Novel Topoisomerase II alpha isoform as a drug resistance determinant

DNA拓扑异构酶IIα（TOP2α; 170 kDa）是抗癌药物的重要靶点，其临床 由于获得性的化学抗性，功效经常受到损害。虽然TOP2α的突变形式已经被发现， 在耐药性模型中报告，来自患者样本的证据强烈表明， TOP2α是耐药的主要决定因素。 我们报道了在对依托泊苷耐药的人白血病K/VP. 5细胞中，170 kDa TOP 2 α（TOP 2 α/170） 与亲本K562细胞相比，其C末端截短的90 kDa TOP2α亚型 (TOP2α/90）显著增加。TOP2α/90是交替处理的预处理的翻译产物， 保留内含子19的mRNA;通过cDNA末端的3 '-快速扩增、PCR和测序确认。 TOP2α/90 mRNA的内含子19含有一个符合读框的终止密码子和两个共有的poly（A）位点， 将加工的转录物聚腺苷酸化。TOP2α/90 mRNA被翻译成缺失C- T0 P2 α/170末端770个氨基酸，缺少活性位点Tyr 805。TOP2α/90含有25个独特的氨基酸 通过翻译外显子19/内含子19“通读”，允许产生抗血清以检测 这个isoform使用该抗血清和C-末端抗体检测TOP 2 α/170，细胞实验显示， TOP 2 α/90与TOP 2 α/170共免疫沉淀。TOP 2 α/90在K562细胞中的强制表达 抑制，而siRNA介导的K/VP.5细胞中TOP2α/90的敲低增强了依托泊苷介导的 DNA链断裂。总之，结果强烈表明，TOP 2 α/90的表达是一个决定因素， 通过与TOP2α/170异源二聚化相关的显性负效应产生耐药性。 这一背景为后天免疫的主要机制这一假设奠定了基础。 对TOP2α靶向药物的耐药性是由于选择性RNA加工/剪接。进一步假设 恢复典型的RNA剪接将能够避免抗药性。为了测试 这些假设将追求两个具体目标：1）建立TOP2α/90作为决定因素的作用， 通过与TOP 2 α/170的相互作用获得抗性; 2）确定替代RNA的机制 TOP2α前体mRNA的加工，并开发易于处理的策略来规避耐药性。 成功地实现这些目标将在两个领域产生重要影响。第一，完整 TOP2α的替代RNA加工特性将推动规避获得性药物的策略 阻力获得的结果可用于肿瘤细胞/活检评价TOP 2 α/90作为药物生物标志物 耐药性、预后和/或指导未来的TOP2α靶向治疗。其次，我们的战略将揭示 关于剪接体功能的基本新信息作为一个过程，可以用于调节 TOP2α和/或其他已知可替代加工的重要抗癌药物靶标的表达 作为耐药性的决定因素。 1

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. 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. In order to test these hypotheses two specific aims will be pursued to: 1) establish the role of TOP2α/90 as a determinant of acquired resistance through its interaction with TOP2α/170; 2) determine the mechanism(s) of alternative RNA processing of TOP2α 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 TOP2α will drive strategies to circumvent acquired drug resistance. Results obtained may allow for tumor cell/biopsy evaluation of TOP2α/90 as a biomarker for drug resistance, prognosis, and/or direct future TOP2α-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 TOP2α and/or other important anticancer drug targets known to be alternatively processed as determinants of drug resistance. 1