Microdissected "cuboids" for microfluidic drug testing of intact tissues

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or “cuboids” with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.The National Cancer Institute; Juno Therapeutics; CoMotion at the University of Washington; a Hong Kong Research Grant Council; an International Scholars award from the Consejo Nacional de Ciencia y Tecnología of Mexico; a Department of Defense Prostate Cancer Research Program and the National Science Foundation Graduate Research Fellowship Program.http://pubs.rsc.org/en/Journals/JournalIssues/LChj2022Mechanical and Aeronautical Engineerin

A System-Wide Investigation of the Dynamics of Wnt Signaling Reveals Novel Phases of Transcriptional Regulation

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression

KInhibition: A Kinase Inhibitor Selection Portal

Summary: Protein kinases constitute a large class of signaling molecules frequently targeted in research and clinical uses. However, kinase inhibitors are notoriously non-specific, making it difficult to select an appropriate inhibitor for a given kinase. Available data from large-scale kinase inhibitor screens are often difficult to query. Here, we present KInhibition (https://kinhibition.fredhutch.org), an online portal that allows users to search publicly available datasets to find selective inhibitors for a chosen kinase or group of kinases. Compounds are sorted by a KInhibition Selectivity Score, calculated based on compounds' activity against the selected kinase(s) versus activity against all other kinases for which that compound has been profiled. The current version allows users to query four datasets, with a framework that can easily accommodate additional datasets. KInhibition represents a powerful platform through which researchers from broad areas of biology, chemistry, and pharmacology can easily interrogate large datasets to help guide their selection of kinase inhibitors. : Molecular Biology; Bioinformatics; Software Engineering Subject Areas: Molecular Biology, Bioinformatics, Software Engineerin

Olverembatinib inhibits SARS‐CoV‐2‐Omicron variant‐mediated cytokine release in human peripheral blood mononuclear cells

The N‐terminus domain (NTD) of the SARS‐CoV‐2 Omicron variant spike protien strongly induces multiple inflammatory molecules in human peripheral blood mononuclear cells, unaffected by the mutations observed in the NTD. Olverembatinib, a clinical‐stage multi‐kinase inhibitor, potently inhibits Omicron NTD‐mediated cytokine release.[Image: see text

Proteomic and Transcriptomic Profiling Reveals Mitochondrial Oxidative Phosphorylation as Therapeutic Vulnerability in Androgen Receptor Pathway Active Prostate Tumors

Metastatic prostate cancer (PC) is the second leading cause of cancer deaths in males and has limited therapeutic options. The lack of preclinical models for advanced stage PC represents one of the primary barriers in understanding the key genetic drivers of aggressive subsets, including androgen receptor (AR) pathway active and AR-null castration-resistant prostate cancers (CRPC). In our studies, we described a series of LuCaP patient-derived xenograft (PDX) models representing the major genomic and phenotypic features of human disease. To fully exploit the potential of these preclinical models, we carried out a comprehensive transcriptomic and proteomic profiling of 42 LuCaP PDX prostate tumors. The collected proteomic data (~6000 data points) based on 71 antibodies revealed many of the previously known molecular markers associated with AR-positive and AR-null CRPC. Genomic analysis indicated subtype-specific activation of pathways such as Wnt/beta-catenin signaling, mTOR, and oxidative phosphorylation for AR-positive CRPC and upregulation of carbohydrate metabolism and glucose metabolism for AR-null CRPC. Of these, we functionally confirmed the role of mitochondrial metabolism in AR-positive CRPC cell lines. Our data highlight how the integration of transcriptomic and proteomic approaches and PDX systems as preclinical models can potentially map the connectivity of poorly understood signaling pathways in metastatic prostate cancer

A kinase to cytokine explorer to identify molecular regulators and potential therapeutic opportunities

Cytokines and chemokines are secreted proteins that regulate various biological processes, such as inflammation, immune response, and cell differentiation. Therefore, disruption of signaling pathways involving these proteins has been linked to a range of diseases, including cancer. However, targeting individual cytokines, chemokines, or their receptors is challenging due to their regulatory redundancy and incomplete understanding of their signaling networks. To transform these difficult-to-drug targets into a pharmacologically manageable class, we developed a web-based platform called KinCytE. This platform was designed to link the effects of kinase inhibitors, a well-established class of drugs, with cytokine and chemokine release and signaling networks. The resulting KinCytE platform enables users to investigate protein kinases that regulate specific cytokines or chemokines, generate a ranked list of FDA-approved kinase inhibitors that affect cytokine/chemokine activity, and explore and visualize cytokine signaling network thus facilitating drugging this challenging target class. KinCytE is freely accessible via https://atlas.fredhutch.org/kincyte

