Abstract

An essential function of DNA topoisomerase IIα (170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double-strand breaks (DSBs). TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/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 TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was utilized to make changes (GAG//GTAAACàGAG//GTAAGT) in the TOP2α gene's suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene edited clones were identified by qPCR and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein. Sensitivity to etoposide-induced DNA damage (γH2AX, comet assays) and growth inhibition was restored to the levels comparable to those in parental K562 cells. Together results indicate that our gene editing strategy for optimizing the TOP2α 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 E19/I19 5′ SS in the TOP2α 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 thereby decreasing formation of a truncated TOP2α/90 isoform and increasing expression of full-length TOP2α/170 in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.