靶向抑制CDK12/13在高级别胶质瘤中的体外治疗效果和作用分子机制探究

doi:10.3969/j.issn.1674-8115.2023.05.005

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (5): 545-559.doi: 10.3969/j.issn.1674-8115.2023.05.005

• 创新团队成果专栏 • 上一篇

靶向抑制CDK12/13在高级别胶质瘤中的体外治疗效果和作用分子机制探究

梅艳青¹(), 韩雨洁¹, 翁文筠¹, 张蕾¹^,², 唐玉杰¹^,²()

^1.上海交通大学医学院病理生理学系，上海 200025
^2.上海交通大学医学院组织胚胎学与遗传发育学系，上海 200025

收稿日期:2023-03-08 接受日期:2023-04-10 出版日期:2023-05-28 发布日期:2023-07-11
通讯作者: 唐玉杰 E-mail:yanqingmei@sjtu.edu.cn;yujietang@shsmu.edu.cn
作者简介:梅艳青（1998—），女，硕士生；电子信箱：yanqingmei@sjtu.edu.cn。
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(SHSMU-ZDCX20212700)

In vitro therapeutic effects and molecular mechanisms of targeted inhibition of CDK12/13 in high-grade gliomas

MEI Yanqing¹(), HAN Yujie¹, WENG Wenyun¹, ZHANG Lei¹^,², TANG Yujie¹^,²()

^1.Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Department of Histoembryology and Genetic Development, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2023-03-08 Accepted:2023-04-10 Online:2023-05-28 Published:2023-07-11
Contact: TANG Yujie E-mail:yanqingmei@sjtu.edu.cn;yujietang@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81772655);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212700)

摘要/Abstract

摘要：

目的·筛选高级别胶质瘤（high-grade gliomas，HGGs）共有的表观转录靶向治疗新策略，并进行体外治疗效果的测试与相关分子机制的探究。方法·对HGGs中恶性程度和致死率均较高的多个胶质母细胞瘤（glioblastoma，GBM）和弥漫性内生性脑桥胶质瘤（diffuse intrinsic pontine glioma，DIPG）细胞系进行表观转录相关的靶向小分子药物库筛选和基于CRISPR-Cas9系统的功能基因组筛选，以寻找在GBM和DIPG中共同的表观转录靶向治疗新策略。然后针对筛选得到的目标表观转录调控因子，分别测试经CRISPR-Cas9方法敲除该基因以及对应的靶向小分子处理对GBM和DIPG细胞系的体外生长、细胞增殖与凋亡的影响。继而对靶向小分子处理的GBM和DIPG细胞系进行RNA-seq转录组分析，解析其抗肿瘤分子机制。基于此分析结果，通过实时荧光定量PCR（quantitative real-time PCR，RT-qPCR）、蛋白质印迹法以及流式细胞术进一步验证目标表观转录调控因子的抗肿瘤分子机制。结果·针对表观转录调控因子的靶向小分子药物库筛选和功能基因组筛选鉴定出CDK12和CDK13（CDK12/13）是GBM和DIPG共同的潜在治疗新靶点。在多个GBM和DIPG细胞系中，通过CRISPR-Cas9方法敲除CDK12能够显著降低其体外细胞活性。CDK12/13抑制剂SR-4835或THZ531也均能够通过拮抗细胞增殖和促进细胞凋亡造成这2类HGGs细胞系的体外生长受到显著抑制。SR-4835处理GBM和DIPG细胞之后的RNA-seq转录组分析结果表明，HGGs细胞中受CDK12/13抑制剂作用而表达显著下调的基因主要富集在转录调控、DNA损伤反应（DNA damage response，DDR）通路、泛素-蛋白酶体通路以及细胞周期等生物学过程。进一步通过一系列实验方法验证了在DIPG和GBM中，靶向抑制CDK12/13显著下调DDR相关基因的转录进而引起DNA损伤的累积，并诱导细胞发生G2-M细胞周期阻滞。结论·CDK12/13是GBM和DIPG这2类HGGs共同的潜在表观转录靶向治疗靶标；该发现为后续体内模型验证、组合治疗测试提供了理论支持，也为未来进一步的临床转化应用奠定了基础。

