Publications by authors named "Joel A Schick"

11 Publications

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Evolutionary and functional analyses demonstrate conserved ferroptosis protection by Arabidopsis GPXs in mammalian cells.

FASEB J 2021 Jun;35(6):e21550

Ministry of Education Key Laboratory for Ecology of Tropical Islands, Key Laboratory of Tropical Animal and Plant Ecology of Hainan Province, College of Life Sciences, Hainan Normal University, Haikou, China.

Species have evolved unique mechanisms to combat the effects of oxidative stress inside cells. A particularly devastating consequence of an unhindered oxidation of membrane lipids in the presence of iron results in cell death, known as ferroptosis. Hallmarks of ferroptosis, including peroxidation of polyunsaturated fatty acids, are conserved among animals and plants, however, early divergence of an ancestral mammalian GPX4 (mGPX4) has complicated our understanding of mechanistic similarities between species. To this end, we performed a comprehensive phylogenetic analysis and identified that orthologous Arabidopsis GPXs (AtGPXs) are more highly related to mGPX4 than mGPX4 is to other mammalian GPXs. This high degree of conservation suggested that experimental substitution may be possible. We, therefore, ectopically expressed AtGPX1-8 in ferroptosis-sensitive mouse fibroblasts. This substitution experiment revealed highest protection against ferroptosis induction by AtGPX5, as well as moderate protection by AtGPX2, -7, and -8. Further analysis of these cells revealed substantial abatement of lipid peroxidation in response to pharmacological challenge. The results suggest that the presence of ancestral GPX4 resulted in later functional divergence and specialization of GPXs in plants. The results also challenge a strict requirement for selenocysteine activity and suggest thioredoxin as a potent parallel antioxidant system in both plants and mammals.
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http://dx.doi.org/10.1096/fj.202000856RDOI Listing
June 2021

Cannabidiol converts NFκB into a tumor suppressor in glioblastoma with defined antioxidative properties.

Neuro Oncol 2021 Apr 17. Epub 2021 Apr 17.

Neurosurgical Research, Department of Neurosurgery, University Hospital, LMU Munich, Germany.

Background: The transcription factor NFκB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFκB activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive.

Methods: In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFκB can be converted into a tumor suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM-datasets.

Results: We found that CBD promotes DNA binding of the NFκB subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA binding by RELA lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC death. Consequently, ROS levels served as predictive biomarker for CBD-sensitive tumors.

Conclusions: This evidence demonstrates how a clinically approved drug can convert NFκB into a tumor suppressor and suggests a promising repurposing option for GBM-therapy.
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http://dx.doi.org/10.1093/neuonc/noab095DOI Listing
April 2021

Genome-Wide Analysis of MDHAR Gene Family in Four Cotton Species Provides Insights into Fiber Development via Regulating AsA Redox Homeostasis.

Plants (Basel) 2021 Jan 25;10(2). Epub 2021 Jan 25.

Key Laboratory of Xinjiang Phytomedicine Resource and Utilization of Ministry of Education, College of Life Sciences, Shihezi University, Shihezi 832003, China.

Monodehydroasorbate reductase (MDHAR) (EC1.6.5.4), a key enzyme in ascorbate-glutathione recycling, plays important roles in cell growth, plant development and physiological response to environmental stress via control of ascorbic acid (AsA)-mediated reduction/oxidation (redox) regulation. Until now, information regarding function and regulatory mechanism in have been limited. Herein, a genome-wide identification and comprehensive bioinformatic analysis of 36 family genes in four species, , , and , were performed, indicating their close evolutionary relationship. Expression analysis of in different cotton tissues and under abiotic stress and phytohormone treatment revealed diverse expression features. Fiber-specific expression analysis showed that , and were preferentially expressed in fiber fast elongating stages to reach peak values in 15-DPA fibers, with corresponding coincident observances of MDHAR enzyme activity, AsA content and ascorbic acid/dehydroascorbic acid (AsA/DHA) ratio. Meanwhile, there was a close positive correlation between the increase of AsA content and AsA/DHA ratio catalyzed by MDHAR and fiber elongation development in different fiber-length cotton cultivars, suggesting the potential important function of MDHAR for fiber growth. Following HO stimulation, demonstrated immediate responses at the levels of mRNA, enzyme, the product of AsA and corresponding AsA/DHA value, and antioxidative activity. These results for the first time provide a comprehensive systemic analysis of the gene family in plants and the four cotton species and demonstrate the contribution of MDHAR to fiber elongation development by controlling AsA-recycling-mediated cellular redox homeostasis.
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http://dx.doi.org/10.3390/plants10020227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912408PMC
January 2021

Nonsense-mediated decay factor SMG7 sensitizes cells to TNFα-induced apoptosis via CYLD tumor suppressor and the noncoding oncogene Pvt1.

Mol Oncol 2020 10 13;14(10):2420-2435. Epub 2020 Jul 13.

Genetics and Cellular Engineering Group, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum Munich GmbH, German Research Center for Environmental Health, Neuherberg, Germany.

Nonsense-mediated decay (NMD) proteins are responsible for the surveillance and degradation of aberrant RNAs. Suppressor with morphogenetic effect on genitalia 7 (SMG7) is an NMD complex protein and a regulator of tumor necrosis factor (TNF)-induced extrinsic apoptosis; however, this unique function has not been explored in detail. In this study, we show that loss of Smg7 leads to unrestricted expression of long noncoding RNAs (lncRNAs) in addition to NMD targets. Functional analysis of Smg7 cells showed downregulation of the tumor suppressor cylindromatosis (CYLD) and diminished caspase activity, thereby switching cells to nuclear factor-κB (NF-κB)-mediated protection. This positive relationship between SMG7 and CYLD was found to be widely conserved in human cancer cell lines and renal carcinoma samples from The Cancer Genome Atlas. In addition to CYLD suppression, upregulation of lncRNAs Pvt1 and Adapt33 rendered cells resistant to TNF, while pharmacologic inhibition of NF-κB in Pvt1-overexpressing TNF-resistant cells and Smg7-deficient spheroids re-established TNF-induced lethality. Thus, loss of SMG7 decouples regulation of two separate oncogenic factors with cumulative downstream effects on the NF-κB pathway. The data highlight a novel and specific regulation of oncogenic factors by SMG7 and pinpoint a composite tumor suppressor role in response to TNF.
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http://dx.doi.org/10.1002/1878-0261.12754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530794PMC
October 2020

GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling.

ACS Cent Sci 2020 Jan 27;6(1):41-53. Epub 2019 Dec 27.

Institute of Molecular Toxicology and Pharmacology, Genetics and Cellular Engineering Group, HelmholtzZentrum Muenchen, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany.

Ferroptosis is an iron-dependent form of regulated cell death linking iron, lipid, and glutathione levels to degenerative processes and tumor suppression. By performing a genome-wide activation screen, we identified a cohort of genes antagonizing ferroptotic cell death, including GTP cyclohydrolase-1 (GCH1) and its metabolic derivatives tetrahydrobiopterin/dihydrobiopterin (BH/BH). Synthesis of BH/BH by GCH1-expressing cells caused lipid remodeling, suppressing ferroptosis by selectively preventing depletion of phospholipids with two polyunsaturated fatty acyl tails. GCH1 expression level in cancer cell lines stratified susceptibility to ferroptosis, in accordance with its expression in human tumor samples. The GCH1-BH-phospholipid axis acts as a master regulator of ferroptosis resistance, controlling endogenous production of the antioxidant BH, abundance of CoQ, and peroxidation of unusual phospholipids with two polyunsaturated fatty acyl tails. This demonstrates a unique mechanism of ferroptosis protection that is independent of the GPX4/glutathione system.
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http://dx.doi.org/10.1021/acscentsci.9b01063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978838PMC
January 2020

ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis.

BMC Genomics 2017 Nov 25;18(1):905. Epub 2017 Nov 25.

Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.

Background: As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import.

Results: We demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling.

Conclusions: In summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.
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http://dx.doi.org/10.1186/s12864-017-4285-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702081PMC
November 2017

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

Nat Chem Biol 2017 Jan 14;13(1):91-98. Epub 2016 Nov 14.

Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.
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http://dx.doi.org/10.1038/nchembio.2239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610546PMC
January 2017

CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

Sci Rep 2016 09 1;6:32326. Epub 2016 Sep 1.

Institute of Developmental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg/Munich, Germany.

The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods.
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http://dx.doi.org/10.1038/srep32326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007477PMC
September 2016

Limitations of In Vivo Reprogramming to Dopaminergic Neurons via a Tricistronic Strategy.

Hum Gene Ther Methods 2015 Aug 29;26(4):107-22. Epub 2015 Jul 29.

1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.

Parkinson's disease is one of the most common neurodegenerative disorders characterized by cell death of dopaminergic neurons in the substantia nigra. Recent research has focused on cellular replacement through lineage reprogramming as a potential therapeutic strategy. This study sought to use genetics to define somatic cell types in vivo amenable to reprogramming. To stimulate in vivo reprogramming to dopaminergic neurons, we generated a Rosa26 knock-in mouse line conditionally overexpressing Mash1, Lmx1a, and Nurr1. These proteins are characterized by their role in neuronal commitment and development of midbrain dopaminergic neurons and have previously been shown to convert fibroblasts to dopaminergic neurons in vitro. We show that a tricistronic construct containing these transcription factors can reprogram astrocytes and fibroblasts in vitro. However, cassette overexpression triggered cell death in vivo, in part through endoplasmic reticulum stress, while we also detected "uncleaved" forms of the polyprotein, suggesting poor "cleavage" efficiency of the 2A peptides. Based on our results, the cassette overexpression induced apoptosis and precluded reprogramming in our mouse model. Therefore, we suggest that alternatives must be explored to balance construct design with efficacious reprogramming. It is evident that there are still biological obstacles to overcome for in vivo reprogramming to dopaminergic neurons.
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http://dx.doi.org/10.1089/hgtb.2014.152DOI Listing
August 2015

Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.

Nat Cell Biol 2014 Dec 17;16(12):1180-91. Epub 2014 Nov 17.

Helmholtz Zentrum München, Institute of Developmental Genetics, Ingolstädter Landstr. 1 85764 Neuherberg, Germany.

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.
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http://dx.doi.org/10.1038/ncb3064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894846PMC
December 2014

Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

PLoS One 2013 13;8(9):e74207. Epub 2013 Sep 13.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074207PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772885PMC
June 2014