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

• Published in: Molecular pharmacology 2021-03, Vol.99 (3), p.226-241
• Main Authors: Hernandez, Victor A., Carvajal-Moreno, Jessika, Papa, Jonathan L., Shkolnikov, Nicholas, Li, Junan, Ozer, Hatice Gulcin, Yalowich, Jack C., Elton, Terry S.
• Format: Article
• Language: English
• Subjects: Cell Survival; CRISPR-Cas Systems; DNA Topoisomerases, Type II - genetics; Down-Regulation; Drug Resistance, Neoplasm; Etoposide - pharmacology; Gene Editing - methods; Humans; Introns; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - drug therapy; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - enzymology; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics; Poly-ADP-Ribose Binding Proteins - genetics; RNA Splice Sites
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/molpharm.120.000173

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. 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.