Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases
- The Institute of Cancer Research, United Kingdom
- Merck KGaA, Germany
- Cardiff University, United Kingdom
Abstract
Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.
Data availability
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Assessing the mechanism and therapeutic potential of modulators of the human mediator complex-associated protein kinasesPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE80472).
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Effect of cortistatin A (CA) on enhancer occupancy in CA-sensitive and -insensitive human cell linesPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE65138).
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
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Molecular Signatures Database v5.1Available at the Gene Set Enrichment Analysis site (http://software.broadinstitute.org/gsea/msigdb/). Users are required to register to view the MSigDB gene sets and/or download the GSEA software.
Article and author information
Author details
Paul Andrew Clarke
Funding
Cancer Research UK (C309/A11566, C368/A6743, A368/A7990)
- Paul Workman
Breast Cancer Now (2008MayPR16)
- Trevor C Dale
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: In the UK, all animal work was conducted in accordance with National Institute for Cancer Research guidelines, with the research programme and procedures approved by the local Animal Welfare and Ethical Review Boards and subject to UK Government Home Office regulations (Licence PPL 70/7635 & PPL 30/3279). In Germany the animal work was carried out in accordance with the German Law on the Protection of Animals (Article 8a) and the pertaining files at the at the local animal welfare authorities in Darmstadt and Freiburg bear the references DA/375, DA4/1003, DA4/1004 and G13/13 respectively. The studies were designed in accordance with presently valid international study guidelines (e.g. ICH guideline M3 R2) and performed in compliance with animal health and welfare guidelines.The Institute of Cancer Research does not use non-rodent species in research and, where this is deemed essential, requires ethical approval for use by organizations with whom we collaborate. Pharmacokinetic and tolerability analysis of compounds in dogs, necessary for prediction of human pharmacokinetics, was approved by the ICR Animal Welfare and Ethical Review Board. Studies were sponsored and conducted in full compliance with national regulations at an Association for Assessment and Accreditation of Laboratory Animal Care accredited site of Merck Biopharma.
Copyright
© 2016, Clarke et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases
eLife 5:e20722.
https://doi.org/10.7554/eLife.20722
Further reading
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- Cancer Biology
- Genetics and Genomics
Interpretation of variants identified during genetic testing is a significant clinical challenge. In this study, we developed a high-throughput CDKN2A functional assay and characterized all possible human CDKN2A missense variants. We found that 17.7% of all missense variants were functionally deleterious. We also used our functional classifications to assess the performance of in silico models that predict the effect of variants, including recently reported models based on machine learning. Notably, we found that all in silico models performed similarly when compared to our functional classifications with accuracies of 39.5–85.4%. Furthermore, while we found that functionally deleterious variants were enriched within ankyrin repeats, we did not identify any residues where all missense variants were functionally deleterious. Our functional classifications are a resource to aid the interpretation of CDKN2A variants and have important implications for the application of variant interpretation guidelines, particularly the use of in silico models for clinical variant interpretation.
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- Cancer Biology
- Developmental Biology
Missense ‘hotspot’ mutations localized in six p53 codons account for 20% of TP53 mutations in human cancers. Hotspot p53 mutants have lost the tumor suppressive functions of the wildtype protein, but whether and how they may gain additional functions promoting tumorigenesis remain controversial. Here, we generated Trp53Y217C, a mouse model of the human hotspot mutant TP53Y220C. DNA damage responses were lost in Trp53Y217C/Y217C (Trp53YC/YC) cells, and Trp53YC/YC fibroblasts exhibited increased chromosome instability compared to Trp53-/- cells. Furthermore, Trp53YC/YC male mice died earlier than Trp53-/- males, with more aggressive thymic lymphomas. This correlated with an increased expression of inflammation-related genes in Trp53YC/YC thymic cells compared to Trp53-/- cells. Surprisingly, we recovered only one Trp53YC/YC female for 22 Trp53YC/YC males at weaning, a skewed distribution explained by a high frequency of Trp53YC/YC female embryos with exencephaly and the death of most Trp53YC/YC female neonates. Strikingly, however, when we treated pregnant females with the anti-inflammatory drug supformin (LCC-12), we observed a fivefold increase in the proportion of viable Trp53YC/YC weaned females in their progeny. Together, these data suggest that the p53Y217C mutation not only abrogates wildtype p53 functions but also promotes inflammation, with oncogenic effects in males and teratogenic effects in females.
