Therapeutic targeting of splicing in cancer

Abstract

Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer.

Figure 1. Diverse mechanisms by which alterations in splicing promote cancer and treatment resistance

(A) The cis-regulatory sequences and trans-acting splicing factors affected by mutations in cancer are indicated by stars in the figure. Additional splicing regulatory factors whose upregulation have been shown to promote tumorigenesis are shown in the red box while those that have been demonstrated to function as tumor suppressors are shown in the blue box. (B) Examples of therapeutically important splicing alterations that promote cancer and/or resistance to cancer therapy. On the left is an example of mutations affecting splicing of MET exon 14 to promote a specific isoform of MET which lacks the juxtamembrane domain. Mutations affecting intronic sequences at the 5′ or 3′ splice sites of MET result in a form of MET which lacks the residue within exon 14 required for CBL-mediated downregulation. Expression of MET exon 14Δ drives lung adenocarcinomas and sensitizes them to MET inhibitors. On the right is a recently described event affecting expression of CD19. Acquired mutations affecting exon 2 of CD19 result in a stable form of CD19 lacking exon 2 (“CD19 exon 2Δ”) which is not recognized by anti-CD19 chimeric antigen receptor T-cells. Moreover, SRSF3 promotes inclusion of exon 2 and downregulation of SRSF3 expression has similarly been suggested to result in expression of CD19 exon 2Δ.

Figure 2. Methods by which splicing may be modulated for cancer therapy

These include (1) use of a series of compounds that inhibit early spliceosome assembly by inhibiting SF3B1 and (2) inhibition of phosphorylation of Serine/Arginine-rich (SR) proteins through inhibition of CLKs (CDC2-like kinases) and/or SRPKs (SR protein kinases). In addition, use of oligonucleotides to (3) target the RNA binding activity of splicing regulatory proteins mutated or overexpressed in cancer, or (4) directly alter splicing of individual downstream mRNA's critical for cancer pathogenesis, may provide more selective tumor targeting. Alteration of specific events may be achieved by oligonucleotides that alter splicing by promoting or impairing the use of key splicing regulatory sequences (such as 5′/3′ splice sites, exonic splicing silencers (ESSs), exonic splicing enhancers (ESEs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs)). In addition, identification of novel proteins stably produced by aberrant splicing in cancer may result in therapeutic strategies to target these downstream pathologic products and pathways (as depicted in (5)). Additional therapeutic strategies that have been shown to affect splicing only in cell-free in vitro assays to date are not shown above. Inhibition of U2 snRNP by inhibition of SF3B1 function or methylation of Sm proteins is shown in more detail in Figure 3.

Figure 3. Pharmacologic methods to disrupt core spliceosome function

Current methods to directly inhibit spliceosome function include a series of compounds that bind to the SF3B component of U2 snRNP and inhibit early spliceosome assembly. While the precise biochemical interactions between SF3B1 inhibitors and U2 snRNP are not well understood, the effects of each of these drugs on cell toxicity is nearly completely abrogated by mutation of a single residue of SF3B1 (SF3B1 R1074H), suggesting that each of these compounds specifically functions through interactions with SF3B1. Inhibition of U2 snRNP function has been shown to result in widespread intron retention and cassette exon skipping in a time- and dose-dependent manner in a variety of cell types. While this inhibition of splicing results in an accumulation of pre-mRNAs in the nucleus, pharmacologic inhibition of U2 snRNP is also associated with leakage of pre-mRNA into the cytoplasm. Although most unspliced mRNAs are expected to become substrates for nonsense-mediated decay, a portion of these mRNAs may undergo translation to generate aberrant protein products which themselves may have cellular toxicity. For example, a functionally active form of the cell cycle inhibitory protein p27 (termed “p27*”) which lacks the C-terminal domains required for degradation is generated following exposure to several SF3B1 inhibitory compounds. In addition to SF3B1 inhibitory compounds, recent data suggests that inhibition of Sm protein methylation through downregulation of PRMT5 (protein arginine methyltransferase-5) may also inhibit splicing. PRMT5 symmetrically methylates arginine residues of SmB/B′, SmD1, and SmD3.