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

描述（由申请人提供）：耐药肿瘤细胞的出现是成功治疗癌症的主要限制。跨病灶合成（trans -病变Synthesis， TLS）是癌细胞获得DNA损伤耐受性和对顺铂等一线基因毒性化疗产生耐药性的主要机制。顺铂诱导大体积的链内DNA加合物，导致DNA复制叉停滞并导致细胞死亡。TLS涉及招募专门的TLS DNA聚合酶来阻止复制分叉。由于具有灵活的活性位点，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叉的进展，从而赋予DNA损伤耐受性。因为波尔吗?-介导的TLS是癌细胞逃避化疗的主要机制，长期目标是开发TLS的小分子抑制剂，以改善临床癌症治疗方法。本项目的具体目的是：(1)验证新的TLS小分子抑制剂用于癌症化疗[(2)定义癌细胞DNA损伤耐受和化疗耐药的新机制。为了支持SA1，我们开发了一种新颖灵敏的高通量筛选（HTS）方法来测量Pol？和泛素化的PCNA（一种相互作用，对肿瘤细胞的TLS和顺铂耐受性至关重要）。我们对化合物文库进行了HTS，并确定了Pol？pcna交互。在提议的工作中，我们将验证这些候选小分子作为Pol？- PCNA相互作用和评估这些抑制剂在培养癌细胞中作为化学增敏剂的效用。[为了支持SA2，我们已经确定了一种称为MAGEA4的癌症/睾丸抗原，作为肺癌细胞TLS途径的近端激活成分。因此，SA2实验试图确定MAGEA4在癌症中促进TLS并赋予DNA损伤耐受性的令人兴奋的新机制。我们将验证MAGEA4通过与E3泛素连接酶Rad18 (Pol？活动。MAGEA4介导DNA损伤耐受性和化疗耐药的假设是一个范式转移的发现，如果得到证实，将推进表达MAGEA4的化疗耐药肿瘤细胞的治疗方法。由于MAGEA4的癌细胞特异性表达，MAGEA4- rad18 - Pol？信号轴代表一个有吸引力的，可药物的目标。这项提议的工作意义重大，因为它寻求通过对DNA抗损伤癌细胞的个性化医疗的创新应用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.