Journal of Shanghai Jiao Tong University (Medical Science) >

2023 , Vol. 43 >Issue 5: 545 - 559

DOI: https://doi.org/10.3969/j.issn.1674-8115.2023.05.005

Innovative research team achievement column

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

  • Yanqing MEI ,
  • Yujie HAN ,
  • Wenyun WENG ,
  • Lei ZHANG ,
  • Yujie TANG
Expand
  • 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
TANG Yujie, E-mail: yujietang@shsmu.edu.cn.

Received date: 2023-03-08

  Accepted date: 2023-04-10

  Online published: 2023-07-11

Supported by

National Natural Science Foundation of China(81772655);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212700)

Fold

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

Cite this article

Yanqing MEI , Yujie HAN , Wenyun WENG , Lei ZHANG , Yujie TANG . 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 . DOI: 10.3969/j.issn.1674-8115.2023.05.005

References

1 MILLER K D, OSTROM Q T, KRUCHKO C, et al. Brain and other central nervous system tumor statistics, 2021[J]. CA Cancer J Clin, 2021, 71(5): 381-406.
2 CHOI S, YU Y, GRIMMER M R, et al. Temozolomide-associated hypermutation in gliomas[J]. Neuro Oncol, 2018, 20(10): 1300-1309.
3 OSTROM Q T, PRICE M, NEFF C, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015—2019[J]. Neuro Oncol, 2022, 24(Suppl 5): v1-v95.
4 MCKINNON C, NANDHABALAN M, MURRAY S A, et al. Glioblastoma: clinical presentation, diagnosis, and management[J]. BMJ, 2021, 374: n1560.
5 BRENNAN C W, VERHAAK R G, MCKENNA A, et al. The somatic genomic landscape of glioblastoma[J]. Cell, 2013, 155(2): 462-477.
6 MENG W, WANG J J, WANG B C, et al. CDK7 inhibition is a novel therapeutic strategy against GBM both in vitro and in vivo[J]. Cancer Manag Res, 2018, 10: 5747-5758.
7 YAN G, WANG Y F, CHEN J C, et al. Advances in drug development for targeted therapies for glioblastoma[J]. Med Res Rev, 2020, 40(5): 1950-1972.
8 NJONKOU R, JACKSON C M, WOODWORTH G F, et al. Pediatric glioblastoma: mechanisms of immune evasion and potential therapeutic opportunities[J]. Cancer Immunol Immunother, 2022, 71(8): 1813-1822.
9 BERGER T R, WEN P Y, LANG-ORSINI M, et al. World Health Organization 2021 classification of central nervous system tumors and implications for therapy for adult-type gliomas: a review[J]. JAMA Oncol, 2022, 8(10): 1493-1501.
10 ?LEDZI?SKA P, BEBYN M G, FURTAK J, et al. Prognostic and predictive biomarkers in gliomas[J]. Int J Mol Sci, 2021, 22(19): 10373.
11 PATHANIA M, DE JAY N, MAESTRO N, et al. H3.3K27M cooperates with Trp53 loss and PDGFRA gain in mouse embryonic neural progenitor cells to induce invasive high-grade gliomas[J]. Cancer Cell, 2017, 32(5): 684-700.e9.
12 HOFFMAN L M, VELDHUIJZEN VAN ZANTEN S E M, COLDITZ N, et al. Clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (DIPG): a collaborative report from the international and European society for pediatric oncology DIPG registries[J]. J Clin Oncol, 2018, 36(19): 1963-1972.
13 AZIZ-BOSE R, MONJE M. Diffuse intrinsic pontine glioma: molecular landscape and emerging therapeutic targets[J]. Curr Opin Oncol, 2019, 31(6): 522-530.
14 RASHED W M, MAHER E, ADEL M, et al. Pediatric diffuse intrinsic pontine glioma: where do we stand?[J]. Cancer Metastasis Rev, 2019, 38(4): 759-770.
15 COONEY T M, LUBANSZKY E, PRASAD R, et al. Diffuse midline glioma: review of epigenetics[J]. J Neurooncol, 2020, 150(1): 27-34.
16 MENG W, WANG B C, MAO W W, et al. Enhanced efficacy of histone deacetylase inhibitor combined with bromodomain inhibitor in glioblastoma[J]. J Exp Clin Cancer Res, 2018, 37(1): 241.
17 MO J L, TAN K Z, DONG Y, et al. Therapeutic targeting the oncogenic driver EWSR1-FLI1 in Ewing sarcoma through inhibition of the FACT complex[J]. Oncogene, 2023, 42(1): 11-25.
18 NAGARAJA S, VITANZA N A, WOO P J, et al. Transcriptional dependencies in diffuse intrinsic pontine glioma[J]. Cancer Cell, 2017, 31(5): 635-652.e6.
19 NGUYEN T T T, ZHANG Y R, SHANG E Y, et al. HDAC inhibitors elicit metabolic reprogramming by targeting super-enhancers in glioblastoma models[J]. J Clin Invest, 2020, 130(7): 3699-3716.
