Circumvention of Topoisomerase II Intron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant Human Leukemia K562 Cells.

DNA topoisomerase II (TOP2; 170 kDa, TOP2/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2 poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2 isoform (90 kDa; TOP2/90), which heterodimerized with TOP2/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2/90 as the translation product of a TOP2 mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. 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 was used to hybridize/block the PAS in TOP2 pre-mRNA in K/VP.5 cells, resulting in decreased TOP2/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (AAA→AAA) to prevent IPA. Gene-edited clones exhibited increased TOP2/170 and decreased TOP2/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2-targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance.