(541.10) 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

Poster Board Number: B148

Victor Hernandez (Ohio State University), Jessika Carvajal-Moreno (Ohio State University), Xinyi Wang (Ohio State University), Maciej Pietrzak (Ohio State University), Jack Yalowich (Ohio State University), Terry Elton (Ohio State University)

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.