Crispr/Cas9-Mediated ASXL1knockout in U937 Human Leukemic Cells Perturbs Myeloid Differentiation

• Published in: Blood Vol. 128; no. 22; p. 1529
• Main Authors: Wu, Zhijie, Zhao, Xin, Banaszak, Lauren G., Keyvanfar, Keyvan, Kajigaya, Sachiko, Young, Neal S.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 02-12-2016
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.1529.1529

Introduction.Mutations in the Additional Sex Combs Like1 (ASXL1) gene are frequent in myeloid malignances (myelodysplastic syndromes [MDS], myeloproliferative neoplasms, chronic myelomonocytic leukemia and acute myeloid leukemia [AML]), and they predict poor survival. As we recently published (Yoshizato T, et al., N Engl J Med 2015), mutations in ASXL1 as well as BCOR/BCORL1, PIGA, and DNMT3A are most prevalent in patients with aplastic anemia (AA). ASXL1 grouped with "unfavorable mutations" that conferred poor survival and increased risk of "clonal evolution" of AA to MDS and AML. As an epigenetic modifier, ASXL1mutations may be involved in myeloid malignant transformation (Davies C, et al., Br J Haematol 2013; Abdel-Wahab O, et al., Cancer Cell 2012) but precise mechanisms have not been delineated.
Methods.The CRISPR/Cas9 system was employed to generate ASXL1-knockout clones from the U937 human leukemic cell line. Single cell clones were sorted by flow cytometry, and ASXL1 mutations were assessed by Sanger sequencing. Clones with mutations that encoded truncated proteins were used for further experiments. Characteristic features of both mutated and wild-type (WT) clones were examined: cell morphology by Wright-Giemsa staining; karyotype analysis by G-banding; and cell cycle, apoptosis, and cell differentiation by flow cytometry. RNA sequencing (RNA-Seq) was performed to screen differentially expressed genes between WT and ASXL1-knockout clones, followed by validation of gene expression using reverse transcription quantitative PCR (RT-qPCR).
Results.Of 88 single cell clones, 23 clones exhibited frame-shift and nonsense mutations of ASXL1, resulting in truncated proteins. Among them, 17 clones with single nucleotide insertion (c.594insA, heterozygous or homozygous) that encoded truncated proteins Ser199Glufsx55 were used for further experiments. Karyotype analysis revealed no significant differences between WT and ASXL1-knockout clones, suggesting that neither CRISPR/Cas9 itself nor ASXL1 mutations caused chromosomal instability. No significant differences between WT and ASXL1-knockout clones were observed in cell morphology, cell proliferation, cell cycle, or 5-fluoruracil-induced cell apoptosis. When monocyte/macrophage differentiation was induced chemically by exposure to phorbol 12-myristate 13-acetate, CD11b cell surface expression was much lower in ASXL1-knockout clones than in WT. By RNA-Seq, several genes (BIRC7, CACNA2D3, CTSG, CYBB, NAIP, NTNG1, OXR1, and ACTL8)involved in pathways related to cell death and survival (including TNFR1 signaling pathway, TNFR2 signaling pathway, TWEAK signaling pathway, apoptosis signaling pathway, death receptor signaling pathway, Rac signaling pathway, and phagosome maturation pathway) were down-regulated, but were not correlated to functional deficits in cell growth or apoptosis, which was indistinguishable between ASXL1-knockout and WT clones. RT-qPCR confirmed down-regulation of genes in ASXL1-knockout compared to WT clones, including CYBB (restrictedly expressed in terminally differentiating myeloid cells) and CLEC5A (involved in granulocytic differentiation), which may contribute to disturbance in myeloid differentiation of ASXL1-knockout U937 cells. Downregulaton of NAIP, which inhibits CASP3, CASP7, and CASP9 activities, may be related to deregulation of apoptosis in ASXL1-knockout U937.
Conclusion.ASXL1 mutations are frequent in both malignant and non-malignant blood diseases, and may be involved in myeloid neoplastic transformation. Using gene editing of a human leukemic cell line, we show that ASXL1-knockout perturbs myeloid differentiation and down regulates multiple genes associated with myeloid development such as CYBB and CLEC5A, providing potential mechanisms for its role in bone marrow failure and neoplastic transformation.
Young:Novartis: Research Funding.