PO.ET07.02 · 实验与分子治疗
肝细胞对肿瘤学药物化合物毒性的高通量高多重蛋白质组学分析：一个用于毒性评估、免疫风险预测和加速药物开发的平台
High-throughput high-plex proteomic profiling of hepatocyte toxicity to oncologic drug compounds: A platform for toxicity assessment, immune risk prediction, and drug development acceleration
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摘要 Abstract
中文摘要
癌症药物开发的快速且必要的扩展需要可及且可扩展的策略用于早期毒性筛查和免疫风险分析。肝脏在药物代谢、解毒和免疫调节中发挥核心作用。对肝脏特异性响应肿瘤学治疗药物的稳健检测对于识别和减轻可能限制临床开发的肝毒性和免疫相关不良事件（irAEs）至关重要。在此，我们展示了一个高通量、高多重蛋白质组学平台，作为评估肝细胞在一系列癌症药物作用下功能扰动的强大方法。利用基于nELISA技术的新型Omni 1000蛋白质组学解决方案，我们在超过550种不同药物化合物（每种化合物3个剂量）暴露后的>6500份原代人肝细胞样本中分析了1,000种蛋白质。Omni 1000蛋白质靶点内容可同时定量应激反应标志物、炎症细胞因子、代谢酶和信号通路效应因子。这使得能够评估细胞毒性和免疫调节两方面的特征。用tripolide、doxorubicin和cobimetinib进行扰动导致剂量依赖性细胞毒性，通过检测释放到上清液中的细胞内蛋白（如GAPDH和Casp3）水平升高而发现，这与临床观察到的肝毒性一致。我们还在非毒性扰动剂量下检测到Galectin-3、Galectin-1和VEGFA升高，提示细胞应激和在更高剂量下潜在的毒性。此外，我们同时观察到与抗凋亡保护相关的蛋白质丢失，如ST6GAL1，一种FAS介导凋亡的强效抑制剂。在筛选的510种化合物中，68种显示出类似的毒性迹象，包括在临床上具有剂量限制性肝毒性的化合物。特别值得注意的是PHA-793887，一种潜在的治疗候选药物，由于意外的肝毒性在临床上失败，而此处在临床前体外模型中捕获到了这一点。我们进一步证明了该平台通过检测关键细胞因子的剂量依赖性增加来识别可能的irAEs的能力。用Imatinib和Pomalidomide扰动的肝细胞显示IL-6、IL-10和IL-2增加，提示潜在的irAE并与这些药物的已发表文献一致。肝细胞反应的高通量高多重蛋白质组学分析在临床前毒理学和免疫安全性方面提供了重大进展，并能够对药物性肝损伤（DILI）风险和免疫毒性潜力进行早期指示，这是肿瘤学药物开发中的两大主要障碍。
查看英文原文 English abstract
The rapid and necessary expansion of cancer drug development requires accessible and scalable strategies for early toxicity screening and immune risk profiling. Liver plays a central role in drug metabolism, detoxification, and immune modulation. Robust detection of liver-specific responses to oncologic therapeutics is essential to identify and mitigate hepatotoxicity and immune-related adverse events (irAEs) which could limit clinical development. Here, we demonstrate a high-throughput, high-plex proteomic platform as a powerful approach to evaluate functional perturbations of hepatocytes across a spectrum of cancer drugs.Utilizing the novel Omni 1000 proteomic solution, based on nELISA technology, we profiled 1,000 proteins in >6500 samples of primary human hepatocytes following exposure to a diverse panel of over 550 distinct drug compounds at 3 doses per compound. Omni 1000 protein target content enables simultaneous quantification of stress response markers, inflammatory cytokines, metabolic enzymes, and signaling pathway effectors. This allows for evaluation of both cytotoxic and immunomodulatory signatures. Perturbation with tripolide, doxorubicin, and cobimetinib resulted in dose dependent cell toxicity, detected via increased levels of intracellular proteins such as GAPDH and Casp3 released into the supernatant, consistent with clinically observed hepatotoxicity. We also detect increased Galectin-3, Galectin-1, and VEGFA at non-toxic perturbation doses, indicative of cell stress and potential toxicity at higher doses. In addition, we concurrently observe loss of proteins associated with protection against apoptosis, such as ST6GAL1, a potent inhibitor of FAS-mediated apoptosis. Across the 510 compounds screened, 68 showed similar signs of toxicity, including compounds with dose-limiting liver toxicity in the clinic. Of particular note is PHA-793887, a potential therapeutic candidate, which failed clinically due to unexpected hepatotoxicity, which here was captured in a pre-clinical in vitro model.We further demonstrated the platform's ability to identify likely irAEs through detection of dose dependent increases in key cytokines. Hepatocytes perturbed with Imatinib and Pomalidomide demonstrated increases in IL-6, IL-10, and IL-2, indicative of potential irAE and consistent with published literature for these drugs.High-throughput high-plex proteomic analysis of hepatocyte responses offers a significant advance in preclinical toxicology and immune safety, and enables an early stage indication of drug-induced liver injury (DILI) risk and immunotoxic potential, two major barriers in oncology drug development.
利益披露 Disclosure
N. Paul, None.
N. Chang,
Nomic Bio Employment.