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.
Reviewing Editor
- Benjamin F Cravatt, The Scripps Research Institute, United States
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.
Version history
- Received: August 21, 2016
- Accepted: November 29, 2016
- Accepted Manuscript published: December 9, 2016 (version 1)
- Version of Record published: January 10, 2017 (version 2)
- Version of Record updated: January 31, 2017 (version 3)
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
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Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.
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- Cancer Biology
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Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.
