Increasing the Efficacy of Cancer Therapy Via Inhibition of DNA Damage Tolerance

描述(申请人提供)：抗药性肿瘤细胞的出现是成功治疗癌症的主要限制因素。跨损伤合成(TLS)是肿瘤细胞获得DNA损伤耐受性和对顺铂等一线基因毒性化疗药物耐药的主要机制。顺铂诱导巨大的链内DNA加合物，导致DNA复制分叉停滞并导致细胞死亡。TLS涉及将专门的TLS DNA聚合酶招募到停滞不前的复制分叉上。由于活性部位灵活，TLS DNA聚合酶(如DNA聚合酶ETA或Pol？)可以复制受损的基因组并维持复制分叉进程，从而增强DNA损伤耐受性。由于POL？介导的TLS是癌细胞逃避化疗的主要机制，因此长期目标是开发TLS的小分子抑制剂，以改进癌症治疗的临床方法。本项目的具体目标是：(1)验证用于癌症化疗的新型TLS小分子抑制剂[(2)确定癌细胞对DNA损伤耐受和化疗耐药的新机制。]为了支持SA1，我们开发了一种新的、灵敏的高通量筛选(HTS)方法，用于测量POL？以及泛素化的增殖细胞核抗原(这种相互作用对于TLS和顺铂在癌细胞中的耐受是必不可少的)。我们已经对化合物文库进行了HTS，并确定了POL？-增殖细胞核抗原相互作用的候选抑制剂。在这项拟议的工作中，我们将验证这些候选小分子作为POL？-增殖细胞核抗原相互作用的抑制剂，并评估这些抑制剂在培养的癌细胞中作为化学增敏剂的有效性。[为了支持SA2，我们已经确定了一种名为MAGEA4的癌症/睾丸抗原，它是肺癌细胞中TLS途径的近端激活成分。因此，在SA2中的实验试图确定MAGEA4促进TLS并赋予癌症DNA损伤耐受性的令人兴奋的新机制。我们将检验MAGEA4通过与E3泛素连接酶Rad18的相互作用来刺激TLS的假设，Rad18是Pol？活动。MAGEA4介导DNA损伤耐受和化疗耐药的假设是一个范式转换的发现，如果得到证实，将推动MAGEA4表达的化疗耐药肿瘤细胞的治疗方法。由于MAGEA4在癌细胞中的特异性表达，MAGEA4-Rad18-Pol？信号轴代表了一个有吸引力的、可下药的靶子。这项拟议的工作意义重大，因为它寻求通过创新地应用抗DNA损伤的癌细胞的个性化药物来改进传统的癌症治疗方法。

DESCRIPTION (provided by applicant): The emergence of drug-resistant tumor cells is a major limitation to the successful treatment of cancer. Trans-Lesion Synthesis (TLS) is a major mechanism by which cancer cells acquire DNA damage tolerance and resistance to front line genotoxic chemotherapies such as cisplatin. Cisplatin induces bulky intra-strand DNA adducts that cause DNA replication fork stalling and lead to cell death. TLS involves the recruitment of specialized TLS DNA polymerases to stalled replication forks. Due to flexible active sites, TLS DNA polymerases (such as DNA polymerase eta or Pol?) can replicate damaged genomes and maintains replication fork progression, thereby conferring DNA damage tolerance. Because Pol?-mediated TLS is a major mechanism by which cancer cells evade chemotherapy, the long-term goal is to develop small molecule inhibitors of TLS to improve clinical approaches to cancer treatment. The Specific Aims of this project are: (1) To validate novel small molecule inhibitors of TLS for cancer chemotherapy [(2) To define a novel mechanism of DNA damage tolerance and chemoresistance in cancer cells.] In support of SA1 we have developed a novel and sensitive High-Throughput Screening (HTS) assay for measuring association between Pol? and ubiquitinated PCNA (an interaction that is essential for TLS and cisplatin tolerance in cancer cells). We have performed HTS of compound libraries and have identified candidate inhibitors of the Pol?-PCNA interaction. In the proposed work we will validate those candidate small molecules as inhibitors of the Pol?- PCNA interaction and evaluate the utility of these inhibitors as chemosensitizers in cultured cancer cells. [ In support of SA2 we have identified a Cancer/Testes Antigen termed MAGEA4 as a proximal activating component of the TLS pathway in lung cancer cells. Therefore, experiments in SA2 seek to define the exciting, new mechanism by which MAGEA4 promotes TLS and confers DNA damage tolerance in cancer. We will test the hypothesis that MAGEA4 stimulates TLS via an interaction with the E3 ubiquitin ligase, Rad18, an upstream regulator of Pol? activity. The hypothesis that MAGEA4 mediates DNA damage tolerance and chemoresistance is a paradigm-shifting discovery and if proven will advance the therapeutic approaches in MAGEA4-expressing, chemoresistant tumor cells. Owing to the cancer cell-specific expression of MAGEA4, the MAGEA4-Rad18- Pol? signaling axis represents an attractive, druggable target. The proposed work is significant because it seeks to improve traditional cancer therapies through the innovative application of personalized medicine for DNA damage-resistant cancer cells.