Novel DNA damage response therapeutics targeting replication protein A

针对复制蛋白A的新型DNA损伤反应疗法 摘要 DNA损伤反应(DDR)现在被认为是癌症治疗靶点的一条容易处理的途径。这个 DDR和DNA修复途径也适用于通过利用合成致死物质进行个性化治疗 PARP抑制剂在同源重组(HR)中的临床成功证明了相互作用 有缺陷的癌症。DDR是通过参与PI3激酶相关的ATR、ATR和DNA- PK。这些激酶和下游的检查点激酶CHK1和CHK2是临床验证的靶点 正在多个临床试验中进行积极研究。DDR途径中的一个新靶点是人类单细胞 链DNA结合蛋白、复制蛋白A(RPA)在DDR检测中起关键作用 复制应激(RS)和信号传递到ATR激酶。RS是癌细胞的共同特征，它提供了 DDR靶向药物抗癌活性的治疗窗口。RS加上DDR封锁结果 在复制灾难(RC)和最终细胞死亡中。RPA是防止RC和耗尽的关键保护器 当单链DNA结合不足时，RPA或RPA耗竭可导致RC和细胞死亡 保护基因组DNA的能力。我们已经确定了一种有效的、选择性的小分子RPA抑制剂 (RPAI)，NERX 329，具有生化RPA抑制和细胞参与RPA的作用。NERX 329 在体内也没有明显的毒性，并具有单独和与DNA结合的抗癌活性 破坏性的化疗药物和某些DDR靶向药物。而抗癌活性可能是 抑制RPA在单个修复或复制途径中的作用的结果，我们已发表的初步数据 指出一种更全球化的行动机制。我们最重要的假设是化学抑制 RPA可以模拟RPA的耗竭来抑制DDR，并通过新的 肺癌和卵巢癌中常见的基因交互作用。为了解决这一假设，我们将阐明 我们的新型RPAI的细胞抗癌活性的机制和决定因素。我们将重点关注两个方面 癌症类型，高级别浆液性卵巢上皮癌(EOC)和非小细胞肺癌(NSCLC)，AS 我们对临床生存数据的分析揭示了RPA在这些癌症中的重要作用。在目标1中，我们将 询问RPA的化学耗竭如何影响肺癌和卵巢癌的RS和DDR。目标2将 重点阐明对RPAI的敏感性的遗传决定因素。我们将确定新的化学遗传 以DDR为重点的CRISPR筛查中的相互作用评估RPAI活性。临床相关的遗传交互作用 将在我们开发的一系列独特的PD-EOC椭球线中识别。全基因组测序 将确定可能与RPAI反应相关的基因改变和染色体重排 心理治疗。从这些研究中，影响RPAI敏感性的特定基因改变将在 肺癌和卵巢癌的体内PDX模型和DDR靶向开发治疗药物。完成 这些目标将定义RPAI的作用机制和决定RPAI敏感性的遗传交互作用。 这些数据对于开发新的DDR靶向癌症疗法和确定相关的 最大限度地提高患者反应和疗效的遗传学特征。

Novel DNA damage response therapeutics targeting replication protein A Abstract The DNA damage response (DDR) is now considered a tractable pathway to target for cancer therapy. The DDR and DNA repair pathways are also amenable to personalized therapies by exploiting synthetic lethal interactions as evidenced by the clinical success of PARP inhibitors in homologous recombination (HR) deficient cancers. The DDR is initiated by engagement of the PI3 kinase-related kinases ATM, ATR, and DNA- PK. These kinases and the downstream checkpoint kinases CHK1 and CHK2, are clinically validated targets being actively investigated in multiple clinical trials. A novel target in the DDR pathway is the human single stranded DNA binding protein, replication protein A (RPA) which plays a critical role in the DDR to detect replication stress (RS) and signal to the ATR kinase. RS is a common feature in cancer cells and provides the therapeutic window for the anticancer activity of DDR targeted drugs. RS coupled with a DDR blockade results in replication catastrophe (RC) and eventually cell death. RPA is a critical protector from RC and depletion of RPA or “RPA exhaustion” can elicit RC and cell death when there is insufficient single-strand DNA binding capacity to protect the genomic DNA. We have identified a potent and selective small molecule RPA inhibitor (RPAi), NERx 329, which possesses biochemical RPA inhibition and cellular engagement of RPA. NERx 329 also displays no overt toxicity in vivo and possesses anticancer activity alone and in combination with DNA damaging chemotherapeutics and certain DDR targeted agents. While the anticancer activity could be the result of inhibiting RPA’s role in individual repair or replication pathways, our published and preliminary data point to a more global mechanism of action. Our overarching hypothesis is that chemical inhibition of RPA can mimic RPA exhaustion to inhibit the DDR and provide selective anticancer activity via novel genetic interactions common in lung and ovarian cancer. To address this hypothesis, we will elucidate the mechanisms and determinants of the cellular anticancer activity of our novel RPAi. We will focus on two cancers types, high grade serous epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC), as our analysis of clinical survival data reveals an important role for RPA in these cancers. In Aim 1 we will interrogate how chemical exhaustion of RPA impacts RS and the DDR in lung and ovarian cancer. Aim 2 will focus on elucidating the genetic determinants of sensitivity to RPAi’s. We will identify novel chemical-genetic interactions in a DDR focused CRISPR screen assessing RPAi activity. Clinically relevant genetic interactions will be identified in a unique series of PD-EOC spheroid lines we have developed. Whole genome sequencing will identify genetic alterations and chromosomal rearrangements which may correlate with response to RPAi therapy. From these studies, specific genetic alterations that impact RPAi sensitivity will be validated with in vivo PDX models of lung and ovarian cancer and DDR targeted developmental therapeutic agents. Completion of these aims will define RPAi mechanism of action and the genetic interactions that dictate RPAi sensitivity. These data are crucial towards developing novel DDR targeted cancer therapeutics and identifying the relevant genetics characteristics to maximize patient response and efficacy.