CRISPR editing of sftb-1/SF3B1 in C. elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing

Abstract

SF3B1 is the most frequently mutated splicing factor in cancer. Mutations in SF3B1 confer growth advantages to cancer cells but they may also confer vulnerabilities that can be therapeutically targeted. In contrast to other animal models, SF3B1 cancer mutations can be maintained in homozygosis in C. elegans, allowing synthetic lethal screens with a homogeneous population of animals. These mutations cause alternative splicing (AS) defects in C. elegans, as it occurs in SF3B1-mutated human cells. In an RNAi screen, we identified U2 snRNP components as causing synthetic lethality with sftb-1/SF3B1 mutations.We also detected synthetic interactions between sftb-1 mutants and cancerrelated mutations in uaf-2/U2AF1 or rsp-4/SRSF2, demonstrating that this model can identify mutations that are mutually exclusive in human tumors.Thus, we have established a multicellular model amenable for high-throughput screening to uncover synthetic lethal interactions that could selectively kill cancer cells harboring SF3B1 mutations. 4 genetic manipulation of C. elegans, we established a multicellular model to study SF3B1 cancer-related mutations. Since the spliceosome components, and particularly SF3B1, are being intensively studied as a target of antitumor drugs (Bonnal, Vigevani and Valcárcel, 2012;Effenberger, Urabe and Jurica, 2017;DeNicola and Tang, 2019), the multicellular model of SF3B1 mutations described in this study would provide a convenient pre-clinical platform for identifying new targets and small molecules for use in cancer therapies. 5 RESULTS sftb-1, the C. elegans ortholog of SF3B1, is an ubiquitously expressed gene essential for developmentThe C. elegans protein SFTB-1 is 66% identical to human SF3B1 in terms of amino acid composition. Homology is particularly high at the HEAT domain, reaching 89% identity, and the most frequently mutated amino acids in cancer are conserved. The SFTB-1 sequence also conserves some of the U2AF ligand motifs (ULMs) that bind U2AF homology motifs (UHMs) present in other splicing factors (Figure 1A and S1) (Loerch et al., 2018).As expected for a core splicing factor, the CRISPR-engineered endogenous fluorescent reporter mCherry::SFTB-1 showed ubiquitous expression in somatic and germ cells, being absent in mature sperm only (Figure 1b and 1c). We also used CRISPR to generate the sftb-1(cer6) mutation, a deletion allele that produces a premature stop codon (Figure S2a) and causes an arrest at early larval stages, confirming that sftb-1 is essential for development (Figures 1d and 1e). sftb-1(cer6) homozygous animals completed embryonic development but arrested as larvae that could undergo few germ cell divisions (Figure S2b), suggesting that maternal wild-type (WT) product can still be present in the early larva.

Splicing-related Retinitis Pigmentosa mutations mimicked in C. elegans allow the identification of disease modifiers and drug screens

“…Alternatively, if the functional replacement cannot be complete, a fraction of the protein could be replaced. Thus, we recently humanized the pladienolide binding domain of sftb-1/SF3B1 to make C. elegans sensitive to this drug without causing any other effect in the animal (32).…”