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

基本信息

批准号：
10057231
负责人：
TERRY S ELTON
金额：
$ 35.69万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10057231
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

项目摘要

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，其临床虽然 TOP2α 的突变形式已被证实，但其疗效常常因获得性化学耐药性而受到损害。在耐药模型中报告，来自患者样本的证据强烈表明， TOP2α是耐药性的主要决定因素。我们报道，在依托泊苷耐药的人白血病 K/VP.5 细胞中，170 kDa TOP2α (TOP2α/170) 与亲本 K562 细胞进行比较，而新型 C 端截短的 90 kDa TOP2α 亚型 (TOP2α/90) 显着增加。 TOP2α/90 是替代处理预处理的翻译产物。保留内含子 19 的 mRNA；通过 cDNA 末端的 3' 快速扩增、PCR 和测序得到证实。 TOP2α/90 mRNA 中的内含子 19 包含一个框内终止密码子和两个共有的 Poly(A) 位点，可实现加工后的转录物被聚腺苷酸化，TOP2α/90 mRNA 被翻译成缺少 C- 的蛋白质。 TOP2α/170 的末端 770 个氨基酸，缺少活性位点 Tyr805，包含 25 个独特的氨基酸。通过外显子 19/内含子 19 的翻译“通读”，允许产生抗血清以进行检测细胞实验表明，使用这种抗血清和 C 端抗体来检测 TOP2α/170。 TOP2α/90 与 TOP2α/170 共免疫沉淀，在 K562 细胞中强制表达 TOP2α/90。抑制，而 siRNA 介导的 K/VP.5 细胞中 TOP2α/90 的敲低则增强依托泊苷介导的总之，结果强烈表明 TOP2α/90 的表达是 DNA 链断裂的决定因素。通过与 TOP2α/170 异二聚化相关的显性负面效应来产生化学耐药性。这一背景是以下假设的基础：获得性的主要机制对 TOP2α 靶向药物的耐药性是由于替代性 RNA 加工/剪接造成的。恢复规范的 RNA 剪接将能够规避耐药性，以便进行测试。这些假设将追求两个具体目标：1）确立 TOP2α/90 作为决定因素的作用通过与 TOP2α/170 相互作用获得耐药性；2) 确定替代 RNA 的机制； TOP2α 前 mRNA 的加工并开发易于处理的策略来规避耐药性。这些目标的顺利完成将在两个方面产生重要影响。 TOP2α 替代 RNA 加工的表征将推动规避获得性药物的策略获得的结果可用于肿瘤细胞/活检评估 TOP2α/90 作为药物的生物标志物。其次，我们的策略将揭示耐药性、预后和/或未来直接的 TOP2α 靶向治疗。关于剪接体功能作为可用于调节的过程的基本新信息 TOP2α 和/或已知可替代处理的其他重要抗癌药物靶标的表达作为耐药性的决定因素。 1