20 ANASTAS J N, ZEE B M, KALIN J H, et al. Re-programing chromatin with a bifunctional LSD1/HDAC inhibitor induces therapeutic differentiation in DIPG[J]. Cancer Cell, 2019, 36(5): 528-544.e10.
21 DAHL N A, DANIS E, BALAKRISHNAN I, et al. Super elongation complex as a targetable dependency in diffuse midline glioma[J]. Cell Rep, 2020, 31(1): 107485.
22 RANJAN A, PANG Y, BUTLER M, et al. Targeting CDK9 for the treatment of glioblastoma[J]. Cancers (Basel), 2021, 13(12): 3039.
23 CHOU J, QUIGLEY D A, ROBINSON T M, et al. Transcription-associated cyclin-dependent kinases as targets and biomarkers for cancer therapy[J]. Cancer Discov, 2020, 10(3): 351-370.
24 LIANG K W, GAO X, GILMORE J M, et al. Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing[J]. Mol Cell Biol, 2015, 35(6): 928-938.
25 CHOI S H, KIM S, JONES K A. Gene expression regulation by CDK12: a versatile kinase in cancer with functions beyond CTD phosphorylation[J]. Exp Mol Med, 2020, 52(5): 762-771.
26 QUEREDA V, BAYLE S, VENA F, et al. Therapeutic targeting of CDK12/CDK13 in triple-negative breast cancer[J]. Cancer Cell, 2019, 36(5): 545-558.e7.
27 WANG C, WANG H, LIEFTINK C, et al. CDK12 inhibition mediates DNA damage and is synergistic with sorafenib treatment in hepatocellular carcinoma[J]. Gut, 2020, 69(4): 727-736.
28 DIETER S M, SIEGL C, CODó P L, et al. Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancer[J]. Cell Rep, 2021, 36(3): 109394.
29 LIU H, SHIN S H, CHEN H Y, et al. CDK12 and PAK2 as novel therapeutic targets for human gastric cancer[J]. Theranostics, 2020, 10(14): 6201-6215.
30 JIANG B S, JIANG J, KALTHEUNER I H, et al. Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma[J]. Eur J Med Chem, 2021, 221: 113481.
31 BLAZEK D, KOHOUTEK J, BARTHOLOMEEUSEN K, et al. The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes[J]. Genes Dev, 2011, 25(20): 2158-2172.
32 DUBBURY S J, BOUTZ P L, SHARP P A. CDK12 regulates DNA repair genes by suppressing intronic polyadenylation[J]. Nature, 2018, 564(7734): 141-145.
33 ZHANG T H, KWIATKOWSKI N, OLSON C M, et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors[J]. Nat Chem Biol, 2016, 12(10): 876-884.
34 FENG J X, MEYER C A, WANG Q, et al. GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data[J]. Bioinformatics, 2012, 28(21): 2782-2788.
35 INIGUEZ A B, STOLTE B, WANG E J, et al. EWS/FLI confers tumor cell synthetic lethality to CDK12 inhibition in Ewing sarcoma[J]. Cancer Cell, 2018, 33(2): 202-216.e6.
36 COLLINS P L, PURMAN C, PORTER S I, et al. DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner[J]. Nat Commun, 2020, 11(1): 3158.
37 LI R, OKADA H, YAMASHITA T, et al. FOXM1 is a novel molecular target of AFP-positive hepatocellular carcinoma abrogated by proteasome inhibition[J]. Int J Mol Sci, 2022, 23(15): 8305.
38 HOPKINS J L, ZOU L. Induction of BRCAness in triple-negative breast cancer by a CDK12/13 inhibitor improves chemotherapy[J]. Cancer Cell, 2019, 36(5): 461-463.
39 NIU T, LI K L, JIANG L, et al. Noncovalent CDK12/13 dual inhibitors-based PROTACs degrade CDK12-cyclin K complex and induce synthetic lethality with PARP inhibitor[J]. Eur J Med Chem, 2022, 228: 114012.
40 YAM C Q X, LIM H H, SURANA U. DNA damage checkpoint execution and the rules of its disengagement[J]. Front Cell Dev Biol, 2022, 10: 1020643.
41 XIE J B, SHEN Z Y, ANRAKU Y, et al. Nanomaterial-based blood-brain-barrier (BBB) crossing strategies[J]. Biomaterials, 2019, 224: 119491.
42 BOBO R H, LASKE D W, AKBASAK A, et al. Convection-enhanced delivery of macromolecules in the brain[J]. Proc Natl Acad Sci U S A, 1994, 91(6): 2076-2080.
43 MADANI F, LINDBERG S, LANGEL U, et al. Mechanisms of cellular uptake of cell-penetrating peptides[J]. J Biophys, 2011, 2011: 414729.
44 HERAVI SHARGH V, LUCKETT J, BOUZINAB K, et al. Chemosensitization of temozolomide-resistant pediatric diffuse midline glioma using potent nanoencapsulated forms of a N(3)-propargyl analogue[J]. ACS Appl Mater Interfaces, 2021, 13(30): 35266-35280.
45 WU T T, LIU Y, CAO Y, et al. Engineering macrophage exosome disguised biodegradable nanoplatform for enhanced sonodynamic therapy of glioblastoma[J]. Adv Mater, 2022, 34(15): e2110364.
Options
Outlines

/

〈 〉