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

Oncogene volume 33, pages 5675–5687 (2014)Cite this article

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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 and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation 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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Abbreviations

BSA:

bovine serum albumin

CDC2:

cell division cycle 2

CDK:

cyclin-dependent kinase

CHX:

cycloheximide

CK1:

casein kinase 1

CLK:

cdc2-like kinase

CRKRS:

cdc2-related protein kinase 7 (CDK12)

DMEM:

Dulbecco/Vogt modified Eagle's minimal essential medium

DMSO:

dimethylsulfoxide

DTT:

dithiothreitol

DYRK:

dual specificity, tyrosine phosphorylation regulated kinase

FADK:

focal adhesion kinase

FCS:

fetal calf serum

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GSK-3:

glycogen synthase kinase-3

PBS:

phosphate-buffered saline

PDXK:

pyridoxal kinase

RT:

room temperature.

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Acknowledgements

This article is dedicated to the memory of Jill Lahti and Vincent Kidd. We are grateful to Jacint Boix and Jean Bénard, for the neuroblastoma cell lines. This research was supported by grants from the EEC (FP6 Life Sciences & Health PRO-KINASE and TEMPO Research Projects), the ‘Cancéropole Grand-Ouest’, the ‘Association France-Alzheimer Finistère’, the ‘Association pour la Recherche sur le Cancer’ (ARC-1092), the ‘Ligue Nationale contre le Cancer (Comité Grand-Ouest)’, the Polycystic Kidney Disease Foundation, the Fondation Jérôme Lejeune, the ‘Conseil Régional de Bretagne’ (‘Fonds de Maturation’ 2009) and the ‘Institut National contre le Cancer’ (INCa) GLIOMER and CCCDK8 programs.

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Authors and Affiliations

  1. ManRos Therapeutics, Hôtel de Recherche, Centre de Perharidy, Roscoff, France

    C Delehouzé, N Loaëc, C Bruyère, N Oumata & L Meijer

  2. Evotec (München) GmbH, Am Klopferspitz 19a, Martinsried, Germany

    K Godl

  3. C.N.R.S., ‘Protein Phosphorylation & Human Disease’ Group, Station Biologique, B.P. 74, Roscoff cedex, Bretagne, France

    N Loaëc, N Desban & L Meijer

  4. Laboratoire de Chimie Organique 2, INSERM U 648, Université Paris—Descartes, 4 avenue de l'Observatoire, Paris cedex, France

    H Galons

  5. Cancer Sciences & Clinical and Experimental Medicine, University of Southampton, Institute for Life Sciences, Center for Proteomics & Metabolomics Research, Highfield Campus, Southampton, UK

    T I Roumeliotis & S D Garbis

  6. Institute for Computational Biomedicine, Weil Cornell Medical College, New York, NY, USA

    E G Giannopoulou

  7. Department of Tumor Cell Biology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, USA

    J Grenet, D Twitchell & J Lahti

  8. CNRS UMR 6290-Institut de Génétique et Développement de Rennes, Equipe Expression des Gènes et Oncogenèse, Université de Rennes1, SFR Biosit, Faculté de Médecine, 2 avenue du Pr. Léon Bernard, Rennes cedex, France

    N Mouchet & M-D Galibert

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Corresponding authors

Correspondence to H Galons, S D Garbis or L Meijer.

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Competing interests

Dr Meijer is the co-inventor on the roscovitine patent. Dr Galons, Dr Oumata and Dr Meijer are co-inventors on the CR8 patent. Dr Meijer and Dr Galons are co-founders of ManRos Therapeutics. Dr Meijer is the President and CSO of ManRos Therapeutics.

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Supplementary Information accompanies this paper on the Oncogene website

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Delehouzé, C., Godl, K., Loaëc, N. et al. CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells. Oncogene 33, 5675–5687 (2014). https://doi.org/10.1038/onc.2013.513

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  • DOI: https://doi.org/10.1038/onc.2013.513

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