RT Journal Article
SR Electronic
T1 CRISPR/Cas9-Mediated Induction of Relapse-Specific <em>NT5C2</em> and <em>PRPS1</em> Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 199
OP 210
DO 10.1124/molpharm.122.000546
VO 103
IS 4
A1 Thao Thu Thi Nguyen
A1 Yoichi Tanaka
A1 Masashi Sanada
A1 Masumi Hosaka
A1 Minori Tamai
A1 Keiko Kagami
A1 Chiaki Komatsu
A1 Shinpei Somazu
A1 Daisuke Harama
A1 Shin Kasai
A1 Atsushi Watanabe
A1 Koushi Akahane
A1 Kumiko Goi
A1 Takeshi Inukai
YR 2023
UL http://molpharm.aspetjournals.org/content/103/4/199.abstract
AB 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.