The Mediator complex is a regulator of gene expression, influencing chromatin structure and RNA polymerase II-mediated transcription. Its activity is controlled by a protein kinase module, which includes cyclin-dependent kinases 8 and 19, that phosphorylates RNA polymerase II and transcription factors to regulate gene expression. Using orthogonal approaches combining chemical and genetic tools, we demonstrated the selectivity of our small-molecule inhibitors derived from 3,4,5-trisubstituted pyridine and 3-methyl-1H-pyrazolo[3,4-b]pyridine chemical series in human colorectal cell culture and tumor xenograft models. The lack of activity of our inhibitors in CDK8/19 double knockout models, with respect to molecular, proliferative, and antitumor end points, revealed their specificity and dependence on these kinases. Using our chemical probes and knockout models, we explored Mediator kinase function in human colorectal cancer cells. Phospho-proteome profiling revealed substrates enriched with transcription and chromatin regulators, while promoter reporter experiments identified transcription factor binding sites, including TCF/LEF and AP1, regulated by Mediator kinases. Additionally, altered phosphorylation of several Mediator subunits suggests a mechanism for the rapid regulation of the Mediator complex. Overall, our results demonstrate that CDK8 and CDK19 play pivotal roles in regulating gene expression associated with oncogene activation and signaling pathways. Further studies are warranted to elucidate their broader cellular roles and regulatory mechanisms. The selective inhibitors validated in this study will provide valuable tools for such mechanistic investigations into Mediator kinase functions and their potential therapeutic exploitation.

Adobe PDF - MCT-07-0149--Suppl_Data.pdf from Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Changes in candidate plasma metabolite pharmacodynamic biomarker concentrations over first 2 weeks in 17 patients treated with {greater than or equal to}330 mg once-daily of pictilisib. Cells with p-values <0.05 are shaded dark grey and those with p-values <0.10 and {greater than or equal to}0.05 are shaded light grey.

Metabolite changes that constitute the Venn diagram in Figure 2. Significant changes listed are reductions (labelled "-1") or increases (labelled "1") in concentrations relative to the relevant controls.

Changes in plasma metabolites across timepoints by patient/sample.

Supplementary Figure Legends 1-4 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

Supplementary Figure 1C from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Materials, Figure Legends 1-6, Tables 1-5 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

PDF file, 63KB, Kinase inhibitory profile of SAR-020106 at 10 μMa.

Supplementary Figure 6 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 3A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Summary of baseline reproducibility of candidate plasma metabolite biomarkers (n=17 patients).

Time of day variation in metabolites in healthy volunteers and in patients treated with the MEK inhibitor.

Supplementary Figure S1. Schematic of the MR elastography platform used in this study.

Supplementary Figure 2 from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Supplementary Table 4 from CHR-2797: An Antiproliferative Aminopeptidase Inhibitor that Leads to Amino Acid Deprivation in Human Leukemic Cells

Heat map of changes (relative to vehicle control) in candidate plasma and tumor metabolite biomarkers at 8 hours post-dosing of mice bearing U87MG xenografts with pictilisib 75mg/kg or buparlisib 60mg/kg (a, acyl; aa, acyl-acyl; ae, acyl-alkyl; Cx:y, where x is the number of carbons in the fatty acid side chain; y is the number of double bonds in the fatty acid side chain; AC, acylcarnitine; DC, decarboxyl; M, methyl; OH, hydroxyl; PC, phosphatidylcholine)

Characteristics of dose-dependent changes in candidate plasma metabolite pharmacodynamic biomarker concentrations in 41 patients, as determined on day 1 of pictilisib clinical trial. Cells with p-values <0.05 are shaded dark grey and those with p-values <0.10 and {greater than or equal to}0.05 are shaded light grey.

<p>Supplementary Figure S3. Representative Luxol Fast Blue staining for myelin fibres within intracranial U-87 MG, RG2 and MDA-MB-231 tumors.</p>