Inhibition of cyclin-dependent kinase 7 suppresses human hepatocellular carcinoma by inducing apoptosis.

Increasing evidence has shown that THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, exhibits therapeutic effects in various tumors. However, the possible effect of THZ1 on hepatocellular carcinoma (HCC) remains unknown. Our study was to investigate the roles of THZ1 in HCC cells and in subcutaneous HCC model and illustrate the molecular mechanisms. The phosphorylation levels of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII) C-terminal domain (CTD) and the expression levels of Ki67, Mcl-1, survivin, XIAP, and p53 in HCC cells under different conditions were examined by Western blot analysis. Cell growth and apoptosis were assessed via 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Tumor volume was assessed in HCC mice with THZ1 or vehicle treatment and immunohistochemical (IHC) analysis was conducted on excised tumors. THZ1 significantly inhibited the phosphorylation of Ser2, Ser5, and Ser7 within RNAPII-CTD in the dose-dependent and irreversible manner. MTT assay and flow cytometry analysis showed that THZ1 inhibited HCC cell proliferation and induced apoptosis, respectively. Western blot analysis indicated THZ1 significantly upregulated p53 expression and downregulated the expressions of Mcl-1, survivin, XIAP, and Ki67. THZ1 suppressed tumor growth in Hep3B xenografted mice in a time-dependent manner. IHC analysis indicated that tumors in THZ1 group had less Ki67+ cells and more cleaved caspase-3+ cells than those in vehicle group. THZ1 exhibited anti-HCC effects through irreversibly inhibiting CDK7 activity, decreasing RNAPII-CTD phosphorylation, inducing p53 expression and inhibiting antiapoptotic gene expressions, which subsequently induced apoptosis and inhibited proliferation of HCC cells.

Zhong L ，Yang S ，Jia Y ，Lei K ... -  《-》

THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are characterized by activating mutations of c-KIT or PDGFRa receptor tyrosine kinases (RTKs). Despite the clinical success of tyrosine kinase inhibitors (TKIs), more than half of GIST patients develop resistance due to a second mutation. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of CDK-activating kinase (CAK), and it plays an important role in the regulation of cell cycle transitions and gene transcription. THZ1, a CDK7 inhibitor, exhibits a dose-dependent inhibitory effect in various cancers. Data from the public GEO database and tissue microarray were used to analyse the gene expression levels of CDKs in GISTs. The impact of CDK7 knockdown and the CDK7 inhibitor THZ1 on GIST progression was investigated in vitro using CCK-8, colony formation, and flow cytometry assays and in vivo using a xenograft mouse model. RNA sequencing was performed to investigate the mechanism of GIST cell viability impairment mediated by THZ1 treatment. Our study demonstrated that CDK7 is relatively overexpressed in high-risk GISTs and predicts a poor outcome. A low concentration of THZ1 exhibited a pronounced antineoplastic effect in GIST cells in vivo and in vitro. Moreover, THZ1 exerted synergistic anticancer effects with imatinib. THZ1 treatment resulted in transcriptional modulation by inhibiting the phosphorylation of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII). c-KIT, an oncogene driver of GIST, was transcriptionally repressed by THZ1 treatment or CDK7 knockdown. Transcriptome sequencing analysis showed that OSR1 acts as a downstream target of CDK7 and regulates c-KIT expression. Taken together, our results highlight elevated CDK7 expression as a predictor of poor outcome in GIST and present the combination of CDK7 and RTK inhibitors as a potent therapeutic strategy to improve the efficacy of GIST treatment. Video abstract.

