CRISPR/Cas9 Genome Editing of the Human Topoisomerase II Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.

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.