Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells

• Published in: PloS one 2022-05, Vol.17 (5), p.e0265794-e0265794
• Main Authors: Hernandez, Victor A, Carvajal-Moreno, Jessika, Wang, Xinyi, Pietrzak, Maciej, Yalowich, Jack C, Elton, Terry S
• Format: Article
• Language: English
• Subjects: Anticancer properties; Antineoplastic drugs; Antitumor agents; Bioassays; Biology and life sciences; Chemoresistance; Cloning; CRISPR; Damage detection; Deoxyribonucleic acid; DNA; DNA damage; DNA topoisomerase; Drug resistance; Efficiency; Enzymes; Etoposide; Gene expression; Gene sequencing; Genetic modification; Genome editing; Genomes; Homology; Isoforms; Laboratories; Leukemia; Mutation; Optimization; Polyadenylation; Polymerase chain reaction; Protein expression; Proteins; Repair; Research and Analysis Methods; Retention; Ribonucleic acid; RNA; RNA processing
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0265794

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5' splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5' splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. 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 important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.