Sun J ，Zhang Q ，Sun X ，Xue A ，Gao X ，Shen K ... -  《Cell Communication and Signaling》

Xylocydine, a novel Cdk inhibitor, is an effective inducer of apoptosis in hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) frequently includes abnormalities in cell cycle regulators, including up-regulated cyclin-dependent kinase (Cdks) activities due to loss or low expression of Cdk inhibitors. In this study, we show that xylocydine, a cyclin-dependent kinase (Cdk) specific inhibitor, is a good anti-cancer drug candidate for HCC treatment. Xylocydine (50muM) selectively down-regulates the activity of Cdk1 and Cdk2, accompanied by significant cell growth inhibition in HCC cells. Xylocydine also strongly inhibits the activity of Cdk7 and Cdk9, in vitro as well as in cell cultures, that is temporally associated with apoptotic cell death in xylocydine-induced HCC cells. This is associated with inhibition of phosphorylation of RNA polymerase II at serine residues 5 and 2, which are targets of Cdk7 and Cdk9, respectively. The effects on apoptosis are concomitant with changes in the levels of anti-apoptotic proteins, Bcl-2, XIAP, and survivin, which are markedly down-regulated, and pro-apoptotic molecules, p53 and Bax, which are elevated in HCC cells after treatment with xylocydine. The up-regulated level of p53 was associated with increased stability of the protein, as levels of Ser15 and Ser392 phsophorylated p53 are similarly elevated in the inhibitor treated cells. We demonstrated that xylocydine can effectively suppress the growth of HCC xenografts in Balb/C-nude mice by preferentially inducing apoptosis in the xenografts, whereas the drug did not cause any apparent toxic effect on other tissues. Taken together, these data suggest that the novel Cdk inhibitor xylocydine is a good candidate for an anti-cancer drug for HCC therapy.

Cho SJ ，Lee SS ，Kim YJ ，Park BD ，Choi JS ，Liu L ，Ham YM ，Moon Kim B ，Lee SK ... -  《-》

The Covalent CDK7 Inhibitor THZ1 Potently Induces Apoptosis in Multiple Myeloma Cells In Vitro and In Vivo.

The goal of this study was to characterize the activity of the covalent CDK7 inhibitor THZ1 in multiple myeloma models. Multiple myeloma lines were exposed to varying THZ1 concentrations alone or with carfilzomib or ABT-199, after which apoptosis was monitored by flow cytometry, protein expression by Western blot analysis, mRNA by RT-PCR. Analogous studies were performed in cells ectopically expressing c-MYC, MCL-1, or BCL-XL, or CRISPER-Cas CDK7 sgRNA knockout. Primary multiple myeloma cells were exposed to THZ1 ± carfilzomib or ABT-199. In vivo effects of THZ1 were examined in a systemic U266 xenograft model. THZ1 markedly diminished multiple myeloma cell proliferation and survival despite bortezomib or stromal cell resistance in association with G2-M arrest, inactivation of CTD RNA Pol II, dephosphorylation of CDKs 7 as well as 1, 2, and 9, and MCL-1, BCL-xL, and c-MYC mRNA or protein downregulation. Ectopic MCL-1, c-MYC, or BCL-XL expression significantly protected cells from THZ1 lethality. Both THZ1 and CRISPR-Cas CDK7 knockout sharply diminished multiple myeloma cell proliferation and significantly increased carfilzomib and ABT-199 lethality. Parallel effects and interactions were observed in primary CD138+ (N = 22) or primitive multiple myeloma cells (CD138-/CD19+/CD20+/CD27+; N = 16). THZ1 administration [10 mg/kg i.p. twice daily (BID), 5 days/week] significantly improved survival in a systemic multiple myeloma xenograft model with minimal toxicity and induced similar events observed in vitro, for example, MCL-1 and c-MYC downregulation. THZ1 potently reduces multiple myeloma cell proliferation through transcriptional downregulation of MCL-1, BCL-XL, and c-MYC in vitro and in vivo. It warrants further attention as a therapeutic agent in multiple myeloma.

Zhang Y ，Zhou L ，Bandyopadhyay D ，Sharma K ，Allen AJ ，Kmieciak M ，Grant S ... -  《-》

Inhibition of anaplastic lymphoma kinase promotes apoptosis and suppresses proliferation in human hepatocellular carcinoma.

Yu Z ，Zhao R 《-》