XLF in double-strand break repair and chemo/radiosensitization

XLF 在双链断裂修复和化疗/放射增敏中的应用

• 批准号: 9031072
• 负责人: Matthew C Hartman
• 金额: $ 30.79万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9031072
• 关键词: Affect; Affinity; Binding; Biological Assay; Breast Cancer Cell; Cell Cycle Checkpoint; Cell Extracts; Cell Line; Cells; Chemosensitization; Clinical; Core Protein; Cyclic Peptides; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Repair Gene; Development; Double Strand Break Repair; Effectiveness; Ensure; Etoposide; Filament; Genetic; Genetic study; Genome; Genotype; Goals; Health; Immunoprecipitation; In Vitro; Incubated; Ionizing radiation; Joint repair; Lesion; Libraries; Ligation; Malignant Neoplasms; Mammalian Cell; Maps; Messenger RNA; Nucleotides; Pathway interactions; Peptide Library; Peptides; Pharmaceutical Preparations; Photoaffinity Labels; Poison; Proteins; Radiation therapy; Radiation-Sensitizing Agents; Radio; Radioresistance; Radiosensitization; Reagent; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Structure; System; Techniques; Testing; Time; Topoisomerase II; Topoisomerase-II Inhibitor; Translations; Treatment Failure; Work; XRCC4 gene; base; cancer cell; cancer therapy; carcinogenesis; cell growth; cell injury; chemosensitizing agent; chemotherapy; cytotoxic; homologous recombination; improved; inhibitor/antagonist; insight; killings; member; mutant; neoplastic cell; novel; protein function; radiosensitizing; repaired; research study; response; screening; success; targeted treatment

DESCRIPTION (provided by applicant): Many of the agents commonly used for cancer therapy kill tumor cells by damaging cellular DNA, and thus DNA repair proteins and their interactions represent potential targets for enhancing such therapy. Double- strand breaks (DSBs) in DNA, which are the major cytotoxic lesions formed by ionizing radiation as well as by topoisomerase II inhibitors, are repaired primarily by nonhomologous end joining (NHEJ) and, to a lesser extent, by homologous recombination (HRR). Thus, the efficiency of these repair systems is a critical factor in the effectiveness of radio- and chemotherapy. XLF is the most recently discovered core protein in the NHEJ pathway. Although genetic studies show that XLF is essential for efficient NHEJ, its precise role in the pathway is poorly understood. In addition, because the interaction between XLF and another NHEJ protein, XRCC4, is critical for NHEJ, but is transient and has only moderate (low ¿M) affinity, it represents an attractive target for chemo/radiosensitization by disruption of NHEJ. The primary objectives of this application are, therefore, to elucidate in more detail the role of XLF in NHEJ, and to develop XLF/XRCC4 interaction inhibitors and evaluate them as chemo/radiosensitizers. In order to better clarify XLF's role and test the hypothesis that XLF and XRCC4 polymerize into a filament that aligns DNA ends, photoactivatable DNA substrates will be added to cell extracts to map the interactions of XLF with DNA near a DSB as a function of time and distance from the break. Experiments with a fluorescent NHEJ reporter integrated into the genome will determine whether alignment-based gap filling at DNA ends is strictly dependent on XLF in intact cells, as has been already demonstrated in cell extracts. For the inhibitor development, the novel in vitro selection technique, mRNA display, will be used to isolate small (Mr 1000-2000), cell-penetrating, drug-like cyclic peptides that bind to XRCC4 at its interface with XLF. Using mRNA display, an extremely diverse library of 10 trillion semi- random peptides fused to the mRNAs that encode them will be prepared using in vitro translation. Those that bind to the XLF binding pocket on XRCC4 will be selectively captured and their mRNA amplified with RT-PCR. After several rounds of iterative translation, selection, and amplification, candidate peptides that bloc the XLF/XRCC4 interaction will be identified. These peptides will be screened for NHEJ inhibition using a cell extract-based assay wherein joining of partially complementary DSB ends is stringently dependent on XLF. The strongest inhibitory peptides will be chemically synthesized with addition of cell-penetrating tags, and tested for sensitization of triple-negative breast tumor cells to two DSB-inducing agents: ionizing radiation and etoposide. The most potent sensitizers will be used in further experiments to determine to what extent DSB repair is inhibited in several cell lines, chosen either for their known radioresistance, or because they are deficient in DSB repair by the alternate HRR pathway. Based on their unique mode of action, these peptides or subsequent derivatives thereof may be clinically useful in sensitizing tumor cells for radio- and chemotherapy.

描述(申请人提供)：许多通常用于癌症治疗的药物通过破坏细胞DNA来杀死肿瘤细胞，因此DNA修复蛋白及其相互作用是加强这种治疗的潜在靶点。DNA双链断裂(DSB)是电离辐射和拓扑异构酶II抑制剂造成的主要细胞毒性损伤，主要通过非同源末端连接(NHEJ)修复，其次是同源重组(HRR)。因此，这些修复系统的效率是放化疗效果的关键因素。XLF是NHEJ途径中最新发现的核心蛋白。虽然遗传学研究表明，XLF对于有效的NHEJ是必不可少的，但它在该途径中的确切作用尚不清楚。此外, 由于XLF与另一种NHEJ蛋白XRCC4之间的相互作用对NHEJ是关键的，但它是暂时的，并且只有中等(低)的亲和力，因此它是一个有吸引力的靶点，可以通过破坏NHEJ进行化疗/放射增敏。因此，本申请的主要目的是更详细地阐明XLF在NHEJ中的作用，开发XLF/XRCC4相互作用抑制剂，并评估它们作为化疗/放射增敏剂的作用。为了更好地阐明XLF的作用并检验XLF和XRCC4聚合成排列DNA末端的细丝的假设，将光激活DNA底物添加到细胞提取液中，以绘制XLF与DSB附近DNA的相互作用随时间和距离断裂的函数关系。将荧光NHEJ报告整合到基因组中的实验将确定DNA末端基于比对的缺口填充是否严格依赖于完整细胞中的XLF，正如已经在细胞提取液中证明的那样。对于抑制剂的开发，将使用一种新的体外选择技术-mRNA显示，以分离小的(MR 1000-2000)、穿透细胞的类药物环肽，这些环肽在XRCC4与XLF的界面结合。利用信使核糖核酸显示技术，我们将利用体外翻译技术来制备一个极其多样化的文库，该文库由10万亿个半随机多肽与编码它们的信使核糖核酸融合而成。与XRCC4上的XLF结合口袋结合的那些将被选择性地捕获，并用RT-PCR扩增它们的mRNA。经过几轮迭代翻译、选择和扩增，将确定阻断XLF/XRCC4相互作用的候选肽。这些多肽将使用基于细胞提取物的试验来筛选NHEJ抑制作用，其中部分互补DSB末端的连接严格依赖于XLF。最强的抑制多肽将通过添加穿透细胞的标签进行化学合成，并测试对三阴性的敏化。 乳腺肿瘤细胞对两种DSB诱导剂：电离辐射和依托泊苷。最有效的增敏剂将用于进一步的实验，以确定在几种细胞系中DSB修复被抑制的程度，选择这些细胞系要么是因为它们已知的辐射抗性，要么是因为它们是 通过交替的HRR途径缺乏DSB修复。基于其独特的作用模式，这些多肽或其后续的衍生物可能在临床上用于增敏肿瘤细胞进行放射治疗和化疗。