CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells

Abstract

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of ‘-omics’ techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: [1] kinase interaction assays, [2] affinity competition on immobilized broad-spectrum kinase inhibitors, [3] affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, [4] whole genome transcriptomics analysis and specific quantitative PCR studies, [5] global quantitative proteomics approach and Western blot analysis of selected proteins. Altogether the results show that the major direct targets of these two molecules belong to the CDKs (1, 2, 5, 7, 9, 12), DYRKs, CLKs, CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in down-regulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors down-regulation. Indeed CDK inhibitors trigger rapid and massive down-regulation of MYCN expression in MYCN amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

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CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses

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Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study

“…The offline HILIC peptide separation has also been reported by the authors (Garbis et al , 2011; Delehouze et al , 2014; Bouchal et al , 2015). The discovery experiment was executed once.…”