Journal Article

Inhibitors of CLK Protein Kinases Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing

Nakayama, Yusuke, Iwatani, Misa, Nakanishi, Osamu, Murai, Aiko, Araki, Shinsuke, Dairiki, Ryo, Nomura, Toshiyuki, Miyashita, Risa

2015

Overview

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Alternative splicing / Analysis / Anticancer properties / Antitumor agents / Apoptosis / Apoptosis - drug effects / Apoptosis - genetics / Arginine / Arginine - antagonists & inhibitors / Arginine - metabolism / Cancer / Cell growth / Cell Line, Tumor / Cell Proliferation - drug effects / Cell Proliferation - genetics / Codons / Compounds / Depletion / Enlargement / Epidermal growth factor receptors / Fluorescence / Gene expression / Gene sequencing / Genes / Genomes / Growth / HCT116 Cells / Health aspects / Humans / Inhibitors / Isoforms / Kinases / Localization / Messenger RNA / Molecular machines / Molecular modelling / mRNA / Nuclear Proteins - antagonists & inhibitors / Nuclear Proteins - metabolism / Pharmaceutical industry / Phosphatase / Phosphorylation - drug effects / Phosphorylation - genetics / Physiological aspects / Poly(ADP-ribose) polymerase / Protein Isoforms - antagonists & inhibitors / Protein Isoforms - metabolism / Protein kinase / Protein Kinase Inhibitors - pharmacology / Protein kinases / Protein-Serine-Threonine Kinases - antagonists & inhibitors / Protein-Serine-Threonine Kinases - metabolism / Protein-Tyrosine Kinases - antagonists & inhibitors / Protein-Tyrosine Kinases - metabolism / Proteins / RNA Precursors - genetics / RNA Precursors - metabolism / RNA Splicing - drug effects / RNA Splicing - genetics / RNA, Messenger - genetics / RNA-Binding Proteins - metabolism / RNA-protein interactions / Serine / Small Molecule Libraries - pharmacology / Survival