Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time.

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein-protein interactions, condensate formation, and chemical degradation.

MicroRNA mimics can distort physiological microRNA effects on immune checkpoints by triggering an antiviral interferon response.

The microRNA-200 family has wide-ranging regulatory functions in cancer development and progression. Above all, it is strongly associated with the epithelial-to-mesenchymal transition (EMT), a process during which cells change their epithelial to a mesenchymal phenotype and acquire invasive characteristics. More recently, miR-200 family members have also been reported to impact the immune evasion of cancer cells by regulating the expression of immunoinhibitory immune checkpoints (ICs) like PD-L1. Therefore, we aimed to comprehensively characterize this miR-200 family as a regulatory interface between EMT and immune evasion mechanisms in biliary tract cancer. Initial correlation analyses and transient overexpression experiments using miRNA mimics suggested miR-200c-3p as a putative regulator of ICs including PD-L1, LGALS9, and IDO1. However, these effects could not be confirmed in stable miR-200c-3p overexpression cell lines, nor in cells transiently transfected with miR-200c-3p mimic from an independent manufacturer. By shifting our efforts towards dissecting the mechanisms leading to these disparate effects, we observed that the initially used miR-200c-3p mimic triggered a double-stranded (ds)RNA-dependent antiviral response. Besides upregulating the ICs, this had substantial cellular consequences including an induction of interferon type I and type III expression, increased levels of intracellular dsRNA sensors, and a significantly altered cellular growth and apoptotic activity.Our study highlights the capability of miRNA mimics to non-specifically induce a dsRNA-mediated antiviral interferon response. Consequently, phenotypic alterations crucially distort physiological miRNA functions and might result in a major misinterpretation of previous and future miRNA studies, especially in the context of IC regulation.

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2-Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

Pooled multicolour tagging for visualizing subcellular protein dynamics.

Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.

Pharmacological perturbation of the phase-separating protein SMNDC1.

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity.

Benefit of second-line systemic chemotherapy for advanced biliary tract cancer: A propensity score analysis.

Whether 2nd-line-chemotherapy (2LCTX) + best-supportive-care (BSC) benefits patients with advanced biliary tract cancer (aBTC) more than BSC alone is unclear. We therefore conducted a propensity-score-based comparative effectiveness analysis of overall survival (OS) outcomes in 80 patients with metastatic, recurrent, or inoperable aBTC, of whom 38 (48%) were treated with BSC + 2LCTX and 42 (52%) with BSC alone. After a median follow-up of 14.8 months and 49 deaths, the crude 6-, 12-, and 18-month Kaplan-Meier OS estimates were 77%, 53% and 23% in the BSC + 2LCTX group, and 29%, 21%, and 14% in patients in the BSC group (p = 0.0003; Hazard ratio (HR) = 0.36, 95%CI:0.20-0.64, p = 0.001). An inverse-probability-of-treatment-weighted (IPTW) analysis was conducted to rigorously account for the higher prevalence of favorable prognostic variables in the 2LCTX + BSC group. After IPTW-weighting, the favorable association between 2LCTX and OS prevailed (adjusted HR = 0.40, 95%CI: 0.17-0.95, p = 0.037). IPTW-weighted 6-, 12-, and 18-month OS estimates were 77%, 58% and 33% in the BSC + 2LCTX group, and 39%, 28% and 22% in the BSC group (p = 0.037). Moreover, the benefit of 2LCTX was consistent across several clinically-relevant subgroups. Within the limitations of an observational study, these findings support the concept that 2LCTX + BSC is associated with an OS benefit over BSC alone in aBTC.

Crosstalk between the Notch signaling pathway and long non-coding RNAs.

The Notch signaling pathway has a key role in cellular development and is involved in initiation and progression of cancer by fundamentally influencing cellular processes such as differentiation, proliferation or migration. The pathway is regulated on many stages, generating diverse outcomes depending on cellular context or signaling dose. Recent studies suggest that long non-coding RNAs (lncRNAs), a class of non-coding RNAs deregulated in many cancers, are involved in regulating the Notch pathway activity by modulating the expression of receptors or ligands on transcriptional or posttranscriptional levels. LncRNAs are also downstream targets of Notch signaling and some of these Notch-induced lncRNAs have been reported to be inducers of its oncogenic effects. This cross-talk between Notch signaling and lncRNAs makes those molecules potential biomarkers for Notch signaling activity and interesting therapeutic RNA-based targets in the future.

miR-196b-5p Regulates Colorectal Cancer Cell Migration and Metastases through Interaction with HOXB7 and GALNT5.

Purpose: miR-196b-5p has been previously implicated in malignant transformation; however, its role in colorectal cancer has not been fully explored. In this study, we examine the clinical and biological relevance of miR-196b-5p, and the molecular pathways regulated by miR-196b-5p in colorectal cancer.Experimental Design: miR-196b-5p expression was quantitated by qRT-PCR in 2 independent cohorts composed of 292 patients with colorectal cancer in total, to explore its biomarker potential. Transient and stable gain- and loss-of-function experiments were conducted in a panel of colorectal cancer cell lines and mice, to evaluate the impact of miR-196b-5p on proliferation, chemosensitivity, migration/invasion, and metastases formation in vitro and in vivo The molecular pathways influenced by miR-196b-5p were characterized using whole transcriptome profiling, in silico target prediction tools, luciferase interaction assays, and phenocopy/rescue gene knockdown experiments.Results: Low miR-196b-5p expression was significantly associated with metastases and poor outcomes in 2 independent colorectal cancer patient cohorts (P < 0.05, log-rank test). miR-196b-5p inhibition led to significantly increased colorectal cancer cell migration/invasion and metastases formation in mice, whereas ectopic overexpression showed the opposite phenotype. Molecular profiling and target confirmation identified an interaction between miR-196b-5p and HOXB7 and GALNT5, which in turn regulated colorectal cancer cell migration.Conclusions: The association of low levels of miR-196b-5p and poor prognosis in patients with colorectal cancer can be explained by its influence on cancer cell migration and metastases formation. miR-196b-5p has an impact on colorectal cancer progression pathways through direct interaction with genes involved in cancer cell migration. Clin Cancer Res; 23(17); 5255-66. ©2017 AACR.