Expression analysis of 32Dcl3 cells expressing ASXL-MT in the presence of IL-3 or G-CSF
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE49117 | GEO | 2013/07/31
SECONDARY ACCESSION(S): PRJNA213052
REPOSITORIES: GEO
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Project description:Recurrent mutations in ASXL1 are found in various hematological malignancies and are associated with poor prognosis. In particular, ASXL1 mutations are frequently found in patients with hematological malignancies associated with myelodysplasia including myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal truncating ASXL1 mutations (ASXL1-MT) inhibit myeloid differentiation and induce MDS-like disease in mice, displaying all the features of human MDS including multi-lineage myelodysplasia, pancytopenia and occasional progression to overt leukemia. Concerning the molecular mechanisms, ASXL1-MT derepressed expression of Hoxa9 and miR-125a through inhibiting PRC2-mediated methylation of H3K27. miR-125a targeted expression of a surface receptor Clec5a, which was found to supports for myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1 mutations while Clec5a expression was generally low in MDS patients. Thus, ASXL1-MT induced MDS-like disease in mice via derepression of Hoxa9 and miR-125a, and Clec5a downregulation. Our data provide evidence for a novel axis of MDS pathogenesis (ASXL1 mutations-upregulation of HoxA9 and miR-125a-downregulation of Clec5a) and implicate both ASXL1 mutants and miR-125a as therapeutic targets in MDS.
2013-07-31 | GSE49118 | GEO
Project description:Recurrent mutations in ASXL1 are found in various hematological malignancies and are associated with poor prognosis. In particular, ASXL1 mutations are frequently found in patients with hematological malignancies associated with myelodysplasia including myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal truncating ASXL1 mutations (ASXL1-MT) inhibit myeloid differentiation and induce MDS-like disease in mice, displaying all the features of human MDS including multi-lineage myelodysplasia, pancytopenia and occasional progression to overt leukemia. Concerning the molecular mechanisms, ASXL1-MT derepressed expression of Hoxa9 and miR-125a through inhibiting PRC2-mediated methylation of H3K27. miR-125a targeted expression of a surface receptor Clec5a, which was found to supports for myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1 mutations while Clec5a expression was generally low in MDS patients. Thus, ASXL1-MT induced MDS-like disease in mice via derepression of Hoxa9 and miR-125a, and Clec5a downregulation. Our data provide evidence for a novel axis of MDS pathogenesis (ASXL1 mutations-upregulation of HoxA9 and miR-125a-downregulation of Clec5a) and implicate both ASXL1 mutants and miR-125a as therapeutic targets in MDS. BM cells derived from mock-transplanted mice and ASXL1-MT transplanted mice were incubated with biotinylated antibodies for CD3e, B220, and TER-119, followed by incubation with streptavidin Micro Beads (Miltenyi Biotec). The marker-negative fraction was separated with LS Columns (Miltenyi Biotec). Using the sorted-BM cells, we compared the expression proliles between 3 sorted-BM cells with mock and 3 sorted-BM cells with ASXL1-MT. Total RNA was extracted by Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Double-stranded cDNA was synthesized from 5 μg of total RNA with oligo (dT)24 T7 primer, amplified with T7 RNA polymerase up to approximately 50 μg of cRNA, and hybridized to Affymetrix Mouse Expression array 430A, which contains 45000 probe sets for 39000 transcripts and variants from over 34000 well-characterized mouse genes (Affymetrix). After washing and staining, the arrays were scanned on the GeneChip system confocal scanner (Affymetrix). The intensity for each feature of the array was captured with Affymetrix Microarray Suite (MAS) Version 5.0 software. Gene set enrichment analysis was performed by using Gene Ontology gene sets from the Molecular Signatures Database (http://www.broad.mit.edu/gsea/msigdb/).
2013-07-31 | E-GEOD-49118 | biostudies-arrayexpress
Project description:Recurrent mutations in ASXL1 are found in various hematological malignancies and are associated with poor prognosis. In particular, ASXL1 mutations are frequently found in patients with hematological malignancies associated with myelodysplasia including myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal truncating ASXL1 mutations (ASXL1-MT) inhibit myeloid differentiation and induce MDS-like disease in mice, displaying all the features of human MDS including multi-lineage myelodysplasia, pancytopenia and occasional progression to overt leukemia. Concerning the molecular mechanisms, ASXL1-MT derepressed expression of Hoxa9 and miR-125a through inhibiting PRC2-mediated methylation of H3K27. miR-125a targeted expression of a surface receptor Clec5a, which was found to supports for myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1 mutations while Clec5a expression was generally low in MDS patients. Thus, ASXL1-MT induced MDS-like disease in mice via derepression of Hoxa9 and miR-125a, and Clec5a downregulation. Our data provide evidence for a novel axis of MDS pathogenesis (ASXL1 mutations-upregulation of HoxA9 and miR-125a-downregulation of Clec5a) and implicate both ASXL1 mutants and miR-125a as therapeutic targets in MDS. Using 32Dcl3 cells transduced with pMYs-IG (mock) and pMYs-FLAG-ASXL1-MT-IG, we compared the expression profiles of 32Dcl3 cells with mock or ASXL1-MT under G-CSF stimulation at 4 time points, including 0hr (IL-3), 2hr, 6hr and 24hrs. Total RNA was extracted by Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Double-stranded cDNA was synthesized from 5 μg of total RNA with oligo (dT)24 T7 primer, amplified with T7 RNA polymerase up to approximately 50 μg of cRNA, and hybridized to Affymetrix Mouse Expression array 430A, which contains 45000 probe sets for 39000 transcripts and variants from over 34000 well-characterized mouse genes (Affymetrix). After washing and staining, the arrays were scanned on the GeneChip system confocal scanner (Affymetrix). The intensity for each feature of the array was captured with Affymetrix Microarray Suite (MAS) Version 5.0 software. Gene set enrichment analysis was performed by using Gene Ontology gene sets from the Molecular Signatures Database (http://www.broad.mit.edu/gsea/msigdb/)
2013-07-31 | E-GEOD-49117 | biostudies-arrayexpress
Project description:Recurrent somatic ASXL1 mutations occur in patients with myelodysplasia (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML), and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here we identify that ASXL1 mutations result in loss of PRC2-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data we identify targets of ASXL1 repression including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the Polycomb repressive complex 2 (PRC2), and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.
2012-08-21 | GSE38861 | GEO
Project description:Recurrent somatic ASXL1 mutations occur in patients with myelodysplasia (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML), and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here we identify that ASXL1 mutations result in loss of PRC2-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data we identify targets of ASXL1 repression including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the Polycomb repressive complex 2 (PRC2), and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis. To assess the genome-wide effects of ASXL1 loss on chromatin state we performed chromatin immunoprecipitation followed by next generation sequencing (CHIP-seq) for histone modifications known to be associated with PcG (histone H3 lysine 27 trimethylation (H3K27me3)) or TxG activity (histone H3 lysine 4 trimethylation (H3K4me3)) in UKE1 cells expressing empty vector (EV) or 2 independent validated shRNAs for ASXL1. This Series represents the ChIP-Seq data (not the microarray data referenced in the summary above). The related micorarray data are available in GEO as GSE38692.
2012-08-21 | E-GEOD-38861 | biostudies-arrayexpress
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:Recent studies have shown that epigenetic regulator are mutated in myelodysplastic syndrome (MDS) patients. Ezh2 is frequently mutated in hematopoietic malignant patients, however, the pathophysiological role of Ezh2 mutations has not been elucidated yet. In this study, we examined the function of Ezh2 in murine hematopoietic disease. Ezh2 deleted mice with myeloid malignancies including MDS, Myelodysplasia/myeloneoplasm(MDS/MPN), Myelodysplasia/myeloneoplasm_thrombocytosis(MDS/MPN_TC)
2015-03-14 | GSE66897 | GEO
Project description:BACKGROUND: Our previous studies showed that RUNX1 and ASXL1 mutations were frequently co-existed in chronic myelomonocytic leukemia (CMML) and clonal evolution of RUNX1 and/or ASXL1 occurred most frequently in chronic myeloid leukemia (CML) with myeloid blastic crisis. The molecular pathogenesis of cooperation of RUNX1 and ASXL1 mutations has not been reported yet. METHODS: Lentiviral-mediated stable transduction of RUNX1-WT/MT (R135T) in K562 cells which harboring ASXL1-MT (Y591X). RNA was extracted from stable cell line and used for gene-expression microarray analysis. RESULTS: For in vitro study, we overexpressed RUNX1-WT/MT (R135T) in K562 cells which harboring ASXL1-MT (Y591X). We found that RUNX1-MT augmented cell proliferation, colony formation, HOXA gene expression and inhibited megakaryocytic differentiation in ASXL1-MT K562 cells compared to RUNX1-WT or empty vector control. We performed gene expression profile of K562 cells overexpressed with EV, RUNX1-WT and RUNX1-R135T mutation. Gene expression microarray data revealed that 147 genes upregulated more than 2-fold in RUNX1-R135T expressing K562 cells compared to EV control cells. From gene expression data analysis, we found that inhibitor of DNA binding 1 (ID1), a key transcriptional regulator of hematopoietic stem cell (HSC) lineage commitment, is upregulated in RUNX1-R135T-transduced K562 cells compared to EV and RUNX1-WT-expressing cells.
2018-06-01 | GSE99640 | GEO
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:Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
2021-02-08 | MSV000086816 | MassIVE