CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation

Wu,Zhi-Jie; Zhao,Xin; Banaszak,Lauren  G.; Gutierrez-Rodrigues,Fernanda; Keyvanfar,Keyvan; Gao,Shou-Guo; Quinones Raffo,Diego; Kajigaya,Sachiko; Young,Neal   S.

doi:10.3892/ijo.2018.4290

CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation

Authors:
- Zhi-Jie Wu
- Xin Zhao
- Lauren G. Banaszak
- Fernanda Gutierrez-Rodrigues
- Keyvan Keyvanfar
- Shou-Guo Gao
- Diego Quinones Raffo
- Sachiko Kajigaya
- Neal S. Young
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Affiliations: Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1202, USA
Published online on: February 28, 2018 https://doi.org/10.3892/ijo.2018.4290
Pages: 1209-1223
Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis.

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