Anesthetics inhibit phosphorylation of the ribosomal protein S6 in mouse cultured cortical cells and developing brain

IntroductionThe development and maintenance of neural circuits is highly sensitive to neural activity. General anesthetics have profound effects on neural activity and, as such, there is concern that these agents may alter cellular integrity and interfere with brain wiring, such as when exposure occurs during the vulnerable period of brain development. Under those conditions, exposure to anesthetics in clinical use today causes changes in synaptic strength and number, widespread apoptosis, and long-lasting cognitive impairment in a variety of animal models. Remarkably, most anesthetics produce these effects despite having differing receptor mechanisms of action. We hypothesized that anesthetic agents mediate these effects by inducing a shared signaling pathway.MethodsWe exposed cultured cortical cells to propofol, etomidate, or dexmedetomidine and assessed the protein levels of dozens of signaling molecules and post-translational modifications using reverse phase protein arrays. To probe the role of neural activity, we performed separate control experiments to alter neural activity with non-anesthetics. Having identified anesthetic-induced changes in vitro, we investigated expression of the target proteins in the cortex of sevoflurane anesthetized postnatal day 7 mice by Western blotting.ResultsAll the anesthetic agents tested in vitro reduced phosphorylation of the ribosomal protein S6, an important member of the mTOR signaling pathway. We found a comparable decrease in cortical S6 phosphorylation by Western blotting in sevoflurane anesthetized neonatal mice. Using a systems approach, we determined that propofol, etomidate, dexmedetomidine, and APV/TTX all similarly modulate a signaling module that includes pS6 and other cell mediators of the mTOR-signaling pathway.DiscussionReduction in S6 phosphorylation and subsequent suppression of the mTOR pathway may be a common and novel signaling event that mediates the impact of general anesthetics on neural circuit development

Machine learning identifies molecular regulators and therapeutics for targeting SARS‐CoV2‐induced cytokine release

Abstract Although 15–20% of COVID‐19 patients experience hyper‐inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N‐terminal domain (NTD) of the SARS‐CoV‐2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning‐based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD‐induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA‐approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD‐mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS‐CoV‐2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide‐mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS‐CoV‐2‐mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID‐19

A Systematic Family-wide Investigation Reveals that ∼30% of Mammalian PDZ Domains Engage in PDZ-PDZ Interactions

SummaryPDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C termini of their target proteins. However, in a few instances, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical-binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify interactions and coaffinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (∼30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand

α-Arrestin ARRDC3 tumor suppressor function is linked to GPCR-induced TAZ activation and breast cancer metastasis.

The α-arrestin domain containing protein 3 (ARRDC3) is a tumor suppressor in triple-negative breast carcinoma (TNBC), a highly metastatic subtype of breast cancer that lacks targeted therapies. Thus, understanding the mechanisms and targets of ARRDC3 in TNBC is important. ARRDC3 regulates trafficking of protease-activated receptor 1 (PAR1, also known as F2R), a G-protein-coupled receptor (GPCR) implicated in breast cancer metastasis. Loss of ARRDC3 causes overexpression of PAR1 and aberrant signaling. Moreover, dysregulation of GPCR-induced Hippo signaling is associated with breast cancer progression. However, the mechanisms responsible for Hippo dysregulation remain unknown. Here, we report that the Hippo pathway transcriptional co-activator TAZ (also known as WWTR1) is the major effector of GPCR signaling and is required for TNBC migration and invasion. Additionally, ARRDC3 suppresses PAR1-induced Hippo signaling via sequestration of TAZ, which occurs independently of ARRDC3-regulated PAR1 trafficking. The ARRDC3 C-terminal PPXY motifs and TAZ WW domain are crucial for this interaction and are required for suppression of TNBC migration and lung metastasis in vivo. These studies are the first to demonstrate a role for ARRDC3 in regulating GPCR-induced TAZ activity in TNBC and reveal multi-faceted tumor suppressor functions of ARRDC3. This article has an associated First Person interview with the first author of the paper