Screening for YAP Inhibitors Identifies Statins as Modulators of Fibrosis.
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ABSTRACT:
SUBMITTER: Santos DM
PROVIDER: S-EPMC7110981 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5? strains show a G1 delay after CG-1521 treatment, however the gcn5? strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5? strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.
| S-EPMC4089024 | biostudies-literature
Project description:Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.
| S-EPMC3479568 | biostudies-other
Project description:TLR3 is one of the major innate immune sensors of dsRNA. The signal transduction pathway activated by TLR3, upon binding to dsRNA, leads to the activation of two major transcription factors: NF-kappaB and IFN regulatory factor (IRF) 3. In an effort to identify specific chemical modulators of TLR3-IRF3 signal transduction pathway, we developed a cell-based readout system. Using the IFN-stimulated gene 56 promoter-driven firefly luciferase gene stably integrated in a TLR3-expressing HEK293 cell line, we were able to generate a cell line where treatment with dsRNA resulted in a dose-dependent induction of luciferase activity. A screen of two pharmacologically active compound libraries using this system identified a number of TLR3-IRF3 signaling pathway modulators. Among them we focused on a subset of inhibitors and characterized their mode of action. Several antipsychotic drugs, such as sertraline, trifluoperazine, and fluphenazine, were found to be direct inhibitors of the innate immune signaling pathway. These inhibitors also showed the ability to inhibit IFN-stimulated gene 56 induction mediated by TLR4 and TLR7/8 pathways. Interestingly, they did not show significant effects on TLR3-, TLR7-, and TLR8-mediated NF-kappaB activation. Detailed analysis of the signaling pathway indicated that these drugs might be exerting their inhibitory effects on IRF3 via PI3K signaling pathway. The data presented in this study provide mechanistic explanation of possible anti-inflammatory roles of some antipsychotic drugs.
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Project description:Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.
| S-EPMC6279925 | biostudies-literature
Project description:Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.
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Project description:BACKGROUND: Inactivation of the NF2 gene predisposes to neurofibromatosis type II and the development of schwannomas. In vitro studies have shown that loss of NF2 leads to the induction of mitogenic signaling mediated by receptor tyrosine kinases (RTKs), MAP kinase, AKT, or Hippo pathways. The goal of our study was to evaluate the expression and activity of these signaling pathways in human schwannomas in order to identify new potential therapeutic targets. METHODS: Large sets of human schwannomas, totaling 68 tumors, were analyzed using complementary proteomic approaches. RTK arrays identified the most frequently activated RTKs. The correlation between the expression and activity of signaling pathways and proliferation of tumor cells using Ki67 marker was investigated by reverse-phase protein array (RRPA). Finally, immunohistochemistry was used to evaluate the expression pattern of signaling effectors in the tumors. RESULTS: We showed that Her2, Her3, PDGFRß, Axl, and Tie2 are frequently activated in the tumors. Furthermore, RRPA demonstrated that Ki67 levels are linked to YAP, p-Her3, and PDGFRß expression levels. In addition, Her2, Her3, and PDGFRß are transcriptional targets of Yes-associated protein (YAP) in schwannoma cells in culture. Finally, we observed that the expression of these signaling effectors is very variable between tumors. CONCLUSIONS: Tumor cell proliferation in human schwannomas is linked to a signaling network controlled by the Hippo effector YAP. Her2, Her3, PDGFRß, Axl, and Tie2, as well as YAP, represent potentially valuable therapeutic targets. However, the variability of their expression between tumors may result in strong differences in the response to targeted therapy.
| S-EPMC4136892 | biostudies-other
Project description:Tubulin modulating agents such as the taxanes are among the most effective antimitotic cancer drugs, although resistance and toxicity present significant problems in their clinical use. However, most tubulin modulators are derived from complex natural products, which can make modification of their structure to address these problems difficult. Here, we report the discovery of new antimitotic compounds with simple structures that can be rapidly synthesized, through the phenotypic screening of a diverse compound library for the induction of mitotic arrest. We first identified a compound, which induced mitotic arrest in human cells at submicromolar concentrations. Its simple structure enabled rapid exploration of activity, defining a biphenylacetamide moiety required for activity, A family of analogues was synthesized, yielding optimized compounds that caused mitotic arrest and cell death in the low nanomolar range, comparable to clinically used antimitotic agents. These compounds can be synthesized in 1-3 steps and good yields. We show that one such compound targets tubulin, partially inhibiting colchicine but not vinblastine binding, suggesting that it acts allosterically to the known colchicine-binding site. Thus, our results exemplify the use of phenotypic screening to identify novel antimitotic compounds from diverse chemical libraries and characterize a family of biphenylacetamides (biphenabulins) that show promise for further development.
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Project description:The prognosis of metastatic melanoma remains poor due to de novo or acquired resistance to immune and targeted therapies. Previous studies have shown that melanoma cells have perturbed metabolism and that cellular metabolic pathways represent potential therapeutic targets. To support the discovery of new drug candidates for melanoma, we examined 180 metabolic modulators, including phytochemicals and anti-diabetic compounds, for their growth-inhibitory activities against melanoma cells, alone and in combination with the BRAF inhibitor vemurafenib. Two positive hits from this screen, 4-methylumbelliferone (4-MU) and ursolic acid (UA), were subjected to validation and further characterization. Metabolic analysis showed that 4-MU affected cellular metabolism through inhibition of glycolysis and enhanced the effect of vemurafenib to reduce the growth of melanoma cells. In contrast, UA reduced mitochondrial respiration, accompanied by an increase in the glycolytic rate. This metabolic switch potentiated the growth-inhibitory effect of the pyruvate dehydrogenase kinase inhibitor dichloroacetate. Both drug combinations led to increased production of reactive oxygen species, suggesting the involvement of oxidative stress in the cellular response. These results support the potential use of metabolic modulators for combination therapies in cancer and may encourage preclinical validation and clinical testing of such treatment strategies in patients with metastatic melanoma.
| S-EPMC7902673 | biostudies-literature
Project description:Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. We used a platform for drug discovery based on systems biology and artificial intelligence, to identify drugs targeting key proteins described to have a role in the microenvironment. The selected compounds were screened in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells. Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib. We proposed that systems biology approaches combined with pharmacological screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.
| S-EPMC7746765 | biostudies-literature
Project description:Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMG-CoA reductase. These results suggest that statins should be re-evaluated as anti-leukemia agents, and illustrate the power of merging physiologically-relevant models with high-throughput screening. Freshly isolated LSCe cells were treated with either 5M-BM-5M BRD7116 or DMSO vehicle control for 6 hours in suspension in IMDM (12440053, Invitrogen), 10% FBS. Total RNA was extracted from the cells, labeled, and hybridized to MouseRef-8 v2.0 Expression BeadChips. Hartwell, K.A. et al., In Press (full citation forthcoming)
2013-09-20 | E-GEOD-51033 | biostudies-arrayexpress