controls liver zonation and size.

Nat Cell Biol 2016 05 18;18(5):467-79. Epub 2016 Apr 18.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland.

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.
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http://dx.doi.org/10.1038/ncb3337DOI Listing
May 2016

CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency.

Science 2016 Mar 4;351(6278):1199-203. Epub 2016 Feb 4.

Developmental Molecular Pathways, Novartis Institutes for Biomedical Research, Basel, Switzerland.

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56β, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.
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http://dx.doi.org/10.1126/science.aad5487DOI Listing
March 2016

Genomic profiling: cDNA arrays and oligoarrays.

Methods Mol Biol 2012 ;823:89-105

Genetics and Biomarkers-Exploratory Medicine, Merck Serono Ivrea, Colleretto Giacosa-TO, Italy.

The introduction of microarray technology, which is a multiplexed hybridization-based process, allows simultaneous analysis of a large number of nucleic acid transcripts. This massively parallel analysis of a cellular genome will become essential for guiding disease diagnosis and molecular profiling of an individual patient's tumor. Nucleic acid based microarrays can be used for: gene expression profiling, single-nucleotide polymorphisms (SNPs) detection, array-comparative genomic hybridizations, comparisons of DNA-methylation status, and microRNA evaluation.A multitude of commercial platforms are available to construct and analyze the microarrays. Typical workflow for a microarray experiment is: preparation of cDNA or gDNA, array construction, hybridization, fluorescent detection, and analysis. Since many sources of variability can affect the outcome of one experiment and there is a multitide of microarray platforms available, microarray standards have been developed to provide industry-wide quality control and information related to each microarray. In this chapter, we review array construction, methodologies, and applications relevant to molecular profiling.
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http://dx.doi.org/10.1007/978-1-60327-216-2_7DOI Listing
April 2012