Alternative RNA Processing as a Determinant of Acquired Resistance to the Anticancer Drug Etoposide in Human Leukemia K562 Cells

Abstract

DNA topoisomerase IIα (170 kDa, TOP2α/170) is a key homodimeric enzyme resolving DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double‐strand breaks (DSB). TOP2α/170 is also a prominent clinical target for anticancer drugs, such as etoposide (VP‐16), whose clinical efficacy is often compromised due to chemoresistance. We recently demonstrated that parental K562 cells and cloned K562 cells (K/VP.5) with acquired resistance to VP‐16 express a novel truncated TOP2α/90 (90 kDa) isoform dramatically overexpressed in drug‐resistant K/VP.5 cells (JPET 360(1): 152–163, 2017). Recent results also demonstrated overexpression of TOP2α/90 in isogenic HL‐60 AML cells resistant to the TOP2α‐targeting agents, mAMSA and mitoxantrone.TOP2α/90 is the C‐terminal truncated translation product of TOP2α mRNA lacking the active‐site Tyr805 required for the generation of DSBs. TOP2α/90 mRNA retains a portion of intron 19 indicating altered RNA processing and an exon‐intron read‐through. TOP2α/90 was detectable in the nucleus and heterodimerizes with TOP2α/170. Forced expression of TOP2α/90 in K562 cells decreased VP‐16–induced DNA damage and cytotoxicity while siRNA knockdown of TOP2α/90 in K/VP.5 cells increased VP‐16 activity (Mol. Pharmacol. 93: 515–525, 2018). Together results suggest that TOP2α/90 is a resistance determinant/biomarker through a dominant negative effect. qPCR analyses of paired/matched AML patient samples (pre‐treatment & relapse) revealed that, in 3 of 4 patients, there was a statistically significant increase in the ratio of expression of TOP2α/90 mRNA compared to TOP2α/170, after relapse.We hypothesize that an intrinsically weak exon 19/intron 19 (E19/I19) splice site (ss) in TOP2α pre‐mRNA results in partial I19 retention and formation of truncated TOP2α/90. We further posit that therapeutic genome to modify I19 will provide a tractable strategy to circumvent TOP2α‐mediated drug resistance.To investigate the influence of a weak TOP2α E19/I19 ss (GAG/gtaaac)in I19 retention, a minigene (MG1+) expression construct with a consensus I19 5′ ss (CAG/gtaagt) was generated. Transfection of K/VP.5 cells with MG1+ resulted in a reduction of I19 retention (assessed by qPCR), accompanied by an increase in the levels of a properly spliced amplicon indicative of TOP2α/170 mRNA. These findings set the stage for CRISPR/Cas9 experiments to determine whether editing the I19 5′ ss of the TOP2α gene will result in decreased I19 retention, diminished TOP2α/90 generation, and increased VP‐16 sensitivity in K/VP.5 cells. To date, CRISPR/Cas9 targeting has resulted in successful targeting/mutation of E19/I19 to a consensus ss sequence. Selection of an edited cell clone is in process. Our results contribute to a better understanding of alternative TOP2α mRNA processing leading to the development of resistance to TOP2α poisons. Characterization of alternative RNA processing of the TOP2α mRNA will lead to strategies to circumvent acquired drug resistance. These and future results may also allow for evaluation of TOP2α/90 as a biomarker for drug resistance, prognosis, and/or guide TOP2α‐targeted therapies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.