XLF in double-strand break repair and chemo/radiosensitization

描述（由申请人提供）：许多常用于癌症治疗的药物通过破坏细胞DNA杀死肿瘤细胞，因此DNA修复蛋白及其相互作用代表了增强这种治疗的潜在靶标。DNA中的双链断裂（DSB）是由电离辐射以及拓扑异构酶II抑制剂形成的主要细胞毒性损伤，其主要通过非同源末端连接（NHEJ）修复，并且在较小程度上通过同源重组（HRR）修复。因此，这些修复系统的效率是放疗和化疗有效性的关键因素。XLF是NHEJ通路中最近发现的核心蛋白。虽然遗传学研究表明XLF对于有效的NHEJ是必不可少的，但其在该途径中的确切作用知之甚少。此外，本发明还提供了一种方法， 由于XLF和另一种NHEJ蛋白XRCC 4之间的相互作用对NHEJ至关重要，但是短暂的，只有中等（低μ M）亲和力，因此它代表了通过破坏NHEJ进行化学/放射增敏的有吸引力的靶点。因此，本申请的主要目的是更详细地阐明XLF在NHEJ中的作用，并开发XLF/XRCC 4相互作用抑制剂并将其作为化学/放射增敏剂进行评估。为了更好地阐明XLF的作用并测试XLF和XRCC 4插入到排列DNA末端的细丝中的假设，将向细胞提取物中加入可光活化的DNA底物，以绘制XLF与DSB附近DNA的相互作用作为时间和距离断裂的函数。将荧光NHEJ报告基因整合到基因组中的实验将确定在DNA末端基于荧光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物中证明的那样。对于抑制剂的开发，新的体外选择技术，mRNA展示，将用于分离小（Mr 1000-2000），细胞穿透，药物样环肽，结合XRCC 4在其界面与XLF。使用mRNA展示，将使用体外翻译制备与编码它们的mRNA融合的10万亿半随机肽的极其多样化的文库。那些结合到XRCC 4上的XLF结合口袋的将被选择性地捕获，并且它们的mRNA用RT-PCR扩增。在几轮迭代翻译、选择和扩增后，将鉴定阻断XLF/XRCC 4相互作用的候选肽。将使用基于细胞提取物的测定筛选这些肽的NHEJ抑制，其中部分互补DSB末端的连接严格依赖于XLF。最强的抑制肽将通过添加细胞穿透标签来化学合成，并测试三阴性的致敏性。 乳腺肿瘤细胞的两个DSB诱导剂：电离辐射和依托泊苷。最有效的敏化剂将用于进一步的实验，以确定在几种细胞系中DSB修复被抑制到什么程度，选择这些细胞系是因为它们已知的辐射抗性，或者因为它们是 缺乏通过替代HRR途径的DSB修复。基于其独特的作用模式，这些肽或其后续衍生物可在临床上用于使肿瘤细胞对放射和化学疗法敏感。

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.