Jack Yalowich, PhD
Professor, Pharmacology
College of Pharmacy
The Ohio State University

Host: Dean Steve Swanson

Alternative RNA Processing of DNA Topoisomerase IIa in Etoposide-Resistant Human Leukemia K562 Cells: Intron Retention Results in a Novel C-Terminal Truncated 90-kDa Isoform; A Determinant of Chemoresistance

DNA Topoisomerase IIα (TOP2α; 170 kDa) is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to acquired chemoresistance. While mutant forms of TOP2α have been reported in resistance models, evidence from patient samples strongly suggests that decreased levels of TOP2α is the major determinant of drug resistance.

We have reported that, in etoposide resistant human leukemia K/VP.5 cells, 170 kDa TOP2α (TOP2α/170) was decreased compared to parental K562 cells, while a novel C-terminal truncated 90 kDa TOP2α isoform (TOP2α/90) was dramatically increased (JPET 360: 152-163, 2017) . TOP2α/90 is the translation product of alternatively processed pre- mRNA which retains intron 19; confirmed by 3′-rapid amplification of cDNA ends, PCR, and sequencing. Intron 19 in TOP2α/90 mRNA harbors an in-frame stop codon, and two consensus poly(A) sites allowing for the processed transcript to be polyadenylated. TOP2α/90 mRNA is translated to a protein missing the C- terminal 770 amino acids of TOP2α/170 and lacks the active site Tyr805. TOP2α/90 contains 25 unique amino acids through translation of the exon 19/intron 19 “read-through” allowing for antisera to be raised to detect this isoform. Using this antisera and a C-terminal antibody to detect TOP2α/170, cellular experiments revealed that TOP2α/90 co-immunoprecipitated with TOP2α/170.

Forced expression of TOP2α/90 in K562 cells suppressed while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced etoposide-mediated DNA strand breaks. Together, results strongly suggest that expression of TOP2α/90 is a determinant of chemoresistance through a dominant negative effect related to heterodimerization with TOP2α/170.

This background serves as the foundation for the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. It is further hypothesized that restoration of canonical RNA splicing will be capable of circumventing drug resistance. Resuts in support of these hypotheses will be presented along with future plans and perspectives.