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

描述（申请人提供）：耐药肿瘤细胞的出现是癌症成功治疗的主要限制。跨病变合成（TLS）是癌细胞获得DNA损伤耐受性和对一线遗传毒性化疗（如顺铂）的抗性的主要机制。顺铂诱导大的链内DNA加合物，其引起DNA复制叉停滞并导致细胞死亡。TLS涉及将专门的TLS DNA聚合酶募集到停滞的复制叉。由于灵活的活性位点，TLS DNA聚合酶（如DNA聚合酶eta或Polη）可以复制受损的基因组并维持复制叉进展，从而赋予DNA损伤耐受性。由于Polη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善癌症治疗的临床方法。本项目的具体目的是：（1）验证用于癌症化疗的新型TLS小分子抑制剂[（2）确定癌细胞中DNA损伤耐受和化疗耐药的新机制。]为了支持SA 1，我们开发了一种新的和敏感的高通量筛选（HTS）测定，用于测量Polv和泛素化PCNA之间的关联（一种对癌细胞中TLS和顺铂耐受性至关重要的相互作用）。我们已经对化合物文库进行了HTS，并鉴定了Polη-PCNA相互作用的候选抑制剂。在所提出的工作中，我们将验证这些候选小分子作为Polη- PCNA相互作用的抑制剂，并评估这些抑制剂作为培养的癌细胞中的化学增敏剂的效用。[为了支持SA 2，我们已经鉴定了称为MAGEA 4的癌症/睾丸抗原作为肺癌细胞中TLS途径的近端活化组分。因此，SA 2的实验试图确定MAGEA 4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MAGEA 4通过与E3泛素连接酶Rad 18（Polη活性的上游调节因子）相互作用刺激TLS的假设。MAGEA 4介导DNA损伤耐受性和化学抗性的假设是一个范式转变的发现，如果得到证实，将推进表达MAGEA 4的化学抗性肿瘤细胞的治疗方法。由于MAGEA 4的癌细胞特异性表达，MAGEA 4-Rad 18- Polη信号传导轴代表了有吸引力的可药用靶标。这项工作意义重大，因为它试图通过创新应用个性化药物治疗DNA损伤抗性癌细胞来改善传统的癌症疗法。

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 Polη) can replicate damaged genomes and maintains replication fork progression, thereby conferring DNA damage tolerance. Because Polη-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 Polv 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 Polη-PCNA interaction. In the proposed work we will validate those candidate small molecules as inhibitors of the Polη- 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 Polη 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- Polη 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.