CRISPR/Cas9-Mediated Induction of Relapse-Specific and Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.

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 and 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 or 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 and 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 gene and S103N mutation of the 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 -R39Q and -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 and genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the and 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 and genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce -R39Q and -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 and gene mutations.