Despite advances in genetic alteration diagnoses and tyrosine kinase inhibitor treatment, the prognosis of patients with GIST remains unsatisfactory, especially for high-risk GISTs. The effectiveness of standard targeted therapy is hampered by secondary resistance following initial responses due to acquired secondary mutations.
CDKs play a significant role in regulating the cell cycle and gene transcription. CDK7 is one of the subunits of the multiprotein transcription factor complex TFIIH and it is critical for facilitating transcription initiation and elongation via phosphorylation of the CTD of RNAPII[19, 20]. CDK7 has been reported to be a potential therapeutic target in transcription-dependent cancers and is associated with a poor prognosis[21, 24–28]. However, the role of CDK7 in GIST tumorigenesis remains unknown.
In our study, we conducted an analysis of the transcriptomic data of 65 GIST patients from the GEO public database. The CDK4, CDK7 and CDK9 mRNA levels were significantly higher than those of other CDKs, and further, we found that the CDK7 mRNA level was significantly elevated in high-risk GISTs, while there was no significant difference in CDK4 and CDK9. Previously, Yu Liu analysed quantitative proteome profiling of GIST and adjacent normal tissue and found that several kinases were significantly upregulated in GIST, including KIT and CDK7. Consistent with this, we validated with tissue microarrays (TMAs) that CDK7 overexpression in GISTs was correlated with tumour progression and an unfavourable prognosis. Inhibition of CDK7 or CDK7 inhibitor treatment significantly inhibited GIST cell proliferation. These results revealed that CDK7 might play an oncogenic role in GIST progression.
GISTs exhibit a homogeneous repertoire of transcription factors, which supports its associated gene expression program throughout all stages of the disease. Several core transcription factors have been revealed to play essential roles in driving GIST cell proliferation and metastasis by binding to enhancers of GIST-associated genes and facilitating KIT gene expression[13–15]. Moreover, a previous study revealed that the KIT-regulated enhancer domain in GISTs could be targeted by BRD4, a key activator of RNAPII transcription at active chromatin marks, and the BET bromodomain inhibitor (BBI) can downregulate KIT transcription[34, 35]. Therefore, characterization of transcription factor deregulation in GIST may provide innovative insights into the pathogenesis mechanisms and offer new therapeutic approaches.
In our study, RNA-seq analysis was used to detect alterations in total transcripts in GIST cells after treatment with THZ1. We observed that a cluster of genes was particularly sensitive to THZ1 treatment and was mainly enriched in biological processes of transcription regulation mediated by RNAPII. Considering the original genetic alteration of GIST, we investigated the transcriptional activity and protein expression of c-kit after CDK7 knockdown or THZ1 treatment. Interestingly, we found that c-kit transcription and protein expression were significantly inhibited after CDK7 knockdown or THZ1 treatment in both GIST T1 and 882 cells. This result indicated that CDK7 might be a key driver of c-kit expression in GIST and that it is a possible therapeutic target.
CDK inhibitors are of great interest to explore as novel therapeutic agents against cancer and several CDK inhibitors have been applied in the clinic. CDK4/6 inhibitors have gained FDA approval for the treatment of hormone receptor-positive breast cancer, and inhibitors targeting other cell cycle CDKs are currently in clinical trials for non-small cell lung cancer and other solid tumours. THZ1, as a CDK7 inhibitor, exerts synergistic anticancer effects when combined with TKIs against neuroblastoma, glioma and non-small-cell lung cancer[32, 37–39]. Therefore, targeting CDK7 may provide an alternative therapeutic option to block the reactivation of receptor tyrosine kinase pathways in RTK-driven neoplasms, especially for TKI-resistant cancer. Our research revealed that a combination of THZ1 and imatinib exerts synergistic antitumour effects in GIST cells. Both THZ1 and imatinib treatment led to c-kit expression inhibition, and the combination treatment enhanced the inhibition of c-kit expression and the downstream AKT and ERK signalling pathways. Taken together, our results indicate that THZ1 has effective antitumour activity against GISTs and may provide an additional therapeutic strategy for GIST patients with a poor response to imatinib.
Super-enhancer (SE) is a large cluster of genomic regulatory elements typically exhibiting an enrichment of histone H3 lysine 27 (H3K27ac) and densely bound by transcription factors and cofactors, playing critical roles in defining cell fate and identity. Interestingly, superenhancers frequently drive the expression of prominent oncogenes in cancer cells. Previous research has used H3K27ac chromatin immunoprecipitation with sequencing (ChIP-seq) of GIST tumour samples and cell lines and identified the SE clusters that drive c-KIT gene expression and are unique to GISTs . Subsequently, studies indicated that the SE domain was essential for c-KIT gene expression and tumorigenesis, including FOXF1, HAND1 and BARX1[13, 14]. Disruption of the SE domain represents a therapeutic vulnerability in GIST. Among the genes screened out by ChIP-seq of GIST tumour samples and cell lines, OSR1 was hypothesized to bind to the c-KIT locus by ATAC sequencing. In our research, we found that CDK7 knockdown significantly inhibited the expression of c-KIT, and we identified the genes downregulated after THZ1 treatment by RNA-seq. Interestingly, we found that OSR1 was the predominantly downregulated gene. Therefore, we hypothesized that CDK7 knockdown inhibited the expression of c-KIT via OSR1. Subsequently, we proved that OSR1 expression was inhibited by THZ1 treatment in a dose-dependent manner and that OSR1 knockdown also significantly inhibited c-KIT expression. Moreover, OSR1 overexpression reversed the inhibition of c-KIT expression induced by CDK7 knockdown. In summary, our research may have revealed the role of OSR1 in c-KIT expression attenuated by CDK7 inhibition.
In summary, our results uncovered the positive correlations between CDK7 and the malignant potential of GISTs and indicated that targeting CDK7 with the selective inhibitor THZ1 may be a promising treatment for GIST patients.