Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors

Aiko Murai,Satoshi Sasaki,Tomohiro Ohashi,Tohru Miyazaki,Shunsuke Ebara,Shinsuke Araki,Toshiyuki Nomura,Irina V Lebedeva

doi:10.1371/journal.pone.0240718

Abstract

Emerging evidence indicates that alternative splicing plays a critical role in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics. CDC-like kinases (CLKs) are central to exon recognition in mRNA splicing and CLK inhibitors exhibit anti-tumour activities. Most importantly, molecular mechanism-based combination strategies for cancer therapy must be considered. However, it remains unclear whether CLK inhibitors modulate expression and splicing of apoptosis-related genes, and whether CLK inhibitors enhance cytotoxicity in combination with apoptosis inducers. Here we report an appropriate mechanism-based drug combination approach. Unexpectedly, we found that the CLK inhibitor T3 rapidly induced apoptosis in A2780 cells and G2/M cell cycle arrest in HCT116 cells. Regardless of the different phenotypes of the two cancer cell types, T3 decreased the levels of anti-apoptotic proteins (cIAP1, cIAP2, XIAP, cFLIP and Mcl-1) for a short period of exposure and altered the splicing of the anti-apoptotic MCL1L and CFLAR isoform in A2780 and HCT116 cells. In contrast, other members of the Bcl-2 family (i.e., Bcl-xL and Bcl-2) were resistant to T3-induced expression and splicing modulation. T3 and a Bcl-xL/Bcl-2 inhibitor synergistically induced apoptosis. Taken together, the use of a CLK inhibitor is a novel therapeutic approach to sensitise cancer cells to Bcl-xL/Bcl-2 inhibitors.

Highlights

Alternative pre-mRNA splicing is a fundamental mechanism that generates multiple mRNAs from a single gene via a mechanism that is tightly regulated to generate proteomic diversity sufficient to maintain physiological homeostasis and processes [1,2,3,4]
Two representative human cancer cell lines were used to investigate the detailed mechanisms underlying the inhibition of cell growth and apoptosis induced by the splicing modulator T3 and its inactive analog T3-1
Caspase 3/7 activity was approximately five-fold greater in A2780 cells, as compared to HCT116 cells, which was consistent with the cell cycle and Annexin V data (Fig 1E)

Summary

Introduction

Alternative pre-mRNA splicing is a fundamental mechanism that generates multiple mRNAs from a single gene via a mechanism that is tightly regulated to generate proteomic diversity sufficient to maintain physiological homeostasis and processes [1,2,3,4]. Dysregulation of alternative splicing leads to the generation of aberrant protein isoforms that contribute to various diseases, including neurodegenerative diseases, muscular dystrophies and various cancers [5]. Specific aberrant splicing of various transcripts, such as Bcl-xL, Cyclin D1, CD44, and VEGF, is known to promote tumour survival and growth, as well as resistance to apoptosis as a consequence of the abnormal expression or mutation of splicing factors [6,7,8,9,10]. Synergistic apoptotic effects in cancer cells by CLK and Bcl-2 family inhibitors

Methods

Results

Conclusion