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Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation., (© 2021. The Author(s).)

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood-brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.

Glioblastoma (GBM) is a highly infiltrative brain cancer, which is thus difficult to operate. GBM cells frequently harbor Epidermal Growth Factor Receptor amplification (EGFRwt) and/or activating mutation (EGFRvIII), generating at least two different cellular subpopulations within the tumor. We examined the relationship between the diffusive architectures of GBM tumors and the paracrine interactions between those subpopulations. Our aim was to shed light on what drives GBM cells to reach large cell-cell distances, and whether this characteristic can be manipulated. We established a methodology that quantifies the infiltration abilities of cancer cells through computation of cell-cell separation distance distributions in 3D. We found that aggressive EGFRvIII cells modulate the migration and infiltrative properties of EGFRwt cells. EGFRvIII cells secrete HGF and IL6, leading to enhanced activity of Src protein in EGFRwt cells, and rendering EGFRwt cells higher velocity and augmented ability to spread. Src inhibitor, dasatinib, at low non-toxic concentrations, reduced the infiltrative properties of EGFRvIII/EGFRwt neurospheres. Furthermore, dasatinib treatment induced compact multicellular microstructure packing of EGFRvIII/EGFRwt cells, impairing their ability to spread. Prevention of cellular infiltration or induction of compact microstructures may assist the detection of GBM tumors and tumor remnants in the brains and improve their surgical removal.

Chordoid glioma (ChG) is a characteristic, slow growing, and well-circumscribed diencephalic tumor, whose mutational landscape is unknown. Here we report the analysis of 16 ChG by whole-exome and RNA-sequencing. We found that 15 ChG harbor the same PRKCA D463H mutation. PRKCA encodes the Protein kinase C (PKC) isozyme alpha (PKCα) and is mutated in a wide range of human cancers. However the hot spot PRKCA D463H mutation was not described in other tumors. PRKCA D463H is strongly associated with the activation of protein translation initiation (EIF2) pathway. PKCα D463H mRNA levels are more abundant than wild-type PKCα transcripts, while PKCα D463H is less stable than the PCKα WT protein. Compared to PCKα WT , the PKCα D463H protein is depleted from the cell membrane. The PKCα D463H mutant enhances proliferation of astrocytes and tanycytes, the cells of origin of ChG. In conclusion, our study identifies the hallmark mutation for chordoid gliomas and provides mechanistic insights on ChG oncogenesis.