Division of Pharmaceutics and Pharmacology, College of Pharmacy (V.A.H., J.C.-M., J.L.P., J.L., J.C.Y., T.S.E., N.S.) and Department of Biomedical Informatics, College of Medicine (H.G.O), The Ohio State University, Columbus, Ohio.
Division of Pharmaceutics and Pharmacology, College of Pharmacy (V.A.H., J.C.-M., J.L.P., J.L., J.C.Y., T.S.E., N.S.) and Department of Biomedical Informatics, College of Medicine (H.G.O), The Ohio State University, Columbus, Ohio
Mol Pharmacol. 2021 Mar;99(3):226-241. doi: 10.1124/molpharm.120.000173. Epub 2021 Jan 14.
An essential function of DNA topoisomerase II (TOP2; 170 kDa, TOP2/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2/170, expresses high levels of a novel C-terminal truncated TOP2 isoform (90 kDa, TOP2/90). TOP2/90, the translation product of a TOP2 mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA →GAG//GTAA ) in the TOP2 gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2 alleles edited revealed improved I19 removal, decreased TOP2/90 mRNA/protein, and increased TOP2/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (H2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2 E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase II (TOP2) gene results in circumvention of acquired drug resistance to etoposide and other TOP2-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2 90 kDa isoform and increasing expression of full-length TOP2 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2-targeted drugs.
DNA 拓扑异构酶 II（TOP2；170 kDa，TOP2/170）的一个基本功能是通过引入瞬时 DNA 双链断裂来解决染色体分离过程中的 DNA 拓扑缠绕。TOP2/170 是 DNA 损伤稳定型抗癌药物的重要靶点，其临床疗效因耐药性而受到影响，耐药性通常与 TOP2/170 表达降低有关。我们最近证明，一种依托泊苷耐药的 K562 克隆亚系 K/VP.5，其 TOP2/170 水平降低，表达高水平的新型 C 端截断 TOP2 同工型（90 kDa，TOP2/90）。TOP2/90 是保留加工内含子 19（I19）的 TOP2 mRNA 的翻译产物，与 TOP2/170 异二聚化，并通过对药物活性的显性负效应成为耐药决定因素。我们假设通过增强 I19 切除的基因组编辑将提供一种可行的策略来规避获得的 TOP2 介导的药物耐药性。为了增强 K/VP.5 细胞中的 I19 切除，使用 CRISPR/Cas9 在 TOP2 基因的次优内含子 19/外显子 19 5' 剪接位点（E19/I19 5' SS）中进行改变（GAG//GTAA →GAG//GTAA ）。通过定量聚合酶链反应鉴定基因编辑克隆，并通过测序验证。对具有所有 TOP2 等位基因编辑的克隆进行表征，结果显示 I19 切除增加，TOP2/90 mRNA/蛋白减少，TOP2/170 mRNA/蛋白增加。与亲本 K562 细胞相比，对依托泊苷诱导的 DNA 损伤（H2AX、彗星试验）和生长抑制的敏感性得到恢复。结果表明，我们在 K/VP.5 细胞中优化 TOP2 E19/I19 5' SS 的基因编辑策略可规避依托泊苷和其他 TOP2 靶向药物的耐药性。 意义：这里呈现的结果表明，通过 CRISPR/Cas9 基因编辑 DNA 拓扑异构酶 II（TOP2）基因的次优内含子 19/外显子 19 5' 剪接位点，可增强内含子 19 的切除，从而减少截断的 TOP2 90 kDa 同工型的形成，并增加这些耐药细胞中全长 TOP2 170 kDa 的表达，从而规避克隆 K562 细胞系中对依托泊苷和其他 TOP2 靶向药物的获得性耐药。结果表明 RNA 加工在 TOP2 靶向药物获得性耐药中的重要性。
