Targeting Rpa3 for cancer treatment

复制蛋白A是由Rpa1、Rpa2和Rpa3组成的异源三聚体复合体，在细胞增殖中起重要作用。作为主要的单链DNA结合蛋白，它在DNA修复、DNA重组和诱导DNA损伤检查点等过程中起着至关重要的作用。在DNA复制期间，RPA保护复制分叉中的单链DNA，并防止复制压力下的复制灾难。通过限制复制起始点的数量，ATR-Chk1途径可以防止复制分叉中的RPA全局耗尽。通过体内RNAi联合阴性选择筛选，我们确定Rpa3是治疗耐药肝癌的有希望的治疗靶点。基于癌细胞中的基本过程可能代表可用于治疗的脆弱性的理论，我们认为确定靶向RPA在治疗耐药实体肿瘤中的潜力和治疗窗口。通过在不同的癌细胞以及基于转座子的小鼠体内模型中应用Rpa3的敲除，我们可以证明抑制Rpa3可以有效地抑制肝癌和胰腺癌的生长。体外实验进一步表明，当Rpa3基因敲除与Chk1抑制相结合时，观察到显著的抗肿瘤活性，导致快速诱导肿瘤细胞死亡，但对正常细胞没有。通过应用shRpa3转基因小鼠品系，我们可以证明小鼠可以耐受9天的Rpa3基因敲除。综上所述，这些数据表明，靶向Rpa3的治疗窗口可能存在，我们开始开发Rpa3的药理抑制剂，其靶向活性和体外治疗效果已经显示出来。在这项建议中，我们的目标是表征肿瘤细胞在同时或不同时抑制Chk1的情况下对Rpa3抑制的细胞应激反应。我们进一步计划描述为什么癌症细胞和非肿瘤细胞的应激反应和治疗反应不同。为了确定靶向Rpa3在癌症治疗中的潜力，我们的目标是在基于转座子的小鼠肝癌和胰腺癌模型中进行临床前治疗研究，这些模型与shRpa3转基因小鼠相结合。这些研究将基于已确定的Rpa3基因敲除的可耐受剂量单独或与Chk1抑制联合进行。此外，我们计划在基于转座子的肝癌小鼠模型中进行体内负选择RNAi筛选，以确定参与DNA复制或DNA损伤的其他基因产物，这些基因产物可以用于治疗，或者确定其被敲除也可能与Rpa3抑制协同作用的候选基因。最后，我们的目标是进一步开发我们的药理Rpa3抑制剂，以获得用于体内研究的工具化合物。新的Rpa3抑制剂将通过详细的ADME和毒性图谱进行分析，并在体内应用于肝癌和胰腺癌的抗癌治疗。

The Replication protein A heterotrimeric complex is composed of Rpa1, 2 and 3 and plays an essential role in proliferation. As the major single-stranded DNA binding protein it is required for DNA repair, DNA recombination and induction of DNA damage checkpoints. During DNA replication Rpa protects the ssDNA in the replication forks and prohibits replication catastrophe upon replicative stress. Global exhaustion of Rpa in the replication forks is prevented by the Atr-Chk1 pathway by confining the amount of replication origins. By applying a pooled negative selection in vivo RNAi screen we identified Rpa3 as a promising therapeutic target for the treatment of therapy resistant liver carcinomas. Based on the rationale that essential processes in cancer cells might represent vulnerabilities that can be exploited therapeutically, we thought to determine the potential and the therapeutic window of targeting Rpa in therapy resistant solid tumors. By applying knockdown of Rpa3 in different cancer cells as well as transposon-based mouse models in vivo we could show that liver cancer- and pancreatic cancer growth can be efficiently decreased by Rpa3 suppression. In vitro experiments furthermore showed that a striking antitumor activity was observed when Rpa3 knockdown was combined with Chk1 inhibition, resulting in rapid induction of cell death specifically in cancer but not in normal cells. By applying a shRpa3 transgenic mouse strain, we could show that a nine-day knockdown of Rpa3 is tolerated by mice. Together, these data indicate that a therapeutic window for targeting Rpa3 may exist and we started to develop pharmacological inhibitors of Rpa3, for which on target activity and in vitro therapeutic efficacy could already be shown. In this proposal, we aim to characterize the cellular stress response of tumor cells upon Rpa3 inhibition with or without simultaneous Chk1 inhibition. We further plan to characterize why the stress- and the therapy response differ between cancerous and non-cancerous cells. To determine the potential of targeting Rpa3 in a cancer therapy, we aim to perform preclinical treatment studies in transposon-based mouse models for liver- and pancreatic cancer which are combined with shRpa3 transgenic mice. These studies will be conducted based on established tolerable doses of Rpa3 knockdown alone or in combination with Chk1 inhibition. In addition, we are planning to perform in vivo negative selection RNAi screening in a transposon-based liver cancer mouse model to either identify other gene products involved in DNA replication or DNA damage that can be exploited therapeutically or to identify candidate genes whose knockdown may also synergize with Rpa3 inhibition. Finally, we aim to further develop our pharmacological Rpa3 inhibitors to obtain a tool compound for in vivo studies. New Rpa3 inhibitors will be analyzed by detailed ADME and toxicity profiling and applied in an anti-cancer therapy of liver- and pancreatic cancer in vivo.