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Functional study on the cooperation of ASXL1 and RUNX1 mutations for myeloid leukemia transformation

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99640 | GEO | 2018/06/01

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. 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/).

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/)

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.

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Functional study on the cooperation of ASXL1 and RUNX1 mutations for myeloid leukemia transformation

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