High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

YAP/TAZ inhibition reduces metastatic potential of Ewing sarcoma cells.

Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.

The YAP/TAZ Pathway in Osteogenesis and Bone Sarcoma Pathogenesis.

YAP and TAZ are intracellular messengers communicating multiple interacting extracellular biophysical and biochemical cues to the transcription apparatus in the nucleus and back to the cell/tissue microenvironment interface through the regulation of cytoskeletal and extracellular matrix components. Their activity is negatively and positively controlled by multiple phosphorylation events. Phenotypically, they serve an important role in cellular plasticity and lineage determination during development. As they regulate self-renewal, proliferation, migration, invasion and differentiation of stem cells, perturbed expression of YAP/TAZ signaling components play important roles in tumorigenesis and metastasis. Despite their high structural similarity, YAP and TAZ are functionally not identical and may play distinct cell type and differentiation stage-specific roles mediated by a diversity of downstream effectors and upstream regulatory molecules. However, YAP and TAZ are frequently looked at as functionally redundant and are not sufficiently discriminated in the scientific literature. As the extracellular matrix composition and mechanosignaling are of particular relevance in bone formation during embryogenesis, post-natal bone elongation and bone regeneration, YAP/TAZ are believed to have critical functions in these processes. Depending on the differentiation stage of mesenchymal stem cells during endochondral bone development, YAP and TAZ serve distinct roles, which are also reflected in bone tumors arising from the mesenchymal lineage at different developmental stages. Efforts to clinically translate the wealth of available knowledge of the pathway for cancer diagnostic and therapeutic purposes focus mainly on YAP and TAZ expression and their role as transcriptional co-activators of TEAD transcription factors but rarely consider the expression and activity of pathway modulatory components and other transcriptional partners of YAP and TAZ. As there is a growing body of evidence for YAP and TAZ as potential therapeutic targets in several cancers, we here interrogate the applicability of this concept to bone tumors. To this end, this review aims to summarize our current knowledge of YAP and TAZ in cell plasticity, normal bone development and bone cancer.

Cell culture models of oral mucosal barriers: A review with a focus on applications, culture conditions and barrier properties.

Understanding the function of oral mucosal epithelial barriers is essential for a plethora of research fields such as tumor biology, inflammation and infection diseases, microbiomics, pharmacology, drug delivery, dental and biomarker research. The barrier properties are comprised by a physical, a transport and a metabolic barrier, and all these barrier components play pivotal roles in the communication between saliva and blood. The sum of all epithelia of the oral cavity and salivary glands is defined as the blood-saliva barrier. The functionality of the barrier is regulated by its microenvironment and often altered during diseases. A huge array of cell culture models have been developed to mimic specific parts of the blood-saliva barrier, but no ultimate standard in vitro models have been established. This review provides a comprehensive overview about developed in vitro models of oral mucosal barriers, their applications, various cultivation protocols and corresponding barrier properties.

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells.

Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl--1, HLF-Axl--2, SNU449-Axl--1, SNU449-Axl--2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl- clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region. However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl--1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl--1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.