RNA Splicing Dysregulation and Cancer

What is RNA Splicing?

RNA splicing is a highly regulated process, carried out by the spliceosome and other splicing regulatory factors. The spliceosome recognizes core regulatory sequences in pre-mRNA, including the 5' and 3' splice sites (5'SS and 3'SS) marking intron-exon boundaries, the branch point site (BPS), and the polypyrimidine tract. Two spliceosome complexes carry out splicing reactions, the U2-type (major spliceosome) or the U12-type (minor spliceosome). They differ primarily in the RNA components used in their respective splicing reactions and the splice site sequences they recognize. The U2-type spliceosome predominantly recognizes GT-AG splice sites, responsible for removing approximately 99% of introns, while the U12-type spliceosome, recognizing AT-AC and GT-AG sites, participates in removing less than 1% of introns and regulates a set of unique splicing events. In addition to nucleotides adjacent to the 5'SS and 3'SS, the core regulatory sequences recognized by the spliceosome exhibit considerable diversity. An additional layer of regulation is thus required, which depends on cis-regulatory sequences and trans-acting splicing factor proteins that can enhance or weaken the spliceosome's recognition of splice sites. Together, these cis-regulatory sequences and trans-acting splicing factors regulate alternative splicing, allowing a single gene to encode multiple different RNA isoforms, which can be translated into functionally distinct protein isoforms. Trans-acting splicing factors that regulate alternative splicing are a class of RNA-binding proteins (RBPs) that recognize and bind cis-regulatory elements on pre-mRNA, namely exonic or intronic splicing enhancers (ESEs or ISEs) or exonic and intronic splicing silencers (ESSs or ISSs), and respectively promote or suppress the inclusion of the exon into mature mRNA.

Principles of RNA splicing. (K, B, Robert.; et al, 2023)

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What is the Connection between RNA Splicing and Cancer?

RNA splicing dysregulation is a molecular characteristic of almost all types of tumors. Cancer-related splicing alterations arise from both recurrent mutations and changes in the expression of trans-acting factors that regulate splicing catalysis. Dysregulation of splicing in cancer can promote tumorigenesis through various mechanisms, leading to increased cell proliferation, decreased apoptosis, enhanced migration and metastasis potential, resistance to chemotherapy, and evasion of immune surveillance. Small molecule drugs that modulate or inhibit RNA splicing have entered clinical development and have become promising anticancer agents.

RNA Splicing Alterations in Tumor

Mutations or expression changes affecting the splicing machinery or components of splicing factors can play a crucial role in the onset and progression of cancer. By inducing splicing alterations that affect many downstream genes, these changes have the potential to disrupt the network of gene products and cancer pathways.

Recurrent Mutations in Splicing Factors

Recurrent somatic mutations in SF3B1, SRSF2, U2AF1, and ZRSR2 often occur in hematologic malignancies, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL). These mutations are commonly referred to as "spliceosome mutations."

Changes in Splicing Factor Expression

The levels and activities of splicing factors are tightly controlled at the epigenetic, transcriptional, and post-transcriptional levels. Any alterations in regulatory pathways may lead to changes in splicing factor expression, resulting in alterations in the selective splicing of downstream targets of splicing factors. Recurrent mutations in splicing factors are common in hematologic malignancies, while changes in splicing factor levels and copy number variations are particularly prominent in solid tumors. A causal relationship between splicing factor dysregulation and various cancer types has been established. It is noteworthy that several upregulated splicing factors in breast tumors have oncogenic functions sufficient to promote tumorigenesis in breast cancer models. Splicing factors can also act as tumor suppressors; thus, downregulation of certain splicing factors may contribute to tumor development.

Aberrant Splicing of RNA Isoforms

Tumors often exhibit more complex splicing patterns than normal tissues, and the tumorigenicity may be associated with cancer-specific alternative splicing events occurring during the transformation process. Cancer-associated alternative splicing isoforms can provide proliferative advantages, improve cell migration and metastasis, protect cells from death, rewire cellular metabolism or signaling, promote favorable microenvironments, alter immune responses, or confer drug resistance.

Cancer Therapy Development Targeting RNA Splicing

Given the crucial role of splicing in tumorigenesis, targeting RNA splicing shows promising prospects as a novel approach for cancer therapy. Various strategies have been developed, ranging from inhibiting key splicing factors or regulating splicing factors to modulating specific alternative splicing events.

Targeting Core Spliceosomes

One approach to targeting splicing in cancer therapy is to inhibit the spliceosome itself. SF3B1 is a crucial spliceosomal component for the selection of BPS and 3'SS, and limiting its function disrupts splicing at the early stage of spliceosome assembly. Various natural and derivative molecules targeting SF3B1 have been identified or developed, including FR901464 and its derivatives, sudemycin E, pladienolide B, and FD-895 and its derivatives. Another broad-spectrum splicing inhibitor is isoginkgetin, which blocks the recruitment of U4/U5/U6 tri-snRNP and leads to stalling of spliceosome pre-A complexes.

Targeting Alternative Splicing Factors

Developing inhibitors targeting specific RBPs and splicing factors has been challenging, partly due to the lack of accessible catalytic active sites targeted by most classical small molecule inhibitor approaches. Several arylsulfonamide class drugs act as molecular glues, leading to the degradation of RBP RBM39 by recruiting the CUL4-DCAF15 ubiquitin ligase complex, and knocking down RBM39 extensively affects alternative splicing events.

Targeting Upstream Regulatory Proteins

Splicing factors undergo extensive post-translational modifications, providing opportunities for cancer therapeutic intervention. For example, splicing factor proteins and splicing factors are widely affected by arginine methylation, hence Type I (PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8) and Type II (PRMT5) protein arginine methyltransferases regulate constitutive splicing and alternative splicing by controlling Sm proteins and splicing factor methylation.

Targeting RNA Splicing Isoforms

As many disease-associated splicing factors currently cannot be therapeutically targeted with small molecule drugs, targeting key downstream mis-spliced RNAs may offer a promising therapeutic avenue.

Oligonucleotide Splicing Modulation

Splice-switching ASOs are short, chemically modified RNA oligonucleotides designed to bind complementary sequences in the target pre-mRNA, thereby preventing their interaction with the splicing machinery. Splice-switching ASOs can be designed to specifically target 5'SS or 3'SS to prevent their usage, target splicing enhancer sequences to prevent binding of splicing factor activators and promote exon skipping, target splicing silencer sequences to prevent binding of splicing factor repressors and promote exon inclusion, and target cryptic splice sites generated by mutations to restore wild-type splice sites.

Targeting Alternative RNA Splicing

Some new approaches targeting splicing factors or specific alternative splicing events have emerged. One example is decoy oligonucleotides, which weaken splicing factor activity by competitively binding to their natural binding targets. Decoy oligonucleotides induce transcriptomic changes similar to splicing factor knockout, and SRSF1 decoys can restrict the growth of glioblastoma cells in vivo.

Reference

1. K, B, Robert.; et al. RNA Splicing Dysregulation and the Hallmarks of Cancer. Nature Reviews Cancer. 2023, 23(3): 135-155.