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Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

ABSTRACT: Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3’ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE144003 | GEO | 2020/01/25

REPOSITORIES: GEO

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Project description:Mutations in spliceosomal genes are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations occur at highly restricted amino acid residues and perturb normal splice site and exon recognition. Spliceosomal mutations are always heterozygous and rarely co-occur with one another, suggesting that cells may only tolerate a partial deviation from normal splicing activity. To test this hypothesis, we generated mice with inducible hemizygous expression of the commonly occurring SRSF2P95H mutation in the hematopoietic system. These mice rapidly developed lethal bone marrow failure upon activation of the Srsf2P95H mutation with concomitant deletion of the wildtype Srsf2 allele, demonstrating that Srsf2-mutant cells depend on the wildtype Srsf2 allele for survival. We next tested whether spliceosomal-mutant leukemias display greater sensitivity to pharmacologic splicing inhibition induced by the small molecule E7107. Treatment of isogenic murine leukemias as well as patient-derived xenograft (PDX) AMLs showed significant reductions in leukemic burden specifically in samples carrying spliceosomal mutations. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal mutations are preferentially susceptible to additional splicing perturbations in vivo compared with wildtype counterparts. Modulation of spliceosome function may provide a novel therapeutic avenue in genetically defined subsets of MDS/AML patients. We created an isogenic murine leukemia model by retroviral overexpression of the MLL-AF9 fusion oncogene in Vav-Cre Srsf2+/+ or Vav-Cre Srsf2P95H/+ BM cells followed by transplantation into lethally irradiated recipient mice. In order to determine the mechanistic origins of the Srsf2 mutant-selective effects of E7107, we analyzed transcriptional changes after five consecutive days of E7107 or vehicle treatment in vivo. GFP+ Cd11b+ cells were purified from the BM of recipient mice exactly three hours after the last dose of E7107 and were subjected to paired-end 2x50bp RNA-seq. For the knock-in/knock-out experiments, we generated inducible, hemizygous Srsf2P95H mice to study the effects of wildtype Srsf2 deletion with concomitant activation of the Srsf2P95H allele. Mx1-cre Srsf2fl/+ mice were crossed to Srsf2P95H/+ mice to generate Srsf2 wildtype (Mx1-cre Srsf2+/+), Srsf2 heterozygous knockout (Mx1-Cre Srsf2+/-), Srsf2 heterozygous P95H mutant (Mx1-Cre Srsf2P95H/+), and Srsf2 hemizygous P95H mutant (Mx1-Cre Srsf2P95H/-) mice, which were subjected to single-end 101bp RNA-seq.

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Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

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