PO.CL06.02 · 临床研究
抑制小儿脑肿瘤中的GSK3B信号通路
Inhibition of GSK3B signaling in pediatric brain tumors
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
基因组分析技术的进步使得对小儿胚胎性脑肿瘤的分子分类日益精确。然而，尽管采用了强化的多模式治疗，非典型畸胎样/横纹肌样瘤（ATRT）和伴多层菊形团的胚胎性肿瘤（ETMR）的五年总生存率仍低于30%。9-ING-41是一种强效GSK3B抑制剂，已被证明能够穿过血脑屏障，对其他脑肿瘤的患者来源颅内异种移植模型具有生物学活性，并在近期的I期人体临床试验中表现出良好的耐受性。此前我们证明9-ING-41通过凋亡降低ETMR和ATRT细胞活力并增强p53信号，但确切机制尚不清楚。本研究的主要目的是确定使用9-ING-41抑制GSK3B是否能通过调节p53信号通路有效抑制ATRT和ETMR的生长。ATRT和ETMR细胞系分别在DMEM和Neurocult培养基中培养24小时，随后进行9-ING-41处理。处理72小时后使用Cell Titer Glo 2.0评估细胞活力。以IC50确定的剂量处理24-72小时的细胞被收集，用于负责凋亡和p53信号分子的关键蛋白的Western blot分析。使用Qiagen RNeasy试剂盒收集细胞进行RNA分离。通过RNA测序建立转录组谱。神经球实验通过在96孔板中每孔接种两个细胞后用9-ING-41处理进行。体内异种移植模型通过颅内立体定向系统将ATRT 2141细胞原位注射至小鼠脑内，随后经腹腔（I/P）注射用溶媒或70mg/Kg 9-ING-41处理9周。使用多种ATRT和ETMR细胞系进行的体外分析显示，在临床相关剂量范围内对GSK3B抑制剂有显著的治疗反应。9-ING-41在BT183、CHLA02-ATRT、ATRT-2187、ATRT 2141中的IC50分别为145.5、481.2、503.1和528.3nM。对处理后细胞的全外显子组和RNA测序显示p53肿瘤抑制基因上调、Shh通路基因下调，支持一种肿瘤抑制机制。p53负调控因子的蛋白表达分析进一步证实，9-ING-41处理通过p53信号通路增强了凋亡通路的活化。体内研究显示，用9-ING-41处理的小鼠总生存率显著改善，生存期从35天延长至55天（P < 0.0001）。对肿瘤组织的机制评估显示p53表达增加，Ki-67显著下调，表明增殖活性降低。
查看英文原文 English abstract
Advances in genomic profiling have enabled increasingly precise molecular classification of pediatric embryonal brain tumors. However, the five-year overall survival rate for atypical teratoid/rhabdoid tumors (ATRT) and embryonal tumors with multilayered rosettes (ETMR) remains below 30%, despite intensive multimodal therapies. 9-ING-41 is a potent GSK3B inhibitor that has been shown to cross the blood-brain barrier and have biological activity against patient-derived intracranial xenograft models of other brain tumors, as well as good tolerability in recent Phase I human clinical trials. Previously we demonstrated that 9-ING-41 decreases ETMR and ATRT cell viability through apoptosis and increases p53 signaling, however the exact mechanism is unknown. The primary objective of this study was to determine whether GSK3B inhibition using 9-ING-41 could effectively suppress ATRT and ETMR growth by modulating p53 signaling pathway. ATRT and ETMR cell lines were grown for 24 hours prior to 9-ING-41 treatment in DMEM and Neurocult medium, respectively. Cell viability was assessed after 72hrs of treatment using Cell Titer Glo 2.0. Cells treated with IC50 determined dosing for 24-72 hours were collected for Western blot analysis of key proteins responsible for apoptosis and p53 signaling molecules. Cells were harvested for RNA isolation using Qiagen RNeasy kit. Transcriptomic profiles were established by RNA seq. Neurosphere assays were done by seeding two cells per well in a 96-well plate followed by treatment with 9-ING-41. In vivo xenograft models were established by orthotopic injection of ATRT 2141 cells into the mice brain through intra cranial stereotaxic system followed by treatment with either vehicle or 70mg/Kg 9-ING-41 by I/P injection for 9 weeks. In vitro analyses using multiple ATRT and ETMR cell lines revealed a significant therapeutic response to GSK3B inhibitor within clinically relevant dosing ranges. The IC50 of 9-ING-41 in BT183, CHLA02-ATRT, ATRT-2187, ATRT 2141 are 145.5, 481.2, 503.1, and 528.3nM respectively. Whole-exome and RNA sequencing of treated cells demonstrated upregulation of p53 tumor suppressor genes and downregulation of Shh pathway genes, supporting a tumor-suppressive mechanism. Protein expression analyses of p53 negative regulator further confirmed that 9-ING-41 treatment enhanced the activation of apoptotic pathways through p53 signaling pathway. In vivo studies showed a significant improvement in overall survival in mice treated with 9-ING-41 increased survival from 35 days to 55 days (P < 0.0001). Mechanistic evaluation of tumor tissues revealed increased p53 expression and marked downregulation of Ki-67, indicating reduced proliferative activity.
利益披露 Disclosure
M. Haque, None..
E. Wert, None.