Enhancing endoplasmic reticulum stress in ovarian cancer

项目总结/摘要 卵巢癌（OCa）是美国所有妇科癌症中最致命的。尽管初步反应， 化疗后，大多数OCa患者变得耐药并进展为转移性疾病。远期预后 OCa令人沮丧，迫切需要新的治疗策略来提高OCa患者的生存率。迅速 增殖性OCa细胞对从头蛋白质合成有很高的需求，这导致基础水平的提高 内质网（ER）应激。事实上，ER-应激相关蛋白GRP 78、PERK和ATF 6是 与正常组织相比，在OCa中过度表达。内质网应激触发代偿性未折叠蛋白反应 (UPR)，它可以恢复体内平衡或激活细胞死亡。我们推断，高基础水平的ER OCa中的压力代表了一种严重的脆弱性，而进一步加剧这一已经参与的系统的药物， OCa可能耗尽其保护功能，并有助于诱导细胞凋亡。为此，我们确定了 一种一流的化合物，低聚苯甲酰胺ERX-208，它可以增强OCa细胞的ER应激，但不能增强OCa细胞的ER应激。 原代上皮细胞ERX-208显著降低OCa异种移植物、患者来源的外植体和 患者来源的异种移植物。无偏CRISPR敲除筛选鉴定了溶酶体酸性脂肪酶A（利帕） 蛋白质作为ERX-208的关键靶标：利帕的敲低消除了对ERX-208的应答，而重建 的利帕恢复ERX-208响应。我们已经表明，LIPA与ERX-208的结合对于LIPA与ERX-208的结合能力至关重要。 ERX-208诱导OCa细胞中的ERS和随后的凋亡。这项建议的目的是进行 机制研究，以确定ERX-208在OCa中的确切作用机制，以优化“先导”ERX-208。 208类似物的药理学和药代动力学特征，并验证ERX-208可以 克服OCa的异质性。我们的首要假设是ERX-208与利帕的结合 增强ERS并诱导OCa的凋亡，并靶向OCa的关键脆弱性。AIM 1研究将建立 使用无偏转录组学和蛋白质组学研究OCa细胞中ERX-208的分子相关性， 在超微结构上，ERX-208与利帕结合如何引起ERS/UPR，定义ERX-208之间的特异性相互作用。 和利帕进行无偏诱变，并确定被ERX-208破坏的利帕结合配偶体及其对 ERS/UPR途径。目的2研究将使用药物化学方法来产生很少的ERX-208类似物， 增强对利帕的特异性，确定最大耐受剂量，ERX-208的剂量范围研究，以及 确定PK、PD和毒性与治疗比。目的3研究将测试ERX-208在阻断 使用临床相关原位异种移植物、同基因小鼠模型、患者源性 外植体、类器官和患者来源的异种移植物模型。我们相信我们的研究将建立一个新的 治疗策略（增强ERS）、新的治疗靶点（利帕）、新的治疗剂（ERX- 208/类似物）和进一步临床转化为晚期OCa患者的临床前基本原理。

Project Summary/Abstract Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Despite initial response to chemotherapy, most OCa patients become resistant and progress to metastatic disease. Long-term prognosis of OCa is dismal, and new therapeutic strategies are desperately needed to improve OCa patient survival. The rapidly proliferative OCa cells have a high demand for de novo protein synthesis, which results in an enhanced basal level of endoplasmic reticulum (ER) stress. Indeed ER-stress associated proteins GRP78, PERK, and ATF6 are overexpressed in OCa compared to normal tissues. ER stress triggers the compensatory unfolded protein response (UPR), which can either restore homeostasis or activate cell death. We reasoned that the high basal level of ER stress in OCa represents a critical vulnerability, and drugs that further aggravate this already engaged system in OCa may exhaust its protective features and contribute to apoptosis induction. Towards that end, we have identified a first-in-class compound, the oligobenzamide ERX-208, which enhances ER stress in OCa cells but not in primary epithelial cells. ERX-208 significantly reduced the growth of OCa xenografts, patient-derived explant, and patient-derived xenografts. Unbiased CRISPR knockdown screens identified the lysosomal acid lipase A (LIPA) protein as the critical target of ERX-208: knockdown of LIPA abrogates response to ERX-208, while reconstitution of LIPA restores ERX-208 response. We have shown that the binding of LIPA to ERX-208 is critical for the ability of ERX-208 to induce ERS and subsequently apoptosis in OCa cells. The objective of this proposal is to conduct mechanistic studies to identify the precise mechanism of action of ERX-208 in OCa, to optimize the “lead” ERX- 208 analog with improved pharmacologic and pharmacokinetic features and to validate that ERX-208 can overcome the heterogeneity of OCa. Our overarching hypothesis is that the binding of ERX-208 to LIPA enhances ERS and induces apoptosis in OCa and targets a critical vulnerability in OCa. Aim1 studies will establish molecular correlates of ERX-208 in OCa cells using unbiased transcriptomics and proteomics, define ultrastructurally how ERX-208 binding to LIPA causes ERS/UPR, define specific interactions between ERX-208 and LIPA with unbiased mutagenesis and define LIPA binding partners disrupted by ERX-208 and their effect on ERS/UPR pathways. Aim 2 studies will use medicinal chemistry approaches to generate few ERX-208 analogs for enhanced specificity for LIPA, define the maximum tolerated dose, dose-ranging studies for ERX-208, and determine PK, PD, and toxicity to therapeutic ratios. Aim 3 studies will test the efficacy of ERX-208 in blocking OCa progression in vivo using clinically relevant orthotopic xenograft, syngeneic mouse models, patient-derived explant, organoids and patient-derived xenograft models. We believe that our studies will establish a novel therapeutic strategy (enhancing ERS), novel therapeutic target (LIPA), novel therapeutic agents (ERX- 208/analogs) and the preclinical rationale for further clinical translation for patients with advanced OCa.