Srsf2P95H/+ mutation exacerbates Asxl1Y588XTg-induced leukemogenesis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE240105 | GEO | 2023/12/01
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.
2016-03-31 | E-GEOD-74064 | biostudies-arrayexpress
Project description:Mutations in the RNA splicing complex member SRSF2 are found frequently in myelodysplastic syndrome and related malignancies such as chronic myelomonocytic leukemia. These mutations cluster on proline 95, with P95H the most frequent. How SRSF2P95H mutations modify hematopoiesis and promote MDS/MPN development is not clear. We have established a conditionally activatable Srsf2P95H/+ knock-in allele which, when expressed within the hematopoietic stem cell populations caused profound myeloid bias, at the expense of erythroid and lymphoid cells, and a reduced frequency and competitive repopulation of HSCs. Long-term aging of Srsf2P95H/+ resulted in the development of MDS/MPN characterised by myeloid dysplasia and monocytosis. Reproducible key phenotypic features make this a mouse model suitable for mechanistic and preclinical MDS sudies.
2018-06-18 | GSE99850 | GEO
Project description:Mutations in the RNA splicing complex member SRSF2 are found frequently in myelodysplastic syndrome and related malignancies such as chronic myelomonocytic leukemia. These mutations cluster on proline 95, with P95H the most frequent. How SRSF2P95H mutations modify hematopoiesis and promote MDS/MPN development is not clear. We have established a conditionally activatable Srsf2P95H/+ knock-in allele which, when expressed within the hematopoietic stem cell populations caused profound myeloid bias, at the expense of erythroid and lymphoid cells, and a reduced frequency and competitive repopulation of HSCs. Long-term aging of Srsf2P95H/+ resulted in the development of MDS/MPN characterised by myeloid dysplasia and monocytosis. Reproducible key phenotypic features make this a mouse model suitable for mechanistic and preclinical MDS sudies.
2018-06-18 | GSE99851 | GEO
Project description:Mutations in the RNA splicing complex member SRSF2 are found frequently in myelodysplastic syndrome and related malignancies such as chronic myelomonocytic leukemia. These mutations cluster on proline 95, with P95H the most frequent. How SRSF2P95H mutations modify hematopoiesis and promote MDS/MPN development is not clear. We have established a conditionally activatable Srsf2P95H/+ knock-in allele which, when expressed within the hematopoietic stem cell populations caused profound myeloid bias, at the expense of erythroid and lymphoid cells, and a reduced frequency and competitive repopulation of HSCs. Long-term aging of Srsf2P95H/+ resulted in the development of MDS/MPN characterised by myeloid dysplasia and monocytosis. Reproducible key phenotypic features make this a mouse model suitable for mechanistic and preclinical MDS sudies.
2018-06-18 | GSE104976 | GEO
Project description:Additional Sex Combs Like 1 (ASXL1) is frequently mutated in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). Although loss of ASXL1 promotes hematopoietic transformation, there is growing evidence that ASXL1 mutations might confer an alteration of function. Here we identify that physiological expression of a C-terminal truncated Asxl1 mutant in vivo using conditional knock-in (KI) results in myeloid skewing, age-dependent anemia, thrombocytosis, and morphologic dysplasia. Although expression of mutant Asxl1 altered the functions of hematopoietic stem cells (HSCs), it maintained their survival in competitive transplantation assays and increased susceptibility to leukemic transformation by co-occurring RUNX1 mutation or viral insertional mutagenesis. KI mice displayed substantial reductions in H3K4me3 and H2AK119Ub without significant reductions in H3K27me3, distinct from the effects of Asxl1 loss. ChIP-seq analysis demonstrated opposing effects of wildtype and mutant Asxl1 on H3K4me3. These findings reveal that ASXL1 mutations confer HSCs with an altered epigenome and increase susceptibility for leukemic transformation, presenting a novel model for CHIP.
2018-02-28 | GSE111067 | GEO
Project description:Primary mielofibrosis (PMF) is a rare chronic myeloproliferative disorder characterized by the accumulation of abnormal megakaryocytes (Mks) in the bone marrow (BM), variable degrees of BM fibrosis, osteosclerosis and angiogenesis, immature myeloid and erythroid cells, and tear-drop erythrocytes in the peripheral blood (PB), and extramedullary hematopoiesis. The identification of the JAK2V617F mutation represented a seminal discovery in the field of Philadelphia-chromosome–negative chronic myeloproliferative neoplasms (MPNs), providing clues to the pathogenesis, prompting a revision of the diagnostic criteria, and culminating in the development of clinical trials with JAK2 (and JAK1) inhibitors. The JAK2V617F mutation occurs in almost all patients with polycythemia vera (PV) and in 50%-70% of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Soon after the identification of the JAK2V617F mutation, mutations in JAK2 exon 12 were described in rare patients with JAK2V617F-negative PV and mutations in MPL were reported in 5%-10% of ET or PMF subjects. The complexity of the molecular pathogenesis of MPNs is reinforced by discovery of additional mutations in TET2, ASXL1, CBL, IDH1/IDH2, EZH2 and IKZF1. These mutations are detected in a minority of patients at different phases of the disorder, including leukemic transformation, and are variably associated each other and with JAK2 or MPL mutations. In order to better characterize biological differences between mutated and wild-type PMF cell populations we performed a gene expression profiling on 9 samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes and 11 wild-type samples using the Affymetrix GeneChip technology. After data preprocessing and filtering a supervised analysis approach was used to define a gene expression signature for mutated samples. PMF samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes exhibit a specific molecular signature as compared with WT samples. Gene expression profile (GEP) of CD34+ cells from 20 PMF patients (1 replicate for each sample). In particular, GEP was performed on 9 samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes and 11 wild-type samples.
2013-07-23 | E-GEOD-41812 | biostudies-arrayexpress
Project description:Asxl1 is one of the most commonly mutated genes in malignancies including myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML). We generated a mouse model that harbors the most common mutation on ASXL1 gene detected in MDS patients: c.1934dupG, p.G646WfsX12 at the endogenous murine Asxl1 locus: the G643WfsX12 mutant, from here on referred as Asxl1G643W. Mutations on Asxl1 co-occur with mutations on CEBPA in AML patients, therefore, in order to understand how Asxl1 and Cebpa co-operate, we set up to cross our Cebpa-p30 mutant mouse model with our newly generated Asxl1 G643Wfs12 mutant. In this study we provided mechanistic information about the cooperation between these two factors. Furthermore, our mouse model proved able to recapitulate the chemotherapy response of human patients with Asxl1 mutations, thus representing a potent tool for future preclinical studies focusing on Asxl1 mutant AML.
2020-05-12 | GSE133314 | GEO
Project description:Purpose: Recurrent ASXL1 mutations are frequently observed in all spectrums of myeloid malignancies and published data suggests that ASXL1 mutations may be involved in leukemic transformation as a tumor suppressor. Yet the molecular mechanisms of cell desitiny regulated by ASXL1 are to be further delineated. Methods: mRNA profiles of wild-type (WT) and CRISPR/Cas9 induced ASXL1 mutated U937 cell lines were generated by next generation sequencing, using Illumina HiSeq2500. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality at the ends. Remaining sequence reads were then aligned to the human reference genome (hg19) using Tophat2. Gene read counts were measured using FeatureCounts and FPKM values were calculated with cufflinks. edgeR was used to identify differentially expressed genes between conditions, and topGO was used for annotation (Alexa, Rahnenfuhrer, and Lengauer, 2006). Sample comparison for differential gene expression was as follows: WTblk and WT1 versus MT2, MT3, MT4, and MT5. Gene enrichment set analysis (GSEA) was conducted with KEGG, Biocarta, and Reactome pathway datasets (Subramanian et al., 2005). Results: ASXL1-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of ASXL1-mutated and WT U937 cells. Transcriptom analysis revealed that ASXL1 mutations decreased the expression of genes essential to myeloid differentiation, including CYBB and CLEC5A genes, which manifested in ASXL1-MT U937 cells as perturbed potential of differentiation compared with WT cells. Also, gene set enrichment analysis revealed that ASXL1 mutations masively affected gene sets relating to cell death and survival. Conclusion: By introduction of mutations into genome using the CRISPR/Cas9 system, we established ASXL1-mutated U937 cell lines. Our results indicated that ASXL1 mutations perturbed monocytic/phagocyte differentiation, which is a hallmark of myeloid malignancies, by down regulating genes essential to myeloid differentiation, including CYBB and CLEC5A, also massively affected multiple gene sets involving in cell survival.
2018-03-14 | GSE98104 | GEO
Project description: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.
2021-09-15 | GSE165505 | GEO
Project description: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.
2021-09-15 | GSE165504 | GEO