ATR inhibitor-mediated reversal of PARP inhibitor resistance in high-grade serous ovarian cancer (HGSOS)

摘要 高级别浆液性卵巢癌(HGSOC)通常存在DNA修复途径缺陷 同源重组(HR)修复缺陷以及停滞的DNA复制的稳定性受损 叉子。这种缺陷经常涉及BRCA改变，并使其对聚(ADP-核糖)的抑制剂敏感。 聚合酶(PARP)抑制剂。这些特工现在已经进入了HGSOC的常规医疗机构， 高级和维护设置。这个项目的首要目标是解决新出现的问题 随着PARP抑制剂使用的增加，PARP抑制剂的耐药性将变得更加重要。该项目 重点是抑制毛细血管扩张性共济失调和RAD3相关(ATR)，以此作为逆转这两种疾病的策略 获得性PARP抑制剂耐药的主要机制，包括HR修复的恢复和 DNA复制分叉。提出了三个具体目标。在目标1中，我们将评估活动和机制 ATR抑制剂AZD6738作为单用药以及与PARP抑制剂奥拉帕利合用的体外和体内实验 体内BRCA突变细胞系、器官培养和患者来源的HGSOC异种移植(PDX)模型 获得性PARP抑制剂耐药性。这项工作将为AIM 2结合AZD6738进行临床试验铺平道路 和奥拉帕利，在耐PARP抑制剂的BRCA患者中最大限度地抑制ATR- 突变的HGSOC。一旦确定了最大耐受量，我们将确认耐受性并进行评估 在12名患者的扩大队列中的初步抗肿瘤活性。配对的活组织检查将用于 用免疫组织化学RAD51评估HR的机制验证药效学研究 在有机物培养中，通过DNA纤维分析来评估检测和复制分叉的稳定性。在《目标3》中，我们将 高度测定ATR单独及联合吉西他滨对HGSOCs的抑制活性 但本质上对PARP抑制没有反应。AZD6738和吉西他滨 在具有高水平CCNE1或MYC扩增的细胞系、有机物和PDX模型中进行了研究。我们将分析 这些模型中的复制应激及其通过ATR抑制和吉西他滨的应用而加剧 ATR途径激活的免疫组织化学、细胞学和基因表达标志物。最后， 我们将利用最近完成的吉西他滨与吉西他滨与ATR抑制剂联合使用的研究 M6620在对铂类耐药的HGSOC患者中的应用，其中联合应用在 无铂间隔不到3个月。我们将检验这一组富含肿瘤的假设 携带由CCNE1或MYC扩增以及生物标记物定义的高度复制压力 在临床前模型中开发。综上所述，这些目标将使我们能够确立抑制ATR的作用 在HGSOC医疗机构，为预后较差的HGSOC人群确定新的治疗途径 并为PARP抑制剂耐药的生物学提供了洞察力。

SUMMARY High-grade serous ovarian cancer (HGSOC) frequently harbors defects in DNA repair pathways that confer homologous recombination (HR) repair deficiency, as well as compromised stability of stalled DNA replication forks. Such defects frequently involve BRCA alterations and confer sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) inhibitors. These agents have now entered the routine HGSOC armamentarium both in the advanced and maintenance settings. An overarching objective of this project is to address the emerging problem of PARP inhibitor resistance that will assume greater importance as PARP inhibitor use increases. The project focuses on inhibition of Ataxia telangiectasia and Rad3-related (ATR) as a strategy designed to reverse the two major mechanisms of acquired PARP inhibitor resistance, including restoration of HR repair and stabilization of DNA replication forks. Three Specific Aims are proposed. In Aim 1, we will assess the activity and mechanisms of the ATR inhibitor AZD6738 as monotherapy and in combination the PARP inhibitor olaparib in in vitro and in vivo BRCA-mutated cell line, organoid culture and patient-derived xenograft (PDX) models of HGSOC with acquired PARP inhibitor resistance. This work will prepare the way for a clinical trial in Aim 2 combining AZD6738 and olaparib with a schedule that maximizes ATR inhibition in patients with PARP inhibitor-resistant BRCA- mutated HGSOC. Once the maximum tolerated doses are established, we will confirm tolerability and assess preliminary antitumor activity in an expansion cohort of twelve patients. Paired biopsies will be procured for proof-of-mechanism pharmacodynamic studies in which HR is assessed with an immunohistochemical RAD51 assay and replication fork stability is assessed with a DNA fiber assay in organoid cultures. In Aim 3, we will determine the activity of ATR inhibition alone and in combination with gemcitabine in HGSOCs with a high degree of replication stress but are intrinsically unresponsive to PARP inhibition. AZD6738 and gemcitabine will be studied in cell line, organoid and PDX models harboring high-level CCNE1 or MYC amplification. We will analyze replication stress in these models and its exacerbation by ATR inhibition and gemcitabine using immunohistochemical, cytological and gene expression signature biomarkers of ATR pathway activation. Lastly, we will leverage a recently completed study of gemcitabine vs. gemcitabine combined with the ATR inhibitor M6620 in platinum-resistant HGSOC patients, in which the combination was superior among patients with a platinum-free interval of less than 3 months. We will test the hypothesis that this group was enriched with tumors carrying a high degree of replicative stress, defined by CCNE1 or MYC amplification, as well as by biomarkers developed in the preclinical models. Taken together, these aims will allow us to establish a role for ATR inhibition in the HGSOC armamentarium, identify new therapeutic avenues for HGSOC populations with poor prognosis and provide insights into the biology of PARP inhibitor resistance.