在肾上腺皮质癌中评估 MELK 和 CDK 抑制剂协同疗效的临床前评估。

BACKGROUND: Adrenocortical cancer (ACC) is a rare and aggressive cancer with dismal 5-year survival due to a lack of effective treatments. We aimed to identify a new effective combination of drugs and investigated their synergistic efficacy in ACC preclinical models. METHODS: A quantitative high-throughput drug screening of 4,991 compounds was performed on two ACC cell lines, SW13 and NCI-H295R, based on antiproliferative effect and caspase-3/7 activity. The top candidate drugs were pairwise combined to identify the most potent combinations. The synergistic efficacy of the selected inhibitors was tested on tumorigenic phenotypes, such as cell proliferation, migration, invasion, spheroid formation, and clonogenicity, with appropriate mechanistic validation by cell cycle and apoptotic assays and protein expression of the involved molecules. We tested the efficacy of the drug combination in mice with luciferase-tagged human ACC xenografts. To study the mRNA expression of target molecules in ACC and their clinical correlations, we analyzed the Gene Expression Omnibus and The Cancer Genome Atlas. RESULTS: We chose the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and cyclin-dependent kinase (CDK) inhibitor (RGB-286638) because of their potent synergy from the pairwise drug combination matrices derived from the top 30 single drugs. Multiple publicly available databases demonstrated overexpression of MELK, CDK1/2, and partnering cyclins mRNA in ACC, which were independently associated with mortality and other adverse clinical features. The drug combination demonstrated a synergistic antiproliferative effect on ACC cells. Compared to the single-agent treatment groups, the combination treatment increased G2/M arrest, caspase-dependent apoptosis, reduced cyclins A2, B1, B2, and E2 expression, and decreased cell migration and invasion with reduced vimentin. Moreover, the combination effectively decreased Foxhead Box M1, Axin2, glycogen synthase kinase 3-beta, and β-catenin. A reduction in p-stathmin from the combination treatment destabilized microtubule assembly by tubulin depolymerization. The drug combination treatment in mice with human ACC xenografts resulted in a significantly lower tumor burden than those treated with single-agents and vehicle control groups. CONCLUSIONS: Our preclinical study revealed a novel synergistic combination of OTS167 and RGB-286638 in ACC that effectively targets multiple molecules associated with ACC aggressiveness. A phase Ib/II clinical trial in patients with advanced ACC is therefore warranted.

背景：肾上腺皮质癌（ACC）是一种罕见且侵袭性的癌症，由于缺乏有效治疗方法，其 5 年生存率较差。我们旨在确定一种新的有效药物组合，并在 ACC 临床前模型中研究它们的协同疗效。

方法：基于增殖抑制和半胱天冬酶-3/7 活性，对两种 ACC 细胞系 SW13 和 NCI-H295R 进行了 4991 种化合物的定量高通量药物筛选。选择候选药物进行两两组合，以确定最有效的组合。通过细胞周期和凋亡测定以及相关分子的蛋白表达，对选定抑制剂的肿瘤发生表型（如细胞增殖、迁移、侵袭、球体形成和集落形成）的协同疗效进行了测试。我们在携带荧光素酶标记的人 ACC 异种移植瘤的小鼠中测试了药物组合的疗效。为了研究 ACC 中靶分子的 mRNA 表达及其与临床的相关性，我们分析了基因表达综合数据库和癌症基因组图谱。

结果：由于从前 30 种单药衍生的药物组合矩阵中，母体胚胎亮氨酸拉链激酶（MELK）抑制剂（OTS167）和细胞周期蛋白依赖性激酶（CDK）抑制剂（RGB-286638）具有很强的协同作用，因此我们选择了这两种药物。多个公开数据库显示，MELK、CDK1/2 和伴侣细胞周期蛋白的 mRNA 在 ACC 中过度表达，这些与死亡率和其他不良临床特征独立相关。药物组合对 ACC 细胞表现出协同的增殖抑制作用。与单药治疗组相比，联合治疗组增加了 G2/M 期阻滞、半胱天冬酶依赖性凋亡、降低了细胞周期蛋白 A2、B1、B2 和 E2 的表达，并降低了细胞迁移和侵袭，同时减少了波形蛋白。此外，联合治疗还能有效降低 Foxhead Box M1、Axin2、糖原合酶激酶 3-β 和β-连环蛋白。组合治疗导致的 p-stathmin 减少通过微管蛋白解聚破坏了微管组装。在携带人 ACC 异种移植瘤的小鼠中，药物联合治疗组的肿瘤负担明显低于单药治疗组和对照组。

结论：我们的临床前研究揭示了 OTS167 和 RGB-286638 在 ACC 中的一种新的协同组合，该组合能有效靶向与 ACC 侵袭性相关的多种分子。因此，在晚期 ACC 患者中进行 Ib/II 期临床试验是必要的。