NOVEL DNA DOUBLE STRAND BREAK REPAIR TARGETING THERAPEUTICS FOR CANCER TREATMENT

描述：癌细胞经常使DNA双链断裂(DSB)修复蛋白失活，导致基因组不稳定和肿瘤的遗传进化。这些蛋白的失活通常不会由于代偿修复途径的活性而导致DNA DSB修复的明显缺陷。我们的理由是，识别削弱这些代偿途径的化合物将是开发治疗药物的第一步，这种药物可以使肿瘤细胞敏感，但不能使正常组织对基因毒性癌症治疗敏感。DNA损伤反应组蛋白H2 AX是一种剂量依赖的肿瘤抑制因子，在许多癌症中表达减少。H_2AX缺乏导致基因组不稳定，但不会导致DNA非同源末端连接修复(NHEJ)的明显缺陷。NHEJ因子XLF的缺陷也不会导致NHEJ明显的缺陷。然而，H_2AX和XLF的联合缺乏导致了NHEJ对DSB修复的严重阻碍。这些发现表明，H2AX和XLF处于代偿性DSB修复途径中，抑制其中一条途径将导致NHEJ的深刻阻断。与新墨西哥大学分子发现中心合作，我们开发了一种用于抑制NHEJ的化合物的流式细胞仪高通量筛查。这个屏幕已经过优化，并用于筛选一个小的化合物库(1,500个)，获得Z‘分数&gt；0.6。在这里，我们建议使用这种方法来筛选更大、更复杂的化学库，以识别在H2AX缺乏的细胞中特异性抑制NHEJ的化合物，但不是野生型细胞。我们将这些化合物称为NHEJ的H_2AX特异性合成抑制剂(SINS)。还将开发一种线性、多步骤方法来验证这些化合物的功能。我们相信，开发在癌细胞中选择性抑制NHEJ的药物最终将使我们能够利用DNA修复途径缺陷来治疗癌症。此外，与目前选择性抑制DSB修复的另一主要途径--癌细胞中的同源重组(HR)相比，这种方法具有显著的优势。虽然HR只在细胞分裂时参与DSB修复，但NHEJ在细胞周期G0-G1期的所有细胞中都需要DSB修复。因此，我们筛选出的抑制NHEJ的药物将有效地治疗所有癌细胞，包括绝大多数不活跃分裂的癌细胞。重要的是，癌症干细胞被认为是许多癌症治疗后复发的基础，它主要存在于G1期，也会被SINS敏化。除了识别特定于H_2AX的SINS外，这些研究的完成还将建立一条线性管道，用于未来识别用于治疗广泛DNA DSB修复蛋白缺陷的癌症的SINS。此外，他们还将识别正常细胞和癌细胞使用的新的DSB修复途径。

DESCRIPTION: Cancer cells frequently inactivate DNA double strand break (DSB) repair proteins causing genomic instability and the genetic evolution of tumors. Inactivation of these proteins usually does not cause overt defects in DNA DSB repair due to the activity of compensatory repair pathways. We reason that identifying compounds that cripple these compensatory pathways will be the first step in developing therapeutic agents that can sensitize tumor cells, but not normal tissues to genotoxic cancer therapies. The DNA damage response histone protein H2AX is a dosage-dependent tumor suppressor that exhibits reduced expression in many cancers. H2AX deficiency leads to genomic instability, but not to overt defects in DNA DSB repair by non-homologous end joining (NHEJ). Deficiency in the NHEJ factor XLF also does not lead to demonstrable defects in NHEJ. However, the combined deficiency of H2AX and XLF leads to a severe block in DSB repair by NHEJ. These findings demonstrate that H2AX and XLF are in compensatory DSB repair pathways and that inhibition of one pathway in cells deficient in the other will lead to a profound block in NHEJ. In conjunction with the University of New Mexico Center for Molecular Discover (UNMCMD), we have developed a flow cytometric high throughput screen for chemical compounds that inhibit NHEJ. This screen has been optimized and used to screen a small library of compounds (1,500) achieving Z' scores of >0.6. Here, we propose to use this approach to screen a larger, more complex chemical library to identify compounds that inhibit NHEJ specifically in H2AX-deficient, but not wild type cells. We refer to these compounds as H2AX-specific Synthetic Inhibitors of NHEJ (SINs). A linear, multistep approach to validate the function of these compounds will also be developed. We believe that developing agents that selectively inhibit NHEJ in cancer cells will ultimately enable us to exploit DNA repair pathway defects to treat cancers. Moreover, this approach has significant advantages over current efforts to selectively inhibit the other major pathway of DSB repair, homologous recombination (HR) in cancer cells. Whereas HR only functions in DSB repair in dividing cells, NHEJ is required for DSB repair in all cells in the G0-G1 phases of the cell cycle. Thus, agents identified by our screen, which inhibit NHEJ will be effective in treating all cancer cells including the vast majority that are not actively dividing. Importantly, cancer stem cells, which are thought to be the basis for relapse of many cancers after treatment, exist primarily in G1 and would also be sensitized by SINs. In addition to identifying H2AX-specific SINs, completion of these studies will establish a linear pipeline for th future identification of SINs for treating cancers defective in a broad range of DNA DSB repair proteins. Moreover, they will also identify novel DSB repair pathways used by normal cells and cancer cells.