TG02 is a novel cyclin‐dependent kinase (CDK) inhibitor and thought to act mainly via CDK‐9 inhibition‐dependent depletion of short‐lived oncoproteins such as MCL‐1 or c‐MYC. We studied the activity of TG02 in 9 human long‐term glioma cell lines (LTC) and 5 glioma‐initiating cell lines (GIC) using various cell death assays in vitro and in the LN‐229 LTC and ZH‐161 GIC models in vivo. TG02 exhibits strong anti‐tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN‐229 and ZH‐161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL‐1 or c‐MYC correlated with sensitivity to TG02. Cdk‐9 or cdk‐5 gene silencing alone did not fully reproduce the effects of TG02. C‐myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan‐caspase inhibitor, zVAD‐fmk, or BCL‐2 or MCL‐1 gene transfer only moderately attenuated TG02‐induced cell death, and caspase inhibition did not prevent loss of MCL‐1 or c‐MYC. TG02 activity was independent of O6‐methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL‐1, loss of c‐MYC, or senescence. TG02 is a highly potent apoptosis‐inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02‐treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.

BackgroundPro-inflammatory cytokines are known to have deleterious effects on Schwann cells (SCs). Interleukin 17 (IL-17) is a potent pro-inflammatory cytokine that exhibits relevant effects during inflammation in the peripheral nervous system (PNS), and IL-17-secreting cells have been reported within the endoneurium in proximity to the SCs.MethodsHere, we analyzed the effects of IL-17 on myelination and the immunological properties of SCs. Dorsal root ganglia (DRG) co-cultures containing neurons and SCs from BL6 mice were used to define the impact of IL-17 on myelination and on SC differentiation; primary SCs were analyzed for RNA and protein expression to define the putative immunological alignment of the SCs.ResultsSCs were found to functionally express the IL-17 receptors A and B. In DRG cultures, stimulation with IL-17 resulted in reduced myelin synthesis, while pro-myelin gene expression was suppressed at the mRNA level. Neuronal outgrowth and SC viability, as well as structural myelin formation, remained unaffected. Co-cultures exhibited SC-relevant pro-inflammatory markers, such as matrix metalloproteinase 9 and SCs significantly increased the expression of the major histocompatibility complex (MHC) I and exhibited a slight, nonsignificant increase in expression of MHCII, and a transporter associated with antigen presentation (TAP) II molecules relevant for antigen processing and presentation.ConclusionsIL-17 may act as a myelin-suppressive mediator in the peripheral nerve, directly propagating SC-mediated demyelination, paralleled by an inflammatory alignment of the SCs. Further analyses are warranted to elucidate the role of IL-17 during inflammation in the PNS in vivo, which could be useful in the development of target therapies.

Novel treatments for glioblastoma, the most common malignant primary brain tumor, are urgently required. Type I interferons (IFN) are natural cytokines primarily involved in the defense against viral infections, which may also serve a role in the control of cancer, notably in the suppression of the cancer stem cell phenotype. TG02 is a novel orally available cyclin-dependent kinase 9 inhibitor which induces glioma cell apoptosis without profound caspase activation, which is currently explored in early clinical trials in newly diagnosed and recurrent glioblastoma. In the present study, human glioma-initiating cell line models were used to explore whether IFN-β modulates the anti-glioma activity of TG02. The present study employed immunoblotting to assess protein levels, several viability assays and gene silencing strategies to assess gene function. Pre-exposure to IFN-β sensitized human glioma models to a subsequent exposure to TG02. Combination treatment was associated with increased DEVD-amc cleaving caspase activity that was blocked by the anti-apoptotic protein, BCL2. However, BCL2 did not protect from the synergistic effects of IFN and TG02 on glioma cell growth. Furthermore, although IFN strongly induced pro-apoptotic XIAP-associated factor (XAF) expression, disrupting XAF expression did not abrogate the synergy with TG02. Consistent with that, caspase 3 gene silencing did not abrogate the effects of TG02 or IFN-β alone or in combination. Finally, it was observed that IFN-β may indeed modulate the effects of TG02 upstream in the signaling cascade since inhibition of RNA polymerase II phosphorylation, a direct readout of the pharmacodynamic activity of TG02, was facilitated when glioma cells were pre-exposed to IFN-β. In summary, these data suggest that type I IFN may be combined with TG02 to limit glioblastoma growth, but that the well characterized effects of IFN and TG02 on apoptotic signaling are dispensable for synergistic tumor growth inhibition. Instead, exploring how IFN signaling primes glioma cells for TG02-mediated direct target inhibition may help to design novel and effective pharmacological approaches to glioblastoma.