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CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.
- Source :
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Molecular pharmacology [Mol Pharmacol] 2023 Apr; Vol. 103 (4), pp. 199-210. Date of Electronic Publication: 2023 Jan 20. - Publication Year :
- 2023
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Abstract
- 6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2 -R39Q and PRPS1 -S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2 -R39Q and PRPS1 -S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.<br /> (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)
- Subjects :
- Humans
CRISPR-Cas Systems genetics
Mutation
Recurrence
5'-Nucleotidase genetics
5'-Nucleotidase metabolism
5'-Nucleotidase therapeutic use
Ribose-Phosphate Pyrophosphokinase genetics
Ribose-Phosphate Pyrophosphokinase metabolism
Mercaptopurine pharmacology
Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy
Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1521-0111
- Volume :
- 103
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Molecular pharmacology
- Publication Type :
- Academic Journal
- Accession number :
- 36669880
- Full Text :
- https://doi.org/10.1124/molpharm.122.000546