In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate ...stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.
High-speed fluorescence image-enabled cell sorting Schraivogel, Daniel; Kuhn, Terra M; Rauscher, Benedikt ...
Science (American Association for the Advancement of Science),
01/2022, Letnik:
375, Številka:
6578
Journal Article
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Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting ...(ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.
The MSAViewer is a quick and easy visualization and analysis JavaScript component for Multiple Sequence Alignment data of any size. Core features include interactive navigation through the alignment, ...application of popular color schemes, sorting, selecting and filtering. The MSAViewer is 'web ready': written entirely in JavaScript, compatible with modern web browsers and does not require any specialized software. The MSAViewer is part of the BioJS collection of components.
The MSAViewer is released as open source software under the Boost Software License 1.0. Documentation, source code and the viewer are available at http://msa.biojs.net/Supplementary information: Supplementary data are available at Bioinformatics online.
msa@bio.sh.
Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow ...spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.
Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship ...to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.
Abstract
Cancer genomes often harbor hundreds of molecular aberrations. Such genetic variants can be drivers or passengers of tumorigenesis and create new vulnerabilities for potential therapeutic ...exploitation. To identify genotype-dependent vulnerabilities, a large number of forward genetic screens in different genetic backgrounds have been conducted. To aggregate and mine functional data from published screens in a standardized format, we recently developed GenomeCRISPR, a curated database for high-throughput CRISPR-Cas9 experiments. We then devised MINGLE, a computational framework that integrates genetic screens in large panels of cancer cell lines to predict genetic interactions between variant and knockout alleles. We showed that our method identifies specific known and previously unknown genetic relationships. For example, we identified a synthetic lethal dependency between RNF43 and members of the Glucosidase II complex. Additional experiments confirmed that the Glucosidase II complex acts as a regulator of Wnt/beta-catenin signalling. In our first study we analyzed a dataset consisting of screens performed in sixty cell lines. Since then the amount of data available for this analysis has increased several fold. We are continuously integrating the new data that become available as we work towards the next iteration of a genetic interaction map in cancer cells. We are specifically interested in using the increased resolution of this new map to understand how genetic networks differ between individual cancer types.
Citation Format: Benedikt Rauscher. Prediction of selective genetic vulnerabilities from large-scale functional screens in cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2457.
Abstract
N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic ...characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.
Graphical Abstract
Graphical Abstract
Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell‐free (cf) DNA offers a noninvasive approach to detect ...mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cfDNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end‐to‐end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom amplicon sequencing. Our approach relies on open‐software tools to select primers suitable for multiplex PCR using minimal cfDNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cfDNA. We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cfDNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient‐specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cfDNA correlate well with disease progression. Finally, we demonstrate that sequencing of cfDNA can reveal mechanisms of resistance to anti‐Epidermal Growth Factor Receptor(EGFR) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cfDNA.
Analysis of cell‐free (cf) DNA offers a noninvasive approach to detect cancer‐associated mutations. In this study, we developed an end‐to‐end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom, multiplexed amplicon panels and detected recurrent and patient‐specific mutational patterns in a cohort of patients with metastatic colorectal cancer.
Im Lebenszyklus einer Prozessanlage ermöglicht die Digitalisierung, Risiken zu minimieren, den Anlagenbau zu beschleunigen sowie Produktion und Qualität zu optimieren. Wesentliche Voraussetzung sind ...eine robuste Infrastruktur für das Feld der Prozessanlage sowie eine digitale Architektur und Beschreibung für alle Anlagenassets – und beides bestenfalls standardisiert.
Over the past years, CRISPR/Cas9 mediated genome editing has developed into a powerful tool for modifying genomes in various organisms. In high-throughput screens, CRISPR/Cas9 mediated gene ...perturbations can be used for the systematic functional analysis of whole genomes. Discoveries from such screens provide a wealth of knowledge about gene to phenotype relationships in various biological model systems. However, a database resource to query results efficiently has been lacking. To this end, we developed GenomeCRISPR (http://genomecrispr.org), a database for genome-scale CRISPR/Cas9 screens. Currently, GenomeCRISPR contains data on more than 550 000 single guide RNAs (sgRNA) derived from 84 different experiments performed in 48 different human cell lines, comprising all screens in human cells using CRISPR/Cas published to date. GenomeCRISPR provides data mining options and tools, such as gene or genomic region search. Phenotypic and genome track views allow users to investigate and compare the results of different screens, or the impact of different sgRNAs on the gene of interest. An Application Programming Interface (API) allows for automated data access and batch download. As more screening data will become available, we also aim at extending the database to include functional genomic data from other organisms and enable cross-species comparisons.
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