Targeting BRCA Deficient Cells for Killing

描述(申请人提供)：同源重组(HR)是促进复制细胞中DNA准确修复的主要细胞途径。因此，该途径的正确功能对于抑制染色体不稳定和癌症是必要的。例如，导致DNA HR修复缺陷的中央HR因子BRCA1或BRCA2(BRCA1/2)的突变极易使女性患乳腺癌和卵巢癌。因此，了解HR的作用机制对于开发新的乳腺癌和卵巢癌治疗方法具有重要意义。以前的研究已经揭示了BRCA1/2缺陷细胞的特殊特征，这些特征为策略提供了如何在保留正常细胞的同时，特定地针对这些细胞进行杀伤。例如，新的研究表明，BRCA1/2基因缺陷的细胞，如乳腺癌和卵巢癌细胞的亚群，依赖RAD52-哺乳动物细胞中的一种备用HR因子-来生长和存活。相比之下，精通BRCA1/2的正常细胞不需要RAD52。事实上，正常的人类细胞和缺乏RAD52的小鼠不会表现出任何明显的表型。另一方面，同时缺乏RAD52和BRCA1/2的细胞无法生长和存活(合成致死)，因为它们不能通过HR充分修复自己的染色体。总之，这些新的研究表明，抑制RAD52活性可以特异性地杀死BRCA1/2缺陷的癌细胞，但对正常细胞没有影响。因此，专门抑制RAD52活性的药物很可能针对BRCA1/2缺陷的癌细胞进行杀伤，同时保留正常细胞。由于这些药物对正常细胞没有影响，它们将使一种无毒的乳腺癌和卵巢癌治疗成为可能。此外，由于BRCA1/2的缺陷已知会促进乳腺癌和卵巢癌的亚群，因此可以开发针对BRCA1/2缺陷的癌前细胞进行杀伤的药物来防止这些疾病的发生。RAD52如何促进BRCA1/2缺陷细胞的存活？最近的研究表明，RAD52与单链DNA(SsDNA)结合是BRCA1/2缺陷细胞生存所必需的。因此，我们的目标是寻找抑制RAD52单链DNA结合的药物，并针对BRCA缺陷细胞进行杀伤：1.进行RAD52抑制剂的高通量筛选；2.使用二级、三级、正交和表面等离子体共振分析验证RAD52抑制剂；3.鉴定和优化特异性靶向BRCA缺陷细胞杀伤的RAD52抑制剂。在预赛中 为了证明这种方法的可行性，我们进行了有限的高通量药物筛选，以确定能够特异性杀死BRCA2缺陷细胞的RAD52抑制剂，同时保留精通BRCA2的细胞。这些积极的初步结果表明，拟议研究的成功完成将导致新的靶向乳腺癌和卵巢癌治疗。

DESCRIPTION (provided by applicant): Homologous recombination (HR) is a major cellular pathway that promotes the accurate repair of DNA in replicating cells. Thus, the proper function of this pathway is necessary for suppressing chromosome instability and cancer. For example, mutations of central HR factors BRCA1 or BRCA2 (BRCA1/2), which cause defects in HR repair of DNA, strongly predispose women to breast and ovarian cancer. Understanding the mechanisms of HR is therefore important for the development of new breast and ovarian cancer therapies. Previous studies have revealed particular characteristics of BRCA1/2 deficient cells that inform strategies how to specifically target these cells for killing, while sparing normal cels. For example, new studies show that cells defective in BRCA1/2, such as subsets of breast and ovarian cancer cells rely upon RAD52-a backup HR factor in mammalian cells-for their growth and survival. In contrast, normal cells that are proficient in BRCA1/2 do not require RAD52. In fact, normal human cells and mice that are deficient in RAD52 do not exhibit any apparent phenotypes. On the other hand, cells that are deficient in both RAD52 and BRCA1/2 are unable to grow and survive (synthetic lethal) since they cannot sufficiently repair their chromosomes via HR. Together, these new studies demonstrate that suppression of RAD52 activity specifically kills BRCA1/2 deficient cancer cells, but has no effect on normal cells. Thus, drugs that specifically inhibit RAD52 activity are likely to target BRCA1/2 deficient cancer cells for killing while sparing normal cells. Since such drugs would have no effect on normal cells, they would enable a non-toxic form of breast and ovarian cancer treatment. Moreover, because defects in BRCA1/2 are known to promote subsets of breast and ovarian cancers, drugs that target BRCA1/2 deficient pre-cancerous cells for killing may be developed to prevent the onset of these diseases. How does RAD52 promote the survival of BRCA1/2 deficient cells? Recent studies demonstrate that RAD52 binding to single-strand DNA (ssDNA) is necessary for the survival of BRCA1/2 deficient cells. We therefore aim to identify drugs that inhibit RAD52 ssDNA binding and target BRCA deficient cells for killing by developing the following Aims: 1. To perform high-throughput screening for RAD52 inhibitors; 2. To validate RAD52 inhibitors using secondary, tertiary, orthogonal, and surface plasmon resonance assays; 3. To identify and optimize RAD52 inhibitors that specifically target BRCA deficient cells for killing. In preliminary studies, we demonstrate the feasibility of this approach by performing limited high- throughput drug screening to identify RAD52 inhibitors that specifically kill BRCA2 deficient cells while sparing cells proficient in BRCA2. These positive preliminary results indicate that successful completion of the proposed research will lead to new targeted breast and ovarian cancer therapies.