CRISPR/Cas9 with Homology‐Directed Repair Effectively Silences the Human Topoisomerase IIα Intron‐19 5′ Splice Site and Results in Etoposide Resistance in Human Leukemia K562 Cells

Abstract

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 a target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170. Our laboratory recently demonstrated that reduced levels of TOP2α/170 in acquired resistance to etoposide is associated with alternative RNA processing of the TOP2α gene and intron retention. Intron 19 retention in an etoposide‐resistant human leukemia K562 subline, K/VP.5, resulted not only in decreased TOP2α/170 but also translation and overexpression of a C‐terminal truncated 90‐kDa isoform of TOP2α, TOP2α/90. We found that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Splice site analysis revealed that the exon 19/intron 19 boundary of the TOP2α gene is suboptimal, providing a potential explanation for intron 19 retention. To further test the hypothesis that intron 19 retention is a determinant of acquired drug resistance, CRISPR/Cas9 with homology‐directed repair (HDR) was used to silence the intron 19 5′ splice site of the TOP2α gene in parental K562 cells (etoposide‐sensitive) by making two nucleotide changes to the splice site (GAG//GTAAAC→GAA//CTAAAC) thereby forcing intron 19 retention by abrogating spliceosome function. Gene‐edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. Clones with TOP2α edited alleles contained decreased TOP2α/170 mRNA/protein resulting in resistance to etoposide as assessed by reduced etoposide‐induced DNA damage (γH2AX, Comet assays) and growth inhibition. Forced expression of TOP2α/90 in the gene‐edited K562 cells further decreased etoposide‐induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the role of alternative TOP2α mRNA splicing as a determinant of resistance to TOP2α‐targeting agents.