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Heterozygous IDH1R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

Oncogenevolume 37pages51605174 (2018) | Download Citation

Abstract

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70–80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called “single base editing”, and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix (ECM), and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin β4 (ITGB4); and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by “single base editing”, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the “single base editing” strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.

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Acknowledgements

We thank Dr. G. Major from NIH/NINDS for SVG cells, and Dr. Mingyao Ying from Kennedy Krieger Institute for YAP constructs. We thank Dr. John Laterra and Dr. Hernando Lopez for comments on this work. This work was supported by grants from NIH R01NS091165 (S.X.), NIH EY024580 (J.Q.), NIH EY023188 (J.Q.), and NIH GM111514 (H.Z. and J.Q.), Ford Foundation pre-doctoral fellowship program (O.O.) and NIH T32 GM007445 (O.O.).

Author contributions:

Shang Wei, O.O., L.K., and Q. X.: conducted experiments, data collection, figure preparation, and manuscript writing; J.W.: bioinformatics analysis, figure preparation, and manuscript writing; D.M., Shuyan Wang, B.L., S. L., H.Z., and Y.L.: conducted experiments and data collection; S.R.S., A.Q.H., and S.L.: sample collection and manuscript writing; L.C.: experimental design and manuscript writing; J.Q.: bioinformatics analysis and financial support; S.X.: experiment design, financial support, figure preparation, and manuscript writing.

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Affiliations

  1. Department of Respiratory and Critical Care Medicine, Tongji Hospital, Tongji Medical College Huazhong University of Science and Technology, 430030, Wuhan, China

    • Shuang Wei
  2. Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD, 21205, USA

    • Shuang Wei
    • , Olutobi Oyinlade
    • , Ding Ma
    • , Shuyan Wang
    • , Lisa Kratz
    • , Bachchu Lal
    • , Qingfu Xu
    • , Yunqing Li
    •  & Shuli Xia
  3. Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Shuang Wei
    • , Ding Ma
    • , Shuyan Wang
    • , Bachchu Lal
    • , Qingfu Xu
    • , Yunqing Li
    •  & Shuli Xia
  4. Department of Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Jie Wang
    •  & Jiang Qian
  5. Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Olutobi Oyinlade
    •  & Heng Zhu
  6. Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Senquan Liu
    •  & Linzhao Cheng
  7. Department of Neurologic Surgery, Mayo Clinic, Jacksonville, FL, 32224, USA

    • Sagar R. Shah
    •  & Alfredo Quiñones-Hinojosa
  8. Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Sagar R. Shah
  9. Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Baltimore, MD, 21205, USA

    • Hao Zhang
  10. Center for High Throughput Biology, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA

    • Heng Zhu
  11. Department of General Surgery, Tongji Hospital, Huazhong University of Science and Technology, 430030, Wuhan, China

    • Zhi-yong Huang

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The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Shuli Xia.

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DOI

https://doi.org/10.1038/s41388-018-0334-9