DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. We previously characterized acquired resistance to etoposide (VP-16) in a cloned human K562 leukemia cell line, K/VP.5, containing reduced TOP2α. In the present study, using an antibody specific for the amino-terminus of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in the etoposide resistant K/VP.5 cells. TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared to parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3’-RACE, PCR, and sequencing. TOP2α/90 mRNA includes retained intron 19 which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076 Da translation product missing the carboxyl-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 ‘readthrough’. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of Complex of Enzyme-to-DNA (ICE) and single cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid, exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared to similarly treated K562 cells transfected with empty vector. Since TOP2α/90 lacks the active site tyrosine (Tyr805) of full length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. . In separate studies the TOP2α/90 mRNA splice variant was found to be expressed in most human tissues suggesting that this novel protein isoform may play a role in both intrinsic chemosensitivity as well as in acquired resistance. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents. In addition, future studies will be directed to examine the RNA processing mechanism(s) operational that suppress intron 19 splicing in TOP2α pre-mRNA.

Citation Format: Ragu Kanagasabai, Lucas Serdar, Soumendrakrishna Karmahapatra, Corey A. Kientz, Mary K. Ritke, Terry S. Elton, Jack C. Yalowich. A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3181. doi:10.1158/1538-7445.AM2017-3181