A global genetic interaction network by single-cell imaging and machine learning.

Cellular and organismal phenotypes are controlled by complex gene regulatory networks. However, reference maps of gene function are still scarce across different organisms. Here, we generated synthetic genetic interaction and cell morphology profiles of more than 6,800 genes in cultured Drosophila cells. The resulting map of genetic interactions was used for machine learning-based gene function discovery, assigning functions to genes in 47 modules. Furthermore, we devised Cytoclass as a method to dissect genetic interactions for discrete cell states at the single-cell resolution. This approach identified an interaction of Cdk2 and the Cop9 signalosome complex, triggering senescence-associated secretory phenotypes and immunogenic conversion in hemocytic cells. Together, our data constitute a genome-scale resource of functional gene profiles to uncover the mechanisms underlying genetic interactions and their plasticity at the single-cell level.

Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

The drug-induced phenotypic landscape of colorectal cancer organoids.

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.

Cancer-Associated Mutations in Normal Colorectal Mucosa Adjacent to Sporadic Neoplasia.

INTRODUCTION: Colorectal cancer arises in a multistep process of carcinogenesis from normal mucosa. The earliest precursor might be a morphologically inconspicuous precancerous field, harboring cancer-associated mutations. METHODS: We systematically analyzed genetic alterations in 77 tissue samples from 30 patients with sporadic colorectal neoplasms (18 large adenomas and 12 adenocarcinomas) and matched adjacent normal mucosa (N = 30), as well as normal rectal tissue (N = 17). We profiled mutations associated with colorectal cancer by targeted sequencing of 46 genetic loci using 157 custom amplicons and a median depth of 42,655 reads per loci. RESULTS: Multiple mutations were found in colorectal neoplasms, most frequently in APC, KRAS, and TP53. In a subgroup of 11 of 30 patients, alterations were also detected in non-neoplastic mucosa. These mutations were divergent from those in matched neoplasms. The total alteration count and the allele frequency of mutations were higher in neoplasms compared with those in adjacent tissues. We found that younger patients (≤70 years) are less likely affected by mutations in non-neoplastic mucosa than older patients (>70 years, P = 0.013), although no association was found for other variables, including type, location and differentiation of neoplasia, and previous history of polyps. DISCUSSION: Our data show that cancer-associated mutations can be found in non-neoplastic tissues in a subgroup of patients with colorectal neoplasms. Further studies are needed to specify the risk of occurrence and recurrence of neoplasia in this patient population.

Time-resolved mapping of genetic interactions to model rewiring of signaling pathways.

Context-dependent changes in genetic interactions are an important feature of cellular pathways and their varying responses under different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic interaction networks over time or in response to external stresses are largely lacking. To analyze the plasticity of genetic interactions, we performed a combinatorial RNAi screen in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, we assessed the effect of 12768 pairwise RNAi perturbations in six different conditions. We found that genetic interactions form in different trajectories and developed an algorithm, termed MODIFI, to analyze how genetic interactions rewire over time. Using this framework, we identified more statistically significant interactions compared to end-point assays and further observed several examples of context-dependent crosstalk between signaling pathways such as an interaction between Ras and Rel which is dependent on MEK activity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The Long Noncoding RNA Cancer Susceptibility 9 and RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein L Form a Complex and Coregulate Genes Linked to AKT Signaling.

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples.

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.