DNA Topoisomerase II? (TOP2?; 170 kDa) is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to acquired chemoresistance. While mutant forms of TOP2? have been reported in resistance models, evidence from patient samples strongly suggests that decreased levels of TOP2? is the major determinant of drug resistance. We have reported that, in etoposide resistant human leukemia K/VP.5 cells, 170 kDa TOP2? (TOP2?/170) was decreased compared to parental K562 cells, while a novel C-terminal truncated 90 kDa TOP2? isoform (TOP2?/90) was dramatically increased. TOP2?/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 TOP2?/90 mRNA harbors an in-frame stop codon, and two consensus poly(A) sites allowing for the processed transcript to be polyadenylated. TOP2?/90 mRNA is translated to a protein missing the C- terminal 770 amino acids of TOP2?/170 and lacks the active site Tyr805. TOP2?/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 TOP2?/170, cellular experiments revealed that TOP2?/90 co-immunoprecipitated with TOP2?/170. Forced expression of TOP2?/90 in K562 cells suppressed while siRNA-mediated knockdown of TOP2?/90 in K/VP.5 cells enhanced etoposide-mediated DNA strand breaks. Together, results strongly suggest that expression of TOP2?/90 is a determinant of chemoresistance through a dominant negative effect related to heterodimerization with TOP2?/170. This background serves as the foundation for the hypothesis that a major mechanism of acquired resistance to TOP2?-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. In order to test these hypotheses two specific aims will be pursued to: 1) establish the role of TOP2?/90 as a determinant of acquired resistance through its interaction with TOP2?/170; 2) determine the mechanism(s) of alternative RNA processing of TOP2? pre-mRNA and develop tractable strategies to circumvent resistance. Successful completion of these Aims will have important impact in two areas. First, complete characterization of alternative RNA processing of TOP2? will drive strategies to circumvent acquired drug resistance. Results obtained may allow for tumor cell/biopsy evaluation of TOP2?/90 as a biomarker for drug resistance, prognosis, and/or direct future TOP2?-targeted therapies. Second, our strategies will reveal fundamental new information regarding spliceosome function as a process that may be utilized for regulating the expression of TOP2? and/or other important anticancer drug targets known to be alternatively processed as determinants of drug resistance. 1
The overall goal of this project is to improve therapeutic outcomes in malignancies treated with DNA topoisomerase II? (TOP2?)-targeted drugs by: 1) characterizing resistance mechanisms associated with alternative RNA processing that leads to production of a truncated form of this enzyme; 2) developing strategies to restore normal RNA processing to circumvent chemoresistance. In addition, results will yield important new fundamental knowledge of RNA processing mechanisms dysregulated in acquired drug resistance to TOP2?- targeted drugs. Finally, truncated TOP2? may be developed as a prognostic biomarker for acquired drug resistance and for therapeutic decision-making.