关键词: 胶质母细胞瘤, 弥漫性内生性脑桥胶质瘤, 分子靶向治疗, 抗肿瘤药物库筛选, CRISPR-Cas9, CDK12, CDK13

Abstract:

Objective ·To find novel and common targeting strategies for high-grade gliomas (HGGs) from the perspective of epigenetic and transcriptional modulators, test the therapeutic effect in vitro and investigate the related molecular mechanisms. Methods ·Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) cell lines with high malignancy and mortality in HGGs were selected for screening of targeted small molecule drug library related to epigenetic transcription and for functional genome screening based on the CRISPR-Cas9 technology. The effect of selected targeted epigenetic transcriptional modulators on growth, proliferation, and apoptosis of GBM and DIPG cell lines were then measured either by CRISPR-Cas9 knockout or treatment with targeted small molecule inhibitors of genes in vitro. Anti-tumor molecular mechanisms of the modulators in corresponding small molecule inhibitors-treated GBM and DIPG cells were explored via RNA-seq transcriptome analysis and further verified by real time quantitative PCR (RT-qPCR), Western blotting and flow cytometry. Results ·Targeted small molecule drug library combined with functional genome screening for epigenetic transcriptional modulators identified CDK12/13 as the novel therapeutic targets for both GBM and DIPG. Knockout out of CDK12 by CRISPR-Cas9 in multiple GBM and DIPG cell lines significantly reduced their in vitro cellular activity. CDK12/13 inhibitors SR-4835 and THZ531 also significantly inhibited the growth of these two types of HGGs cell lines in vitro by antagonizing cell proliferation and promoting cell apoptosis. RNA-seq transcriptome analysis of GBM and DIPG cell lines after SR-4835 treatment showed that genes significantly down-regulated by CDK12/13 inhibitors in HGGs cells were mainly enriched in transcriptional regulation, DNA damage response (DDR) pathway, ubiquitin-proteasome pathway, and cell cycle. Furthermore, a series of experiments demonstrated that targeted inhibition of CDK12/13 significantly down-regulated the transcription of DDR-related genes, resulting in the accumulation of DNA damage, and induced G2-M cell cycle arrest. Conclusion ·CDK12/13 is a common potential therapeutic target of these two types of HGGs, providing theoretical support for the follow-up in vivo verification and combination therapy test. The research also lays the foundation for further clinical application.

Key words: glioblastoma (GBM), diffuse intrinsic pontine glioma (DIPG), molecular targeted therapy, anti-tumor drug screening, CRISPR-Cas9, CDK12, CDK13

中图分类号:

R739.41

梅艳青, 韩雨洁, 翁文筠, 张蕾, 唐玉杰. 靶向抑制CDK12/13在高级别胶质瘤中的体外治疗效果和作用分子机制探究[J]. 上海交通大学学报（医学版）, 2023, 43(5): 545-559.

MEI Yanqing, HAN Yujie, WENG Wenyun, ZHANG Lei, TANG Yujie. In vitro therapeutic effects and molecular mechanisms of targeted inhibition of CDK12/13 in high-grade gliomas[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(5): 545-559.

图/表 8

表1 sgRNA oligo序列

Tab 1 Oligo sequences of sgRNA

Primer	Sequence (5'→3')
sgGFP forward	CACCGGGGCGAGGAGCTGTTCACCG
sgGFP reverse	AAACCGGTGAACAGCTCCTCGCCCC
sgCDK12-2 forward	CACCGACTGACCGACTGCCTTCT
sgCDK12-2 reverse	AAACCGAGAAGGCAGTCGGTCAGTC
sgCDK12-3 forward	CACCGATTCACCAGTTCAGTATCTG
sgCDK12-3 reverse	AAACCAGATACTGAACTGGTGAATC

表2 RT-qPCR引物序列

Tab 2 Primer sequence for RT-qPCR

Primer	Forward (5'→3')	Reverse (5'→3')
ATM	GCTGACAATCATCACCAAGT	GGTTCTCAGCACTATGGGACA
ATR	CGCTGAACTGTACGTGGAAA	CAATTAGTGCCTGGTGAACATC
BRCA1	CTGCTCAGGGCTATCCTCTCA	GCTTCTAGTTCAGCCATTTCCTG
CCNK	AAAGCCATGTTGGTACTGGGA	TGTAGCCCCAAACGTGTGC
E2F1	CATCAGTACCTGGCCGAGAG	CCCGGGGATTTCACACCTTT
FANCD2	CCCAGAACTGATCAACTCTCCT	CCATCATCACACGGAAGAAA
FANCI	CACCACACTTACAGCCCTTG	ATTCCTCCGGAGCTCTGAC
RAD51	GCTGATGAGTTTGGTGTAGCAG	GGAAGACAGGGAGAGTCGTAGA
SMARCC2	GAGGGATAAGCAGGTTCTTCTG	GGGATCCACGTGTCGTAACT
TP53	CCCAAGCAATGGATGATTTGA	GCATTCTGGGAGCTTCATCT
GAPDH	TGACTTCAACAGCGACACCCA	CACCCTGTTGCTGTAGCCAAA
dGAPDH	CGTTCATGCCACCACCGCTA	CCACGTCCATCACGCCACAA

图1 基于表观转录靶向小分子药物库与功能基因组筛选GBM和DIPG共同的潜在药物靶标—— CDK12/13Note： A. Workflow of epigenetic transcription-related targeted small molecule drug library screening in aGBM (U251), pGBM (SF188) and DIPG (DIPG_13F, DIPG_BJ02) cell lines. B. Venn diagram of the epigenetic transcription-related targeted small molecule drug library screening in U251, SF188, DIPG_13F, and DIPG_BJ02 cell lines. C. Heatmap was used to display the screening effects of 17 small molecule classifications. D. Scatter plots showing the dependence of GBM cells on CDK12, CDK13 and CDK9 in DepMap database. E. Epigenetic transcription-related functional genome screening based on CRISPR-Cas9 in vitro for four weeks in DIPG_BJ02 cell line, with the sequencing results analyzed by MAGeCK algorithm. The top 200 genes are thought to be growth-dependent genes in DIPG_BJ02 cell line. F. Box plot of log (fold change) in sgCDK12, sgCDK13 and sgCDK9. NTC—negative control. ①P<0.000 1, ②P=0.001 8, ③P=0.002 9.

Fig 1 Screening for common potential drug targets for GBM and DIPG based on epigenetic transcription-related targeted small molecule drug library and functional genome: CDK12 and CDK13

图2 CDK12 敲除对DIPG和GBM肿瘤细胞体外生长的抑制作用Note： A. Western blotting analysis of CDK12 expression levels in aGBM (U251), pGBM (SF188, KNS42), and DIPG17 cell lines after CDK12 knockout by using CRISPR-Cas9 system. B. Detection of cell viability of U251, SF188 and KNS42 on the fourth day, and DIPG17 on the seventh day after CDK12 knockout by CellTiter-Glo. C. Dot plot (upper) and bar chart (below) of neurosphere assay of DIPG17 cells. Fifty or 100 DIPG17 cells were seeded in each well (96-well plate) following CDK12 knockout. After 14 d of culture, the number of neurosphere was counted and mapped. D. Scanogram (left) and cartogram (right) of colony formation assay of CDK12-knockout GBM cell lines (U251, SF188 and KNS42). Cells were plated at 2 000 per well (six well plate), with half-volume mediumeing changed every 4 d, followed by crystal violet staining, photographing and counting in two weeks. ①P=0.000 2, ②P=0.000 4, ③P=0.000 1, ④P=0.008 7, ⑤P=0.001 4, ⑥P=0.004 0, ⑦P=0.002 9,⑧P<0.000 1.

Fig 2 Inhibitory effect of CDK12 knockout on the growth of DIPG and GBM tumor cells in vitro

图3 CDK12/13抑制剂对DIPG和GBM体外生长的抑制作用Note： A. Dosage-dependent cell viability curves of CDK12/13 inhibitor SR-4835 or THZ531-treated GBM or DIPG cell lines. B. Detection of proliferation and apoptosis of DIPG17 cells treated with DMSO or SR-4835 (0.1, 1 μmol/L) by flow cytometry. C. Detection of proliferation and apoptosis of SF188 cells treated with DMSO or SR-4835 (0.1, 1 μmol/L) by flow cytometry. D. Detection of proliferation and apoptosis of U251 cells treated with DMSO or SR-4835 (0.1, 1 μmol/L) by flow cytometry. Cell proliferation was detected by EdU-flow cytometry on 24 h and apoptosis was detected by Annexin V/PI-flow cytometry on 48 h.

Fig 3 Inhibitory effect of CDK12/13 inhibitors on the growth of DIPG and GBM in vitro

图4 靶向抑制CDK12/13显著降低GBM和DIPG细胞DDR相关基因的富集Note： A. Venn diagram showing differentially expressed genes (Gfold<-1) of 0.1 μmol/L or 1 μmol/L SR-4835 versus DMSO-treated DIPG17 and SF188 cell lines. B. GO analysis on enriched function of down-regulated genes in DIPG17 and SF188 cell lines treated with 0.1 μmol/L or 1 μmol/L SR-4835. C. GSVA analysis of enriched gene sets in DIPG17 and SF188 cell lines. D. Heatmap analysis of RNA-seq data showing transcript level expression alteration of representative DDR-related genes after SR-4835 treatment.

Fig 4 Targeted inhibition of CDK12/13 significantly reduced the enrichment of gene sets involved in DDR in GBM and DIPG cells

图5 靶向抑制CDK12/13通过显著下调GBM和DIPG细胞DDR相关基因的转录引起DNA损伤的积累Note： A. RT-qPCR results showed that SR-4835 or THZ531 treatment for 20 h had effects on the expression of genes related to DNA damage repair in DIPG17, SF188, and U251 cell lines. B. Western blotting results showed the effect of SR-4835 and THZ531 treatment on protein levels of γH2AX and RNA Pol Ⅱ S2P in DIPG17, SF188, and U251 cell lines. ①P=0.001 8, 0.1 μmol/L SR4835 versus DMSO; ②P>0.05, 0.1 μmol/L SR4835 versus DMSO. Except for the P values shown in ① and ②, all other P values are less than 0.000 1 (0.1 μmol/L or 1 μmol/L SR4835 versus DMSO, 0.5 μmol/L or 5 μmol/L THZ531 versus DMSO).

Fig 5 Targeted inhibition of CDK12/13 induced accumulation of DNA damage by significantly down-regulating transcription of genes involved in DDR in GBM and DIPG cells

图6 靶向抑制CDK12/13诱导DIPG和GBM细胞发生G2-M细胞周期阻滞Note： A. GO analysis on enriched function of down-regulated genes in DIPG17 and SF188 cells. B. Heatmap showing GSVA analysis of enriched ubiquitin and cell cycle-related gene sets in DIPG17 and SF188 cells. C. Flow cytometry and bar charts analyses on cell cycle of U251, SF188, and DIPG17 cells treated with DMSO, 0.1 μmol/L SR-4835 or 1 μmol/L SR-4835 for 24 h.

Fig 6 Targeted inhibition of CDK12/13 induced G2-M cell cycle arrest in DIPG and GBM cells

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靶向抑制CDK12/13在高级别胶质瘤中的体外治疗效果和作用分子机制探究

In vitro therapeutic effects and molecular mechanisms of targeted inhibition of CDK12/13 in high-grade gliomas

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