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

doi:10.3969/j.issn.1674-8115.2023.05.005

Abstract

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

CLC Number:

R739.41

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.

Figures/Tables 8

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References 45

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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

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

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

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

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