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Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system

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Abstract

Imatinib and second-generation tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, overcoming TKI resistance due to the T315I gatekeeper mutation of BCR/ABL1 is crucial for further improving the prognosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is appropriate for establishing a human model of Ph+ ALL with the T315I mutation, because it can induce specific mutations via homologous recombination (HR) repair in cells with intact endogenous HR pathway. Here we used CRISPR/Cas9 to introduce the T315I mutation into the Ph+ lymphoid leukemia cell line KOPN55bi, which appeared to have an active HR pathway based on its resistance to a poly (ADP-Ribose) polymerase-1 inhibitor. Single-guide RNA targeting at codon 315 and single-strand oligodeoxynucleotide containing ACT to ATT nucleotide transition at codon 315 were electroporated with recombinant Cas9 protein. Dasatinib-resistant sublines were obtained after one-month selection with the therapeutic concentration of dasatinib, leading to T315I mutation acquisition through HR. T315I-acquired sublines were highly resistant to imatinib and second-generation TKIs but moderately sensitive to the therapeutic concentration of ponatinib. This authentic human model is helpful for developing new therapeutic strategies overcoming TKI resistance in Ph+ ALL due to T315I mutation.

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Acknowledgements

This work was supported by JSPS KAKENHI Grant Numbers JP19H03615 and AMED under Grant Number JP19ck0106253.

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Authors and Affiliations

  1. Department of Pediatrics, School of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan

    Thao T. T. Nguyen, Minori Tamai, Daisuke Harama, Keiko Kagami, Shin Kasai, Atsushi Watanabe, Koshi Akahane, Kumiko Goi & Takeshi Inukai

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  1. Thao T. T. Nguyen
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  2. Minori Tamai
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  3. Daisuke Harama
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  4. Keiko Kagami
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Contributions

TTTN performed research, analyzed data, and wrote the manuscript; MT, DH, and TI suggested the concept of the study and designed the study; KK, SK, AW, KA, and KG analyzed data. TI revised the manuscript and supervised the overall study process.

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Correspondence to Takeshi Inukai.

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Nguyen, T.T.T., Tamai, M., Harama, D. et al. Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system. Int J Hematol 116, 534–543 (2022). https://doi.org/10.1007/s12185-022-03369-x

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