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

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.

CRISPR/Cas9 gene editing: a new approach for overcoming drug resistance in cancer

“…In a recent interesting study of etoposide-resistant leukemia cells, the CRISPR/Cas9 system was designed to target the exon 19/intron 19 5′ splice site and induce mutations as GAG//GTAA AC → GAG// GTAA GT. The results of this study showed that this gene editing significantly reduced the expression of TOP2α/90 and increased the expression of TOP2α/170, which led to increased sensitivity to etoposide [125]. In addition, studies on chronic myeloid leukemia cells have shown that targeting and disrupting the well-known BCR/ABL oncogene is also possible using CRISPR/Cas9 gene editing and can lead to attenuated proliferation and enhanced apoptosis in imatinib-resistant leukemia cells [126].…”

“…Studies have shown that in etoposide-resistant leukemia cells there are two isoforms of this enzyme, TOP2α/170 and TOP2α/90, which appear to be the product of alternative RNA processing. TOP2α/170 is the target of anticancer drugs, but it appears that the expression of this isoform decreases in etoposide-resistant leukemia cells, while the expression of TOP2α/90 increases significantly [124,125]. This increased expression may play an important role in drug resistance.…”