PT - JOURNAL ARTICLE AU - Thao Thu Thi Nguyen AU - Yoichi Tanaka AU - Masashi Sanada AU - Masumi Hosaka AU - Minori Tamai AU - Keiko Kagami AU - Chiaki Komatsu AU - Shinpei Somazu AU - Daisuke Harama AU - Shin Kasai AU - Atsushi Watanabe AU - Koushi Akahane AU - Kumiko Goi AU - Takeshi Inukai TI - <strong>CRISPR/Cas9-mediated induction of relapse-specific <em>NT5C2</em> and <em>PRPS1</em> mutations confers thiopurine resistance as a relapsed lymphoid leukemia model</strong> AID - 10.1124/molpharm.122.000546 DP - 2023 Jan 01 TA - Molecular Pharmacology PG - MOLPHARM-AR-2022-000546 4099 - http://molpharm.aspetjournals.org/content/early/2023/01/20/molpharm.122.000546.short 4100 - http://molpharm.aspetjournals.org/content/early/2023/01/20/molpharm.122.000546.full 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 gene, 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 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 gene as a result of non-homologous 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), we 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.