Abstract
High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase–quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.
Highlights
No effective therapy exists for high-grade glioma (HGG), an incurable brain cancer
Low THZ1 doses plus the spliceosome inactivation of several tumor suppressors such as Cdkn2a/b, inhibitor pladienolide B. These findings further define a promising nanomolar concentrations; Supplementary Figure S8e) resulted in therapeutic approach that, instead of targeting these pathways supra-additive inhibition of HGG cell line growth (Figure 6f). singularly using multiple agents, THZ1 or similar agents may be. These results conclusively show that THZ1 has profound effects used to simultaneously negate several active RTK and oncogenic on the small Cajal body-associated scaRNA population and pathways irrespective of differentially altered induces aberrant mRNA splicing by disrupting nuclear speckle somatic profiles
We show that cyclin-dependent kinase 7 (CDK7) is crucial for gliomagenesis, confirm that CDK7 is a new target for HGG therapy and provide the first description of the cellular processes affected by the CDK7 and nuclear speckles), with irregular spatiotemporal translocation of nucleolar and Cajal body components to identical areas
Summary
No effective therapy exists for high-grade glioma (HGG), an incurable brain cancer. Numerous advanced small molecule inhibitors and antibodies have failed in clinical trials. Two important drivers of gliomagenesis, RNA transcription and the cell cycle, are efficiently negated by THZ1 Another clinically important axis for gliomagenesis is the receptor tyrosine kinase (RTK)/phosphatidylinositol 3 kinase/mitogen-activated protein kinase pathway, which is perturbed in 90% of patient tumors.[10] Further analysis of our microarray data indicated that genes encoding several important and druggable RTKs, including the epidermal growth factor receptor (Egfr) gene, the platelet-derived growth factor receptor-α (Pdgfra) gene and the Met gene (the most commonly altered RTKs in HGG), were expressed at lower levels in THZ1-treated cells (Supplementary Tables S2 and S3). When THZ1 action was compared with actinomycin D (100 nM) and palbociclib (1 μM) for production of snoRNA and scaRNA, we found that the increase in these non-coding RNAs was specific for THZ1 only after 24 h of drug treatment and was not the result of a generalized transcription or cell cycle inhibition (Supplementary Figures S8a and b) These results show that sites of mature spliceosome function are grossly disrupted but remain spatially dissociated from the fibrillarin/dyskerin/coilin-containing foci after THZ1 treatment. We discovered extensive damage to the subnuclear compartments housing these RNAs
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