Dataset Information

Transcriptome analysis of Hoxb8 imortalised GM-CSF cell lines with SRSF2 P95H mutation [RNA-seq]

ABSTRACT: Current strategies to target RNA splicing mutant myeloid cancers propose disruption of normal splicing activity by targeting the splicing apparatus therapeutically. This approach is only modestly sensitizing and is also toxic to non-mutant bearing wild-type cells. To explore potentially exploitable genetic interactions with spliceosome mutations, we combined data mining and functional screening for synthetic lethal interactions with an Srsf2P95H/+ mutation. Analysis of mis-splicing events in a series of both human and murine SRSF2P95H mutant samples across multiple myeloid diseases (AML, MDS, CMML) was first performed to identify conserved mis-splicing events. From this analysis, we identified that the DNA repair and cell cycle pathways were overrepresented within the conserved mis-spliced transcript sets. In parallel, to functionally define pathways essential for survival and proliferation Srsf2P95H/+ cells, we performed a genome-wide pooled CRISPR loss of function screen using Hoxb8 immortalised R26-CreERki/+ Srsf2P95H/+ and R26-CreERki/+ Srsf2+/+ cell lines. We assessed for loss of sgRNA representation at three timepoints: immediately after Srsf2P95H/+ activation, and at one week and two weeks post Srsf2P95H/+ mutation. Pathway analysis demonstrated that the DNA damage response and cell cycle pathways were amongst the top synthetic lethal pathways with Srsf2P95H/+ mutation., Based on the loss of guide RNAs targeting Cdk6, we identified that Palbocilib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary non-immortalised lin-cKIT+Sca-1+ cells compared to wild type controls. Our data strongly suggest that cell cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that Palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant myeloid cancers.

ORGANISM(S): Mus musculus

PROVIDER: GSE165504 | GEO | 2021/09/15

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

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.

Dataset Information

Transcriptome analysis of Hoxb8 imortalised GM-CSF cell lines with SRSF2 P95H mutation [RNA-seq]

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure