Enhancing endoplasmic reticulum stress in ovarian cancer

项目摘要/摘要 卵巢癌(OCA)是美国所有妇科癌症中最致命的。尽管最初的反应是 化疗后，大多数OCA患者变得耐药并进展为转移性疾病。脑血管病的长期预后 OCA是令人沮丧的，迫切需要新的治疗策略来提高OCA患者的存活率。最快的 增殖的OCA细胞对从头合成蛋白质的需求很高，这导致了基础水平的提高。 内质网(ER)应激。事实上，内质网应激相关蛋白GRP78、PERK和ATF6是 与正常组织相比，OCA中高表达。内质网应激触发代偿性未折叠蛋白反应 (UPR)，它既可以恢复内稳态，也可以激活细胞死亡。我们推测内质网的高基础水平 OCA的压力代表着一个严重的脆弱性，而药物进一步加剧了这个已经参与其中的系统 OCA可能耗尽了其保护功能，并有助于诱导细胞凋亡。为此，我们已经确定了 一种一流的化合物，寡苯甲酰胺ERRx-208，它可以增强OCA细胞的内质网应激，但不会 原代上皮细胞。ERX-208显著减少OCA异种移植、患者来源的外植体和 患者来源的异种移植物。无偏CRISPR基因敲除筛选鉴定溶酶体酸性脂肪酶A(LIPA) 作为ERX-208关键靶点的蛋白质：LIPA的敲除取消了对ERX-208的反应，而重建 LIPA恢复ERX-208响应。我们已经证明，LIPA与ERX-208的结合对其能力至关重要 ERX-208在OCA细胞中诱导ERS和随后的细胞凋亡。这项提议的目标是进行 机制研究以确定ERX-208在OCA中的精确作用机制，以优化“铅”ERX-208- 208类似物，具有改进的药理和药代动力学特征，并验证ERX-208可以 克服了OCA的异质性。我们的主要假设是ERX-208与LIPA的结合 增强OCA中的ERS和诱导细胞凋亡，并针对OCA中的一个关键漏洞。AIM1研究将建立 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，定义 在超微结构上，eRx-208如何与LIPA结合导致ERS/UPR，定义eRx-208之间的特定相互作用 和LIPA无偏突变，并定义被eRx-208干扰的LIPA结合伙伴及其对 ERS/UPR途径。目的2研究将使用药物化学方法产生少量的ERX-208类似物用于 增强了LIPA的特异性，定义了最大耐受剂量，ERX-208的剂量范围研究，以及 确定PK、PD和毒性与治疗的比率。目的3项研究将测试ERX-208的阻断效果 临床相关异种原位移植体内OCA进展，同基因小鼠模型，患者来源 外植体、器官和患者来源的异种移植模型。我们相信，我们的研究将建立一部小说 治疗策略(增强ERS)、新的治疗靶点(LIPA)、新的治疗药物(ERX- 208/类似物)，以及晚期OCA患者进一步临床翻译的临床前理论基础。

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.