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In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system, a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Neurofibromatosis Type 1 (NF1) patients develop an array of benign and malignant tumors, of which Malignant Peripheral Nerve Sheath Tumors (MPNST) and High Grade Gliomas (HGG) have a dismal prognosis. About 15-20% of individuals with NF1 develop brain tumors and one third of these occur outside of the optic pathway. These non-optic pathway gliomas are more likely to progress to malignancy, especially in adults. Despite their low frequency, high grade gliomas have a disproportional effect on the morbidity of NF1 patients. In vitro drug combination screens have not been performed on NF1-associated HGG, hindering our ability to develop informed clinical trials. Here we present the first in vitro drug combination screen (21 compounds alone or in combination with MEK or PI3K inhibitors) on the only human NF1 patient derived HGG cell line available and on three mouse glioma cell lines derived from the NF1-P53 genetically engineered mouse model, which sporadically develop HGG. These mouse glioma cell lines were never exposed to serum, grow as spheres and express markers that are consistent with an Oligodendrocyte Precursor Cell (OPC) lineage origin. Importantly, even though the true cell of origin for HGG remains elusive, they are thought to arise from the OPC lineage. We evaluated drug sensitivities of the three murine glioma cell lines in a 3D spheroid growth assay, which more accurately reflects drug sensitivities in vivo. Excitingly, we identified six compounds targeting HDACs, BRD4, CHEK1, BMI-1, CDK1/2/5/9, and the proteasome that potently induced cell death in our NF1-associated HGG. Moreover, several of these inhibitors work synergistically with either MEK or PI3K inhibitors. This study forms the basis for further pre-clinical evaluation of promising targets, with an eventual hope to translate these to the clinic., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dougherty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit., Competing Interests: Declaration of interests W.C.H. is a consultant for Thermo Fisher Scientific, Solasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, iTeos, Frontier Medicines, Jubliant Therapeutics, RAPPTA Therapeutics, and Paraxel. A.J.A. has consulted for Oncorus, Inc., Arrakis Therapeutics, and Merck & Co., Inc., and has research funding from Mirati Therapeutics and Deerfield, Inc. that is unrelated to this project. A.O.G. is a share and option holder of 10X Genomics. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Aims: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions., Approach and Results: Exposure of zebrafish embryos to 17β-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b -/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development., Conclusions: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy., (© 2020 by the American Association for the Study of Liver Diseases.)

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.

Background & Aims: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues., Methods: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry., Results: Exposure to 17β-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish., Conclusions: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment., Transcript Profiling: The accession number in the Gene Expression Omnibus is GSE92544., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML., (© 2018 by The American Society of Hematology.)

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer., Competing Interests: JL, PC, CC, KL, JH, AG, JK, NI, JD, SL, CD, KH, AH, OG, SG, JG, DR, JZ, AB, RW, WH No competing interests declared, (© 2018, Li et al.)

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR., Competing Interests: STATEMENT W.C.H. is a consultant for and receives research support from Novartis., (©2016 American Association for Cancer Research.)

The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.