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ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

ABSTRACT: Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

SUBMITTER: Valletta S

PROVIDER: S-EPMC4792541 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

Valletta Simona S Dolatshad Hamid H Bartenstein Matthias M Yip Bon Ham BH Bello Erica E Gordon Shanisha S Yu Yiting Y Shaw Jacqueline J Roy Swagata S Scifo Laura L Schuh Anna A Pellagatti Andrea A Fulga Tudor A TA Verma Amit A Boultwood Jacqueline J

Oncotarget 20151201 42

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas ...[more]

PMID: 26623729

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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.

Dataset Information

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

Publications

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

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