Circumvention of Topoisomerase IIαIntron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant Human Leukemia K562 Cells

Abstract

DNA topoisomerase IIα (TOP2α, 170kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage stabilizing anti-cancer agents such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposideresistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90) which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3′-Rapid Amplification of cDNA Ends (3′-RACE), we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was utilized. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide (AMO) was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/Cas9 homology-directed repair (HDR) was used to mutate the cryptic I19 PAS (AATAAAACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs.Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs.