Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency.

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

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.

Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma.

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed antiproliferative activity in the SK-N-MC Ewing sarcoma cell line with an IC50 value of 15.9 ± 0.7 µM. The optimised compounds showed enhanced anticancer activity in the SK-N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma.

YB-1 regulates mesothelioma cell migration via snail but not EGFR, MMP1, EPHA5 or PARK2.

Pleural mesothelioma (PM) is characterized by rapid growth, local invasion, and limited therapeutic options. The multifunctional oncoprotein Y-box-binding protein-1 (YB-1) is frequently overexpressed in cancer and its inhibition reduces aggressive behavior in multiple tumor types. Here, we investigated the effects of YB-1 on target gene regulation and PM cell behavior. Whereas siRNA-mediated YB-1 knockdown reduced cell motility, YB-1 overexpression resulted in scattering, increased migration, and intravasation in vitro. Furthermore, YB-1 stimulated PM cell spreading in zebrafish. Combined knockdown and inducible overexpression of YB-1 allowed bidirectional control and rescue of cell migration, the pattern of which was closely followed by the mRNA and protein levels of EGFR and the protein level of snail, whereas the mRNA levels of MMP1, EPHA5, and PARK2 showed partial regulation by YB-1. Finally, we identified snail as a critical regulator of YB-1-mediated cell motility in PM. This study provides insights into the mechanism underlying the aggressive nature of PM and highlights the important role of YB-1 in this cancer. In this context, we found that YB-1 closely regulates EGFR and snail, and, moreover, that YB-1-induced cell migration depends on snail.

Non-destructive characterization of adult zebrafish models using Jones matrix optical coherence tomography.

The zebrafish is a valuable vertebrate animal model in pre-clinical cancer research. A Jones matrix optical coherence tomography (JM-OCT) prototype operating at 1310 nm and an intensity-based spectral-domain OCT setup at 840 nm were utilized to investigate adult wildtype and a tumor-developing zebrafish model. Various anatomical features were characterized based on their inherent scattering and polarization signature. A motorized translation stage in combination with the JM-OCT prototype enabled large field-of-view imaging to investigate adult zebrafish in a non-destructive way. The diseased animals exhibited tumor-related abnormalities in the brain and near the eye region. The scatter intensity, the attenuation coefficients and local polarization parameters such as the birefringence and the degree of polarization uniformity were analyzed to quantify differences in tumor versus control regions. The proof-of-concept study in a limited number of animals revealed a significant decrease in birefringence in tumors found in the brain and near the eye compared to control regions. The presented work showed the potential of OCT and JM-OCT as non-destructive, high-resolution, and real-time imaging modalities for pre-clinical research based on zebrafish.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Preclinical testing of CAR T cells in zebrafish xenografts.

Engineered chimeric antigen receptor T cells (CAR T cells) have emerged as a promising immunotherapy for cancer and have proven to be effective for B cell malignancies. Currently, great efforts are undertaken to expand the application of CAR T cells to other cancer entities, to increase the efficacy of CAR T cell-mediated killing of cancer cells and to reduce possible side effects of CAR T cell therapy. This creates a need for preclinical models to test the many emerging novel CAR designs. Traditionally, mouse xenograft models are applied to investigate the efficacy of CAR T cells in vivo. Here, we describe a complementing xenograft protocol for testing CAR T cells against human leukemia cells in zebrafish embryos. The embryonic zebrafish xenograft promises to be a fast and cost-efficient model and particularly offers live imaging opportunities of CAR T cell distribution and killing of cancer cells in vivo.

Studying the Tumor Microenvironment in Zebrafish.

The tumor microenvironment significantly contributes to tumor initiation, progression, neo-angiogenesis, and metastasis, and a better understanding of the role of the different cellular players would facilitate the development of novel therapeutic strategies for cancer treatment. Towards this goal, intravital imaging is a powerful method to unravel interaction partners of tumor cells. Among vertebrate model organisms, zebrafish is uniquely suited for in vivo imaging studies. In recent years zebrafish has also become a valuable model in cancer research. In this chapter, we will summarize, how zebrafish has been used to characterize cells of the tumor microenvironment. We will cover both genetically engineered cancer models and xenograft models in zebrafish. The majority of work has been done on the role of innate immune cells and their role during tumor initiation and metastasis, but we will also cover studies focusing on adipocytes, fibroblasts, and endothelial cells. Taken together, we will highlight the versatile use of the zebrafish model for in vivo tumor microenvironment studies.

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation.

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.

Toward Quantitative in vivo Label-Free Tracking of Lipid Distribution in a Zebrafish Cancer Model.

Cancer cells often adapt their lipid metabolism to accommodate the increased fatty acid demand for membrane biogenesis and energy production. Upregulation of fatty acid uptake from the environment of cancer cells has also been reported as an alternative mechanism. To investigate the role of lipids in tumor onset and progression and to identify potential diagnostic biomarkers, lipids are ideally imaged directly within the intact tumor tissue in a label-free way. In this study, we investigated lipid accumulation and distribution in living zebrafish larvae developing a tumor by means of coherent anti-Stokes Raman scattering microscopy. Quantitative textural features based on radiomics revealed higher lipid accumulation in oncogene-expressing larvae compared to healthy ones. This high lipid accumulation could reflect an altered lipid metabolism in the hyperproliferating oncogene-expressing cells.

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

Using Zebrafish Larvae as a Xenotransplantation Model to Study Ewing Sarcoma.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Rapid In Vivo Validation of HDAC Inhibitor-Based Treatments in Neuroblastoma Zebrafish Xenografts.

The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations.

A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells In Vivo.

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

LIN28B increases neural crest cell migration and leads to transformation of trunk sympathoadrenal precursors.

The RNA-binding protein LIN28B regulates developmental timing and determines stem cell identity by suppressing the let-7 family of microRNAs. Postembryonic reactivation of LIN28B impairs cell commitment to differentiation, prompting their transformation. In this study, we assessed the extent to which ectopic lin28b expression modulates the physiological behavior of neural crest cells (NCC) and governs their transformation in the trunk region of developing embryos. We provide evidence that the overexpression of lin28b inhibits sympathoadrenal cell differentiation and accelerates NCC migration in two vertebrate models, Xenopus leavis and Danio rerio. Our results highlight the relevance of ITGA5 and ITGA6 in the LIN28B-dependent regulation of the invasive motility of tumor cells. The results also establish that LIN28B overexpression supports neuroblastoma onset and the metastatic potential of malignant cells through let-7a-dependent and let-7a-independent mechanisms.