Exploration of Genome Stability as a Therapeutic Target in Cancer

描述（由申请人提供）：在许多癌症中观察到的基因组不稳定性被认为产生了驱动肿瘤发生的突变（Hanahan和温伯格2000）。然而，导致这种不稳定性的缺陷可能是癌细胞的脆弱性， 特异性靶向并被化疗杀死（D. A. Chan和Giaccia 2011）。在存在导致高于正常水平的DNA损伤的缺陷的情况下，细胞可能变得依赖于补偿途径，该途径减少DNA损伤的影响并防止细胞激活死亡途径。如果这些非必需的补偿机制现在在癌细胞中是必需的，那么灭活这些机制的治疗将以基因型特异性的方式特异性地杀死癌细胞。这种靶向本质上是一种基于治疗的合成致死形式，当两个基因单独失活具有最小影响时观察到，但同时失活两者导致细胞生长减少或细胞死亡。聚（ADP-核糖）聚合酶（PARP）抑制剂对BRCA 1和BRCA 2基因缺陷癌症的成功治疗表明，在双链断裂（DSB）修复缺陷的背景下，合成致死性启发的方法可能是成功的（Bryant et al. 2005; Farmer et al. 2005）。PARP的失活不太可能是BRCA 1和BRCA 2缺陷型癌症的唯一靶向机制。然而，系统地筛选BRCA 1和BRCA 2缺陷型癌细胞存活所需的其他基因或途径既昂贵又具有技术挑战性，这是因为需要高通量实验，难以鉴定真正的致死条件， 以及被敲除的基因的不完全失活。在这里，我将使用与酵母酿酒酵母中DSB修复途径突变的遗传相互作用来鉴定BRCA 1和BRCA 2缺陷癌细胞系存活所需的候选人类基因。考虑到真核DNA修复机制的高度保守性（Aggarwal和Brosh 2012），我将使用以前鉴定的合成致死性相互作用以及新的 鉴定了引起基因组不稳定性增加和对DNA损伤剂敏感性增加的相互作用，以鉴定感兴趣的酵母基因。然后，我将使用siRNA敲除这些酵母基因的人类同源物，以确定是否在人类癌细胞系中存在等效的相互作用。最后，我将探索在人类癌细胞系中验证的这些相互作用是否可以用作诱导细胞死亡的靶点。我希望这些实验将提供一种合理的方法来确定一组高度富集的新靶点，用于未来的治疗开发。

DESCRIPTION (provided by applicant): The genome instability seen in many cancers is thought to generate the mutations that drive tumorigenesis (Hanahan and Weinberg 2000). The defects that lead to this instability, however, may be a vulnerability by which cancer cells can be specifically targeted and killed by chemotherapies (D. A. Chan and Giaccia 2011). In the presence of defects causing higher than normal levels of DNA damage, cells may become reliant upon compensating pathways that reduce the impact of the DNA damage and prevent the cells from activating death pathways. If these non-essential compensating mechanisms are now essential in the cancer cells, then treatments inactivating these mechanisms would specifically kill cancer cells in a genotype-specific fashion. This targeting is essentially a therapy-based form of synthetic lethality, which is observed when inactivation of two genes individually has minimal effect, but simultaneous inactivation of both results in decreased cell growth or cell death. The success of poly(ADP-ribose) polymerase (PARP) inhibitors against cancers with defects in the BRCA1 and BRCA2 genes demonstrates that a synthetic lethality inspired approach can be successful in the context of defects in double-strand break (DSB) repair (Bryant et al. 2005; Farmer et al. 2005). Inactivation of PARP is unlikely to be the only mechanism by which BRCA1 and BRCA2 deficient cancers can be targeted. Systematic screening for additional genes or pathways that are required for BRCA1 and BRCA2 deficient cancer cell survival, however, is both expensive and technically challenging due to the requirements for a high-throughput experiment, difficulties in identifying truly lethal conditions, and incomplete inactivation of genes that are knocked down. Here, I will use genetic interactions with mutations in the DSB repair pathway in the yeast Saccharomyces cerevisiae to identify candidate human genes required for the survival of BRCA1 and BRCA2 deficient cancer cell lines. Given the highly conserved nature of the eukaryotic DNA repair mechanisms (Aggarwal and Brosh 2012), I will use previously identified synthetic lethality interactions as well as newly identified interactions giving rise to increased genome instability and increased sensitivity to DNA damaging agents to identify yeast genes of interest. I will then use siRNA knockdown of human homologs of these yeast genes to determine whether equivalent interactions exist in human cancer cell lines. Finally, I will explore if those interactions validated in human cancer cell lines can be used as a target to induce cell death. I expect these experiments will provide a rational approach to identify a highly enriched set of new targets for future therapeutic development.