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

ABSTRACT: Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, characterized by a lack of therapeutic options. Dysregulation of alternative splicing plays a pivotal role in tumorigenesis and metastasis. Serine/arginine-rich (SR) proteins, essential components of the spliceosome, undergo phosphorylation by Cdc2-like kinases (CLKs). Here we explored the impact of pharmacological inhibition of CLKs using a novel inhibitor, T-025, on TNBC cell proliferation and migration. Methods In this study, 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. Live-cell imaging and photobleaching experiments were conducted to determine the subnuclear localization of SRSF7-GFP and its dynamic mobility. Pulldown/MS was used to uncover the impact of T-025 on SRSF7 interactome. Deep RNA sequencing was performed to unravel the differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) upon T-025 treatment. Results T-025 exhibited a potent anti-proliferative effect, particularly in highly proliferative TNBC 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 4N DNA. T-025 significantly inhibited cell migration in highly migratory TNBC cell lines. T-025 led to the accumulation of SRSF7-GFP at nuclear speckles, restricting its mobility. 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 observed. RNA sequencing uncovered multiple DEGs and ASGs upon T-025 treatment, which were significantly enriched in pathways related to cell division, RNA splicing and cell migration. Conclusions We demonstrated the pivotal role of CLKs inhibition in the proper function of SR proteins and RNA splicing in TNBC cell lines. CLKs inhibition 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 and migration. Our findings provide evidence that CLKs inhibition could be a promising therapeutic approach for TNBC patients.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Nila Overbeek  

LAB HEAD: Sylvia Le Dévédec

PROVIDER: PXD058273 | Pride | 2025-08-04

REPOSITORIES: Pride