Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability

Abstract

Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2 . The most frequently mutated gene is SF3B1 , mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1 -mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.

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

“…SF3B1 mutations have also been reported in solid tumors, including 20% of uveal melanomas (UM) [8–10], 3% of pancreatic ductal adenocarcinomas [11], and 1.8% of breast cancers [12]. RNA-sequencing (RNA-seq) analyses in different tumor types and cell lines with SF3B1 missense mutations, including the most prevalent substitution K700E, identified distinct alternative splicing (AS) defects [13–15].…”

“…Recently, CRISPR/Cas9 genome editing has allowed researchers to faithfully reproduce human pathological mutations in animal models. Human SF3B1 mutations have been introduced in cell lines [15–18], but mimicking these mutations in multicellular organisms has only been achieved by conditional alleles in murine models [19]. Taking advantage of the extraordinary conservation of splicing factors across evolution and the ease of genetic manipulation of C .…”

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

“…First, diverse computational pipelines have been used to assess differences in alternative splicing between WT and SF3B1-mutated samples, resulting in distinct conclusions. For instance, Liberante et al reanalyzed previously published data and found that SF3B1 mutation was mostly associated with differential skipped exon events, rather than A3'SS selection (Liberante et al, 2019). Second, intronic elements directing splicing have evolved differently in C. elegans compared to other organisms.…”

“…Recently, CRISPR/Cas9 genome editing has allowed researchers to faithfully reproduce human pathological mutations in animal models. Human SF3B1 mutations have been introduced in cell lines (Alsafadi et al, 2016;Shiozawa et al, 2018;Gupta et al, 2019;Liberante et al, 2019), but mimicking these mutations in multicellular organisms has only been achieved by conditional alleles in murine models (Xu et al, 2019). Taking advantage of the extraordinary conservation of splicing factors across evolution and the ease of 6 Different sftb-1 missense mutations display additive effects when combined We edited the C. elegans genome to mimic the K700E mutation, the most frequent SF3B1 substitution in tumors.…”

“…SF3B1 mutations have also been reported in solid tumors, including 20% of uveal melanomas (UM) (Furney et al, 2013;Harbour et al, 2013;Martin et al, 2013), 3% of pancreatic ductal adenocarcinomas (Biankin et al, 2012), and 1.8% of breast cancers (Maguire et al, 2015). RNA-sequencing (RNA-seq) analyses in different tumor types and cell lines with SF3B1 missense mutations, including the most prevalent substitution K700E, identified distinct alternative splicing (AS) defects (Darman et al, 2015;DeBoever et al, 2015;Liberante et al, 2019).…”