RT Journal Article SR Electronic T1 CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5′ Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 226 OP 241 DO 10.1124/molpharm.120.000173 VO 99 IS 3 A1 Victor A. Hernandez A1 Jessika Carvajal-Moreno A1 Jonathan L. Papa A1 Nicholas Shkolnikov A1 Junan Li A1 Hatice Gulcin Ozer A1 Jack C. Yalowich A1 Terry S. Elton YR 2021 UL http://molpharm.aspetjournals.org/content/99/3/226.abstract AB An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. 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 used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2α 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 TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, 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 TOP2α E19/I19 5′ SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-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 IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.