Journal Article

Pharmacological CLK inhibition disrupts SR protein function and RNA splicing blocking cell growth and migration in TNBC

van Overbeek, Nila K., Vertegaal, Alfred C. O., Batenburg, Daisy, Liu, Nasi, Le Dévédec, Sylvia E., Koedoot, Esmee, van der Velde, Jurjun J. S., Ramdjielal, Sherien, van de Water, Bob

2025

Overview

Background Dysregulation of alternative splicing plays a pivotal role in tumorigenesis and metastasis in triple-negative breast cancer (TNBC). Serine/arginine-rich (SR) proteins, essential components of the spliceosome, undergo phosphorylation by Cdc2-like kinase (CLK). Here we explored the impact of pharmacological inhibition of CLK using a novel inhibitor, T-025, on the spliceosome complex and transcriptional responses in relation to cell proliferation and migration in TNBC. Methods We evaluated the anti-proliferative and anti-migratory efficacy of T-025 in a spectrum of TNBC cell lines. Fluorescent reporter cell lines and flowcytometry were used to determine the effect of T-025 on cell cycle. Deep RNA sequencing was performed to unravel the differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) upon T-025 treatment. Pulldown/MS was used to uncover the impact of T-025 on SRSF7 interactome. Live-cell imaging and photobleaching experiments were conducted to determine the subnuclear localization of SRSF7-GFP and its dynamic mobility. Results T-025 exhibited a potent anti-proliferative effect in a spectrum of TNBC cell lines, particularly in highly proliferative cell lines. Treatment with T-025 induced cell cycle arrest in the G1-S phase, resulting in an increased proportion of aneuploidy cells and cells with 4 N DNA. T-025 significantly inhibited cell migration in highly migratory TNBC cell lines. Deep RNA sequencing uncovered numerous DEGs and ASGs upon T-025 treatment, which were significantly enriched in pathways related to cell division, RNA splicing and cell migration. Pulldown/MS showed that SRSF7 interacted more with nuclear-speckle-residing proteins, while less with RNA helicases and polymerases upon T-025 treatment. Enhanced interactions between SRSF7 and other phosphorylated SR proteins localized at nuclear speckles were also observed. Live-cell imaging indicated that T-025 treatment induced the accumulation of SRSF7-GFP at nuclear speckles and nuclear speckles’ enlargement, restricting its protein dynamic mobility. Conclusions CLK inhibition using T-025 leads to the accumulation of splicing factors at nuclear speckles and stalls their release to splicing sites, resulting in the RNA splicing reprogramming of a large number of genes involved in cell division, migration and RNA splicing. Our findings provide evidence that T-025 could be a promising therapeutic drug for TNBC patients.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alternative splicing / Alternative Splicing - drug effects / Aneuploidy / Antibodies / Artificial chromosomes / Biomedical and Life Sciences / Biomedicine / Breast cancer / Cancer Research / Cancer therapies / Cdc2 protein / Cdc2-like kinase / Cell adhesion & migration / Cell cycle / Cell division / Cell growth / Cell Line, Tumor / Cell migration / Cell Movement - drug effects / Cell proliferation / Cell Proliferation - drug effects / Chemotherapy / Cloning / Female / Gene Expression Regulation, Neoplastic - drug effects / Genes / Humans / Kinases / Localization / Metastases / Metastasis / Oncology / Phosphorylation / Photobleaching / Protein Kinase Inhibitors - pharmacology / Protein Serine-Threonine Kinases - antagonists & inhibitors / Protein-Tyrosine Kinases - antagonists & inhibitors / Proteins / Ribonucleic acid / RNA / RNA sequencing / RNA Splicing - drug effects / S phase / Serine-Arginine Splicing Factors - genetics / Serine-Arginine Splicing Factors - metabolism / Serine/arginine-rich proteins / Spliceosomes - drug effects / Spliceosomes - metabolism / Splicing factors / Surgical Oncology / Triple Negative Breast Neoplasms - drug therapy / Triple Negative Breast Neoplasms - genetics / Triple Negative Breast Neoplasms - metabolism / Triple Negative Breast Neoplasms - pathology / Triple-negative breast cancer / Tumorigenesis