Novel Topoisomerase II alpha isoform as a drug resistance determinant

新型拓扑异构酶 II α 亚型作为耐药决定因素

• 批准号: 10297850
• 负责人: TERRY S ELTON
• 金额: $ 34.97万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10297850
• 关键词: Active Sites; Algorithms; Alternative Splicing; Amino Acid Sequence; Amino Acids; Antibodies; Antineoplastic Agents; Area; Attenuated; Binding; Biological Assay; Biological Markers; Biopsy; C-terminal; CRISPR/Cas technology; Cell Death; Cells; Chemoresistance; Clinical; Complementary DNA; Complex; Consensus; DNA; DNA Damage; DNA Sequence; DNA Topoisomerases; DNA strand break; DNA topoisomerase II alpha; Decision Making; Deletion Mutation; Dimerization; Dominant-Negative Mutation; Drug Targeting; Drug resistance; Enzymes; Etoposide; Evaluation; Exons; Foundations; Future; Generations; Goals; Heterodimerization; Human; Immune Sera; Introns; K-562; K562 Cells; Knowledge; Length; Leukemic Cell; Malignant Neoplasms; Mediating; Messenger RNA; Methodology; Modeling; Mutate; Newly Diagnosed; Nuclear; Patients; Pharmaceutical Preparations; Poly A; Process; Production; Prognosis; Prognostic Marker; Protein Isoforms; Proteins; Publishing; RNA Processing; RNA Splicing; Reporting; Resistance; Role; Sampling; Site; Small Interfering RNA; Solid Neoplasm; Splice-Site Mutation; Spliceosomes; Terminator Codon; Testing; Therapeutic; Transcript; Translating; Translations; Tumor Cell Line; acquired drug resistance; base; cancer cell; clinical efficacy; clinically relevant; cytotoxicity; experimental study; expression vector; improved; innovation; knock-down; leukemia; mRNA Precursor; mutant; neoplastic cell; novel; premature; relapse patients; resistance mechanism; restoration; targeted agent; targeted treatment; therapy outcome

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

DNA拓扑异构酶IIα（Top2α; 170 kDa）是抗癌药物的重要靶标 由于获得的化学抗性，疗效通常会损害。 top2α的突变形式已经 在阻力模型中报道，来自患者样本的证据强烈表明提高了水平 TOP2α是耐药性的主要决定。 我们报告说，在依托泊苷耐药的人白血病K/VP.5细胞中，170 kDatop2α（top2α/170） 与亲本K562细胞相比，它的进展已进展，而新型的C末端截短了90 kDatop2α同工型 （TOP2α/90）显着增加。 Top2α/90是经过处理的预先处理的乘积 保留内含子19的mRNA；通过对cDNA末端，PCR和测序的3'-rapid扩增确认。 top2α/90 mRNA中的内含子19固定式终止密码子和两个共识poly（a）位点允许 处理的转录本为聚腺苷酸化。 TOP2α/90 mRNA被翻译成缺少c-的蛋白 TOP2α/170的末端770氨基酸，缺乏活性位点Tyr805。 TOP2α/90包含25个独特的氨基 酸通过外显子19/内含子19的“读取”，允许抬高抗血清以检测到酸以检测 使用该抗血清和C末端抗体检测TOP2α/170，细胞实验显示 TOP2α/90与TOP2α/170共免疫沉淀。在K562细胞中TOP2α/90的强制表达 抑制siRNA介导的top2α/90在K/VP.5细胞中抑制了增强的依托泊苷介导的 DNA链断裂。总之，结果强烈表明Top2α/90的表达是确定的 通过与TOP2α/170的异二聚化相关的主要负面影响化学抗性。 这种背景是一个假设的基础，即获得的主要机制 对TOP2α靶向药物的耐药性是由于替代RNA加工/剪接引起的。它是进一步假设的 规范RNA剪接的恢复将能够规避耐药性。为了测试 这些假设将追求两个具体目标：1）确定Top2α/90的作用 通过与TOP2α/170的相互作用获得了电阻； 2）确定替代RNA的机制 加工TOP2α前MRNA并制定可论到耐药性的典型策略。 这些目标的成功完成将在两个领域产生重要影响。首先，完成 TOP2α的替代RNA处理的表征将推动策略避免获得的药物 反抗。获得的结果可能允许对TOP2α/90作为药物的生物标志物进行肿瘤细胞/活检评估 耐药性，预后和/或直接将来的TOP2α靶向疗法。其次，我们的策略将揭示 有关剪接函数的基本新信息，可以用作调节的过程 TOP2α和/或其他重要的抗癌药物的表达已被已知的处理 作为耐药性的确定性。 1