CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron-19 5′ Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells

Results indicate that CRISPR/Cas9 gene editing of a suboptimal E19/I19 5′ splice site 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. 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//GTAAAC→GAG//GTAAGT) in the TOP2α gene’s suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS).