CRISPR/Cas9 genome editing of the human topoisomerase iia intron 19 59 splice site circumvents etoposide resistance in human Leukemia K562 cells

Abstract

An essential function of DNA topoisomerase IIa (TOP2a; 170 kDa, TOP2a/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2a/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2a/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2a/170, expresses high levels of a novel C-terminal truncated TOP2a isoform (90 kDa, TOP2a/90). TOP2a/ 90, the translation product of a TOP2a mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2a/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 TOP2a-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2a gene's suboptimal exon 19/intron 19 59 splice site (E19/I19 59 SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2a alleles edited revealed improved I19 removal, decreased TOP2a/90 mRNA/protein, and increased TOP2a/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (gH2AX, 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 TOP2a E19/I19 59 SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2a-targeted drugs. SIGNIFICANCE STATEMENT Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 59 splice site in the DNA topoisomerase IIa (TOP2a) gene results in circumvention of acquired drug resistance to etoposide and other TOP2a-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2a 90 kDa isoform and increasing expression of full-length TOP2a 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2a-targeted drugs.