There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small molecule WNT-pathway inhibitor discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a Type 1 binding mode involving insertion of the CDK8 C-terminus into the ligand binding site. InReprints and permissions information is available online at http://www.nature.com/reprints/index.html.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

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.DOI: http://dx.doi.org/10.7554/eLife.20722.001

In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT -PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine–piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Purpose: The impact of the presence of a germ-line BRCA1 mutation on gene expression in normal breast fibroblasts after radiation-induced DNA damage has been investigated.Experimental Design: High-density cDNA microarray technology was used to identify differential responses to DNA damage in fibroblasts from nine heterozygous BRCA1 mutation carriers compared with five control samples without personal or family history of any cancer. Fibroblast cultures were irradiated, and their expression profile was compared using intensity ratios of the cDNA microarrays representing 5603 IMAGE clones.Results: Class comparison and class prediction analysis has shown that BRCA1 mutation carriers can be distinguished from controls with high probability (ϳ85%). Significance analysis of microarrays and the support vector machine classifier identified gene sets that discriminate the samples according to their mutation status. These include genes already known to interact with BRCA1 such as CDKN1B, ATR, and RAD51.Conclusions: The results of this initial study suggest that normal cells from heterozygous BRCA1 mutation carriers display a different gene expression profile from controls in response to DNA damage. Adaptations of this pilot result to other cell types could result in the development of a functional assay for BRCA1 mutation status.

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Introduction CDK8 is an oncogenic cyclin-dependent kinase that exists as part of the kinase module within the Mediator complex. This complex interacts with the transcription machinery to regulate transcription; signal transduction pathways, including the WNT pathway; and biological processes, such as cell cycle progression. Recently, we identified a series of 3,4,5-trisubstituted pyridines as inhibitors of CDK8 and, its paralogue, CDK19 in colorectal cancer (CRC). Until now, there have been few validated substrates of CDK8/19. Here, we describe a motif-based phospho-proteomic approach, utilizing our 3,4,5-trisubstituted pyridine inhibitors that we have used to identify substrates of CDK8/19. These substrates could represent useful biomarkers for future drug discovery research. Experimental Outline We used a COLO205 cell line (COLO205 C4) carrying a TCF/LEF reporter construct responsive to CDK8/19 inhibition and CCT251545, a compound we have previously shown to be a potent and selective inhibitor of CDK8/19. Cells were treated with 350 nM CCT251545 (10 x EC50) for 2 or 6 hours. Proteins were then extracted from treated and control cells, trypsin-digested and immunoprecipitated for phospho-peptide enrichment using proline-directed motifs: PXS*P, S*PXR/K, PXS*PXR/K + T*PE + ST*P + K/HS*P. Potential substrates were identified by LC-MS/MS and validated by immunoprecipitation and western blotting. Substrates were validated in another CRC cell line, SW620, which harbors CRISPR knockouts for CDK8/19. Summary of Results LC-MS/MS analyses of COLO205 C4 cell extracts revealed a number of potential CDK8/19 substrates, including some Mediator complex subunits such as MED13, transcriptional coactivators such as HCFC1, and transcription factors such as STAT1. Phosphorylation of STAT1SER727 was the top ranked hit and follow-up studies, in COLO205 C4 cells and xenografts, confirmed repression of STAT1SER727 phosphorylation in the presence of CCT251545. An inactive analogue, CCT251099, and other kinase inhibitors (flavopiridol, KN-93, PD 0325901 and SB 202190) did not block STAT1SER727 phosphorylation. Repression of STAT1SER727 phosphorylation upon treatment with CCT251545 was also observed in SW620 and LS174T CRC cells and xenografts. Conclusion A motif-driven, mass spectroscopy-based phospho-proteomic study identified candidate substrates of CDK8/19. Phosphorylation of STAT1SER727 was validated as a useful marker of target engagement in CRC cell lines both in vitro and in vivo. Citation Format: Olajumoke O. Popoola, Rahul Samant, Maria-Jesus Ortiz-Ruiz, Aurelie Mallinger, Will Court, Steve Hobbs, Robert Te-Poele, Mark Stubbs, Rosemary Burke, Christina Esdar, Kai Schiemann, Dirk Wienke, Sue Eccles, Julian Blagg, Paul Workman, Paul Clarke. Phosphoproteomic-based identification of CDK8/CDK19 substrates in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3869.