1. Introduction of the T315I gatekeeper mutation of BCR/ABL1 into a Philadelphia chromosome-positive lymphoid leukemia cell line using the CRISPR/Cas9 system.
- Author
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Nguyen TTT, Tamai M, Harama D, Kagami K, Kasai S, Watanabe A, Akahane K, Goi K, and Inukai T
- Subjects
- CRISPR-Associated Protein 9 genetics, CRISPR-Associated Protein 9 metabolism, CRISPR-Cas Systems, Cell Line, Dasatinib therapeutic use, Drug Resistance, Neoplasm genetics, Fusion Proteins, bcr-abl, Humans, Imatinib Mesylate pharmacology, Imatinib Mesylate therapeutic use, Mutation, Nucleotides therapeutic use, Oligodeoxyribonucleotides therapeutic use, Philadelphia Chromosome, Poly (ADP-Ribose) Polymerase-1 genetics, Poly (ADP-Ribose) Polymerase-1 metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, RNA, Guide, CRISPR-Cas Systems therapeutic use, Antineoplastic Agents therapeutic use, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics
- 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., (© 2022. Japanese Society of Hematology.)
- Published
- 2022
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