By Samantha Yates
Alternative splicing is a form of messenger RNA (mRNA) splicing by which many different protein isoforms are produced. Alternative splicing rearranges and combines exons in different positions to alter the mRNA coding sequence and is the main source of protein diversity in >90% of human genes. Due to its tightly regulated process (by both cis- and trans-regulatory elements), the dysregulation of alternative splicing is a hallmark of cancer, but is now redeeming itself as a target in alternative splicing-dependent cancer therapy (Yang et al., 2019). Alternative splicing can become oncogenic through mutations in regulatory elements and altered expression of splicing factors. These mutations arise due to different levels of phosphorylation, methylation, and post-translational modifications (Lin, 2017). In this review I will cover the three main methods of alternative splicing-dependent cancer therapy and questions which remain unanswered in the field.
Three methods of alternative splicing-dependent cancer therapy are global splicing inhibition using small molecules blocking the spliceosome and splicing-factor-modifying enzymes, and isoform-specific splice-switching RNA-based therapeutics.
Global splicing inhibition can utilise small molecules to block the assembly of functional spliceosome components and therefore inhibits alternative splicing. (Urbanski et al., 2018). Pladienolides and herboxidienes are small molecules which target SF3B1 (a gene that is commonly mutated in cancer through deleterious point mutations) and prevents its conformation rearrangement, leading to cell cycle arrest (Lin, 2017).
Global splicing inhibition can also be achieved by inhibiting and/or modifying enzymes which regulate the activity of splicing regulatory factors (SF). SRPIN340 is an inhibitor of the serine-rich protein specific kinase (SRPK) family members SRPK1 and SPRK2 as it acts as an ATP binding competitor. This inhibition promotes the production of anti-angiogenic isoforms (Martinez-Montiel et al., 2018).
TG-003 is a protein inhibitor of the Cdc2-like kinase (CLK) family members, CLK1 and CLK4, as it acts as a competitive inhibitor of ATP. TG-003 is known to inhibit cancer cell growth and increase the production of dystrophin protein which can act as a tumour suppressor through regulation of tumour invasion and migration (Araki et al., 2015).
SPRKs and CLKs are vital to the phosphorylation of the variable-length RS domains of SR proteins – which have been found to be crucial in determining the site of alternative splicing and recruitment of complex splicing factors to the pre-mRNA (Lee et al., 2019). Therefore, SRPIN340 and TG-003 indirectly lead to a reduction in phosphorylation of serine/arginine rich (SR) proteins, and the subsequent altered cellular localisation and interactions of the family (Lin, 2017).
Isoform-specific inhibition can be achieved using splice-switching antisense oligonucleotides (SSOs). SSOs are short and modified nucleic acids that bind specifically to sequences of pre-mRNA in an antisense orientation, and sterically block the access of splicing factors and prevent RNA-RNA and RNA-protein interactions (Havens & Hastings, 2016). There are two main groups of SSO: exon skipping, made up of splice site-blocking and splicing enhancer-blocking, and exon inclusion, made up of splicing silencer-blocking SSOs (Martinez-Montinel et al., 2018).
Bcl-x pre-mRNA can be alternatively spliced to form two isoforms: pro-apoptotic Bcl-xS and anti-apoptotic Bcl-xL isoform. An SSO was designed to target the downstream 5’-splice site of Bcl-x which redirected the spliceosome to the upstream alternative splice site. This led to a decrease in Bcl-xL expression and an increase in Bcl-xS expression, leading the cells in the cell line to undergo apoptosis (Li et al., 2015).
Tumours are a heterogeneous mass of tumour cells amongst other cell types, including fibroblasts, immune cells, and endothelial cells. More work needs to be done on the effect of the tumour microenvironment on tumour progression, alternative splicing-targeted drug responses, and alternative splicing itself. It is also unknown whether alternatively spliced protein isoforms are present in all the cell types within tumours or not. (Urbanski et al., 2018). Progression in understanding the regulatory mechanisms involved in cancer-associated alternative splicing will help the development of more specific and personalised treatments for different cancer types (Lin, 2018). How CLK inhibitors, such as TG-003, impact cellular functions and their different selectivity, needs to be investigated further (Araki et al., 2015).
Araki, S., Dairiki, R., Nakayama, Y., Murai, A., Miyashita, R., Iwatani, M., Nomura, T. & Nakanishi, O. (2015) Inhibitors of CLK Protein Kinases Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing. PLoS One. 10(1). Available from: doi:10.1371/journal.pone.0116929
Lee, J. Y., Yun, J.-S., Kim, W.-K., Chun, H.-S., Jin, H., Cho, S. & Chang, J. H. (2019) Structural Basis for the Selective Inhibition of Cdc2-Like Kinases by CX-4945. BioMed Research International. 2019. Available from: doi:10.1155/2019/6125068
Lin, J.-C. (2018) Therapeutic Applications of Targeted Alternative Splicing to Cancer Treatment. International Journal of Molecular Sciences. 19(1). Available from: doi:10.3390/ijms19010075
Havens, M. A. & Hastings, M. L. (2016) Splice-switching antisense oligonucleotides as therapeutic drugs. Nucleic Acids Research. 44(14). Available from: doi:10.1093/nar/gkw533
Martinez-Montiel, N., Rosas-Murrieta, N. H., Ruiz, M. A., Monjaraz-Guzman, E. & Martinez-Contreras, R. (2018) Alternative Splicing as a Target for Cancer Treatment. International Journal of Molecular Sciences. 19(2). Available from: 10.3390/ijms19020545
Urbanski, L., Leclair, N. & Anczuków, O. (2018) Alternative-splicing defects in cancer: splicing regulators and their downstream targets, guiding the way to novel cancer therapeutics. Wiley Interdisciplinary Review RNA. 9(4). Available from: doi:10.1002/wrna.1476Yang, Q., Zhao, J., Zhang, W., Chen, D. & Wang, Y. (2019) Aberrant alternative splicing in breast cancer. Journal of Molecular Cell Biology. 11 (10), 920-929. Available from: doi:10.1093/jmcb/mjz033