Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

A Simple and Efficient CRISPR Technique for Protein Tagging.

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.

Jenkins-CI, an Open-Source Continuous Integration System, as a Scientific Data and Image-Processing Platform.

High-throughput screening generates large volumes of heterogeneous data that require a diverse set of computational tools for management, processing, and analysis. Building integrated, scalable, and robust computational workflows for such applications is challenging but highly valuable. Scientific data integration and pipelining facilitate standardized data processing, collaboration, and reuse of best practices. We describe how Jenkins-CI, an "off-the-shelf," open-source, continuous integration system, is used to build pipelines for processing images and associated data from high-content screening (HCS). Jenkins-CI provides numerous plugins for standard compute tasks, and its design allows the quick integration of external scientific applications. Using Jenkins-CI, we integrated CellProfiler, an open-source image-processing platform, with various HCS utilities and a high-performance Linux cluster. The platform is web-accessible, facilitates access and sharing of high-performance compute resources, and automates previously cumbersome data and image-processing tasks. Imaging pipelines developed using the desktop CellProfiler client can be managed and shared through a centralized Jenkins-CI repository. Pipelines and managed data are annotated to facilitate collaboration and reuse. Limitations with Jenkins-CI (primarily around the user interface) were addressed through the selection of helper plugins from the Jenkins-CI community.

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers.

YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or YAP1 amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

UNLABELLED: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.

Glucocorticoid receptor ligands modulate Cardiovirus encephalomyocarditis virus internal ribosome entry site activity.

The use of small molecules to modulate cellular processes is a powerful approach to investigate gene function as a complement to genetic approaches. The discovery and characterization of compounds that modulate translation initiation, the rate-limiting step of protein synthesis, is important both to provide tool compounds to explore this fundamental biological process and to further evaluate protein synthesis as a therapeutic target. While most messenger ribonucleic acids (mRNAs) recruit ribosomes via their 5' cap, some viral and cellular mRNAs initiate protein synthesis via an alternative "cap-independent" mechanism utilizing internal ribosome entry sites (IRES) elements, which are complex mRNA secondary structures, localized within the 5' nontranslated region of the mRNA upstream of the AUG start codon. This report describes the design of a functional, high throughput screen of small molecules miniaturized into a 1,536-well format and performed using the luciferase reporter gene under control of the viral Cardiovirus encephalomyocarditis virus (EMCV) IRES element to identify nontoxic compounds modulating translation initiated from the EMCV IRES. One activating compound, validated in a dose response manner, has previously been shown to bind the glucocorticoid receptor (GR). Subsequent testing of additional GR modulators further supported this as the possible mechanism of action. Detailed characterization of this compound activity supported the notion that this was due to an effect at the level of translation.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis.

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

JNK is activated but does not mediate hippocampal neuronal apoptosis in experimental neonatal pneumococcal meningitis.

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.