PO.ET03.07 · 实验与分子治疗
确定葡萄膜黑色素瘤中ONC212耐药的机制以开发联合治疗策略
Determining mechanisms of ONC212-resistance in uveal melanoma to develop combination therapy strategies
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
葡萄膜黑色素瘤（UM）是成人中最常见的原发性眼癌，在美国每年影响约2,500人。约50%的UM患者发生转移，主要转移至肝脏（>95%），导致预后不良。转移性UM（mUM）仅有两种FDA批准的疗法，且缓解率较低。迫切需要开发新的治疗策略以实现有效缓解。我们的初步研究表明mUM具有高水平的氧化磷酸化（OXPHOS）。用imipridone类化合物ONC212间接靶向OXPHOS，在原位肝mUM小鼠模型中显著抑制了UM细胞存活、降低了肿瘤负荷并改善了生存。然而，延长治疗导致肿瘤复发，提示获得性耐药。因此，ONC212在mUM中生长抑制效应的成功转化取决于对ONC212耐药机制和新靶点的鉴定。本研究中，我们生成并表征了多个ONC212耐药的UM细胞克隆。通过用高剂量ONC212（0.3 μM）长期处理，从MM28和MP46亲本UM细胞生成了三十多个ONC212耐药克隆。进行细胞活力检测以计算ONC212的IC50和耐药指数（RI），并使用Seahorse Mito Stress Test评估线粒体功能。采用全基因组测序（WGS）鉴定耐药克隆中的获得性突变，同时通过反相蛋白质芯片（RPPA）进行蛋白质组学分析以确定细胞信号通路的改变。完成了高通量药物筛选以鉴定ONC212耐药细胞对已批准抗癌药物的敏感性。用耐药细胞生成原位肝-mUM小鼠模型，并通过生物发光成像监测肿瘤生长。ONC212耐药克隆表现出比亲本细胞更高的IC50和RI，并显示出对其他imipridones（ONC201、ONC206）的交叉耐药，提示共享的耐药机制。耐药克隆对ONC212介导的OXPHOS抑制的敏感性降低。每个耐药克隆的WGS均证实CLPP（ONC212在UM细胞中的作用靶点）存在突变。RPPA数据揭示了多条细胞信号通路的改变。肝mUM体内模型的生物发光成像显示ONC212耐药克隆具有快速增殖能力。利用这些临床前模型和药物筛选中鉴定的化合物，未来将测试和验证联合治疗策略以用于临床研究。重要的是，首个imipridone ONC201最近已被批准用于治疗中线胶质瘤。因此，本研究的经验教训未来也可能使获得ONC201耐药的患者受益。
查看英文原文 English abstract
Uveal melanoma (UM) is the most common primary eye cancer in adults, affecting ~2,500 people annually in USA. Metastasis in ~50% of UM patients is predominantly to the liver (>95%), resulting in poor prognosis. Only two FDA-approved therapies exist for metastatic UM (mUM), with poor response rates. There is an urgent need for developing new therapeutic strategies to achieve effective response. Our preliminary studies indicated that mUM has high Oxidative Phosphorylation (OXPHOS). Indirect targeting of OXPHOS with the imipridone compound ONC212, significantly inhibited UM cell survival, reduced tumor burden and improved survival in orthotopic liver mUM mouse models. However, extended treatment resulted in tumor recurrence, suggesting acquired resistance. Thus, successful translation of the growth inhibitory effect of ONC212 in mUM depends on identification of ONC212 resistance mechanisms and new targets. In this study, we generated and characterized multiple ONC212-resistant UM cell clones. More than thirty ONC212-resistant clones were generated from MM28 and MP46 parental UM cells via prolonged treatment with high-dose ONC212 (0.3 μM). Cell viability assays were done to calculate IC 50 and resistance index (RI) for ONC212, and mitochondrial function was assessed using the Seahorse Mito Stress Test. Whole-genome sequencing (WGS) was used to identify acquired mutations in resistant clones, while proteomic profiling via Reverse Phase Protein Array (RPPA) was used to determine alterations in cell signaling pathways. A high throughput drug screen was completed to identify sensitivities of ONC212-resistant cells to approved anticancer drugs. Orthotopic liver-mUM mouse models were generated with resistant cells and growth of tumors monitored by bioluminescence imaging. ONC212-resistant clones exhibited higher IC 50, and RI compared to parental cells and showed cross-resistance to other imipridones (ONC201, ONC206), suggesting shared mechanisms of resistance. Resistant clones displayed reduced sensitivity to ONC212-mediated OXPHOS inhibition. WGS of each resistant clone confirmed mutations in CLPP, the target of ONC212 action in UM cells. RPPA data revealed alterations in multiple cell signaling pathways. Bioluminescence imaging of liver mUM in vivo models revealed rapid proliferative capacity of ONC212-resistant clones. Using these preclinical models and the compounds identified in drug screen, combination therapy strategies will be tested and validated for clinical studies in future. Importantly, ONC201, the first imipridone was recently approved for therapy of midline gliomas. Therefore, lessons learned from this study may benefit patients acquiring resistance to ONC201 in future as well.
利益披露 Disclosure
M. Alauddin, None..
C. Chattopadhyay, None.