上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 8: 987 - 998

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.08.001

创新团队成果专栏

弥漫内生性脑桥胶质瘤靶向治疗新策略的体外筛选与验证

  • 李瑞 ,
  • 韩雨洁 ,
  • 张蕾 ,
  • 唐玉杰
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  • 上海交通大学医学院病理生理学系，细胞分化与凋亡教育部重点实验室，上海 200025
李瑞（1995―），女，硕士生；电子信箱：lirui_9512@sina.com。
唐玉杰，电子信箱：yujietang@shsmu.edu.cn。

网络出版日期: 2021-08-13

基金资助

国家自然科学基金面上项目(81772655);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180800)

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Invitro screening and validation of novel targeted therapeutic strategy against diffuse intrinsic pontine glioma

  • Rui LI ,
  • Yu-jie HAN ,
  • Lei ZHANG ,
  • Yu-jie TANG
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  • Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
TANG Yu-jie, E-mail: yujietang@shsmu.edu.cn.

Online published: 2021-08-13

Supported by

National Natural Science Foundation of China(81772655);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180800)

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摘要

目的·从表观遗传角度寻找和鉴定弥漫内生性脑桥胶质瘤（diffuse intrinsic pontine glioma，DIPG）的单药治疗和组合靶向治疗新策略。方法·以已发表的基于8例DIPG肿瘤组织和6例正常脑组织的转录组数据为基础选择用于筛选实验的靶向小分子库。在DIPG原代细胞SU_DIPG13中进行单药筛选并寻找新的能显著抑制DIPG细胞生长的靶向小分子。通过实时定量PCR和Western blotting检测药物处理后其靶基因在mRNA和蛋白水平的表达变化。EdU和Annexin V/碘化丙啶染色后利用流式细胞术分别检测药物处理对DIPG原代细胞增殖和凋亡的影响。靶向小分子库中的药物分别与溴结构域和外端家族（bromodomain and extra terminal protein，BET）抑制剂JQ1和panobinostat组蛋白去乙酰化酶（histone deacetylase，HDAC）进行组合筛选，并体外验证对DIPG存在抑制作用的药物组合。结果·选择了包含66个小分子靶向小分子的药物库用于单药和组合筛选。单药筛选鉴定出的YM155能够显著抑制DIPG原代细胞SU_DIPG13和SU_DIPG17的生长，其靶基因BIRC5（baculoviral IAP repeat containing 5；编码survivin）在DIPG肿瘤组织中的表达高于正常组织（P=0.018）。YM155能抑制BIRC5在mRNA和蛋白水平的表达。YM155既能抑制DIPG原代细胞的增殖又能促进其凋亡。靶向小分子库中的CX4945、ABT-737分别与JQ1、panobinostat联用能在体外协同抑制DIPG细胞活性。结论·通过单药和组合药物筛选鉴定出了DIPG的靶向治疗新策略，为后续体内验证这些DIPG的新靶向治疗策略和挖掘治疗机制奠定了基础。

关键词： 弥漫内生性脑桥胶质瘤; 靶向小分子单药筛选; YM155; BIRC5基因; 药物组合筛选

本文引用格式

李瑞 , 韩雨洁 , 张蕾 , 唐玉杰 . 弥漫内生性脑桥胶质瘤靶向治疗新策略的体外筛选与验证[J]. 上海交通大学学报（医学版）, 2021 , 41(8) : 987 -998 . DOI: 10.3969/j.issn.1674-8115.2021.08.001

Abstract

Objective

·To find and identify new targeted therapeutic compounds and combinations for diffuse intrinsic pontine glioma (DIPG) from the perspective of epigenetics.

Methods

·Selection of small molecule compounds was based on the previously published transcriptome analysis of 8 cases of DIPG and 6 cases of normal brain tissues. New inhibitory compounds of DIPG were identified by single agent screening in DIPG primary tumor cells. The changes of target genes at mRNA and protein expression level were detected by real-time PCR and Western blotting after drug treatment. The effects of drug treatment on the proliferation and apoptosis of DIPG primary tumor cells were detected by FACS analyses after EdU and Annexin V/propidium iodide staining, respectively. The combinatory screening of small molecular compounds was performed with bromodomain and extra terminal protein (BET) inhibitor JQ1 or histone deacetylase (HDAC) inhibitor panobinostat, and the drug combination with inhibitory effect on DIPG was verified in vitro.

Results

·Sixty-six small molecules were chosen to be applied to screening. Single agent screening identified that YM155 could significantly inhibit DIPG primary tumor cell growth, and BIRC5 (baculoviral IAP repeat containing 5; gene encoding survivin), a target gene of YM155, was significantly upregulated in DIPG tumor tissues (P=0.018). YM155 could reduce the expression of BICR5 at both mRNA and protein levels. YM155 could repress proliferation and induce apoptosis of DIPG. CX4945 and ABT-737 from the targeted small molecular library were combined with JQ1 (BET inhibitor) and panobinostat (HDAC inhibitor), respectively,which could synergistically inhibit the activity of DIPG cells in vitro.

Conclusion

·Novel targeted therapeutic strategies for DIPG has been identified through single drug and combination drug screening, providing basis for furthervalidation in vivo and therapeutic mechanism exploration.

Key words： diffuse intrinsic pontine glioma (DIPG); single targeted small molecule compounds screening; YM155; baculoviral IAP repeat containing 5 (BIRC5); drug combinatory screening

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