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，其临床应用 由于获得性化疗耐药，疗效往往会受到影响。而Top2α的突变形式已经被 在耐药模型中报道，来自患者样本的证据强烈表明， Top2α是耐药的主要决定因素。 我们已经报道，在对依托泊苷耐药的人白血病K/VP细胞中，170kDa TOP2α(TOP2α/170) 与亲本K562细胞相比减少，而一个新的C末端截短90 kDa TOP2α亚型 (TOP2α/90)显著升高。TOP2α/90是交替加工前处理的翻译产物 经3‘-快速扩增、聚合酶链式反应和测序证实。 TOP2α/90mRNA19内含子含有一个框内终止密码子和两个共有的Poly(A)位点，允许 处理后的转录本将被多聚腺苷化。TOP2α/90mRNA被翻译成一种缺少C- TOP2基因α/170770个氨基酸残基，缺少活性位点Tyr805。TOP2α/90含有25种独特的氨基酸 通过外显子19/内含子19的翻译而产生的酸，允许提高抗血清以检测 这个同源异构体。细胞实验显示，使用该抗血清和一种C末端抗体来检测TOP2α/170 TOP2α/90与TOP2α/170共沉淀。TOP2α/90在K562细胞中的强制表达 小干扰RNA抑制K/VP细胞TOP2α/90的表达增强依托泊苷的作用 DNA链断裂。综上所述，结果有力地表明TOP2α/90的表达是一种决定因素 α/170TOP2通过与异二聚化相关的显性负效应产生化疗耐药性。 这一背景为以下假设奠定了基础，即后天获得的主要机制 对TOP2RNA靶向药物的抗药性是由于替代的α加工/剪接。这是进一步的假设 恢复规范的RNA剪接将能够绕过耐药性。为了测试 这些假设有两个具体的目的：1)建立TOP2α/90作为决定因素的作用 通过与Top2RNA/170RNA的相互作用获得抗性；2)确定交替α的机制(S 处理TOP2α前信使核糖核酸，并制定易处理的策略来规避耐药性。 成功实现这些目标将在两个方面产生重要影响。第一，完成 TOP2RNA替代α加工的特征将推动规避获得性药物的策略 抵抗。所获得的结果可能允许肿瘤细胞/活检评估TOP2α/90作为药物的生物标记物 耐药性、预后和/或未来TOP2TOP2α靶向治疗。第二，我们的战略将揭示 关于剪接体作为调控过程的基本新信息 TOP2α和/或其他已知可交替加工的重要抗癌药物靶点的表达 作为抗药性的决定因素。 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