Background The transcription factor NFκB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFκB activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive. Methods In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFκB can be converted into a tumor suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM-datasets. Results We found that CBD promotes DNA binding of the NFκB subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA binding by RELA lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC death. Consequently, ROS levels served as predictive biomarker for CBD-sensitive tumors. Conclusions This evidence demonstrates how a clinically approved drug can convert NFκB into a tumor suppressor and suggests a promising repurposing option for GBM-therapy.

Highlights d Uncovering brain tumor-associated cells with a myeloid expression profile (TAMEP) d TAMEP appear as a myeloid population but do not derive from microglia or bone marrow d TAMEP originate from local progenitors activated by brain tumors d TAMEP promote tumor expansion by enhancing vessel density

Background The transcription factor NFKB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFKB-activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive. Methods In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFKB can be converted into a tumor-suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene-expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics-analysis of human GBM-datasets. Results We found that CBD promotes DNA-binding of the NFKB-subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA-binding by RELA lacking phospho-serine 311 was found to mediate hGSC-cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC-death. Consequently, ROS-levels served as predictive biomarker for CBD-sensitive tumors. Conclusions This evidence demonstrates how a clinically approved drug can convert NFKB into a tumor-suppressor and suggests a promising repurposing option for GBM-therapy.

Transcriptomic screens of brain tumor (glioblastoma; GBM) parenchymal cells indicated tumor supporting traits of the GBM microenvironment, but largely excluded mitochondrially enriched gene-sets. Here, we show that a mitochondrial transcript of GBM parenchymal cells contributes to therapy resistance. We inspected the non-coding transcriptome of human GBM associated myeloid cells (GAM) and observed an upregulation of the mitochondrial ribosomal subunit MT-RNR2, which contains an open reading frame for the signaling peptide humanin. Immunohistology disclosed that humanin was preponderant in GAM. In vitro assays with a range of human stem-like GBM cells (GSCs) revealed that nanomolar concentrations of humanin can drive tumor cell expansion and chemoresistance to temozolomide. A series of genetic, pharmacological and Western blotting experiments showed that extracellular humanin increased GSC viability via stimulation of the GP130/IL6ST receptor and MAPK (Erk) activation. Humanin responsiveness was subject to inter-individual heterogeneity and predominated in GSC with high expression levels of the IL6ST subunit. Mechanistically, humanin promoted the ATR-dependent DNA-repair machinery in GSCs via induction of the DNA-clamp component HUS1. A slice culture model containing human microglia and GSCs confirmed these observations. Humanin is absent from the mouse brain. Exogenous delivery of humanin into orthotopic GBM mouse models or over-expression specifically of a secreted humanin variant recapitulated the in vitro findings. Blockade of the trimeric interleukin receptor with the brain permeant, FDA-approved drug bazedoxifene blocked humanin-mediated chemoresistance in vitro and in vivo. Overall, we identified a clinically applicable compound together with a predictive marker to prevent chemoresistance in a human-specific GBM model.

OBJECTIVE Glioblastoma (GBM) expansion is accompanied by aberrant tumor vascularization. We demonstrated that the peptide hormone Apelin (APLN) controls GBM neo-vascularization and that the APLN-receptor antagonist Apelin-F13A improved the efficiency and reduced the invasive side effect of established antiangiogenic therapy. Here we investigated if Apelin-F13A blunts the formation of vasogenic edema, which can be monitored by MRI in vivo. METHODS To investigate the role of APLN -signaling in regulating the tightness of the tumor vasculature we performed in vivo leakage assays using Evans-Blue dye and fluorescent dextran. By confocal immunofluorescence, we characterized the maturation of the tumor vasculature with respect to pericyte-coverage and established a dynamic contrast enhanced (DCE) MR imaging protocol to follow vascular edema formation. RESULTS We found that Evans-Blue extravasation is significantly increased by 3-fold in APLNKO tumors compared to controls. Uptake of fluorescent Dextran by CD31+ endothelia was quantified and increased massively from 200μm2 per high magnification field (HMF) in wildtype to 3500μm2 per HMF in APLNKO tumors. Interestingly, intracerebral infusion of Apelin-F13A enhanced pericyte coverage of the tumor vasculature by 50%, decreased Evans-Blue extravasation from 25 μg/ml in controls to 8 μg/ml in treated tumors significantly and efficiently reversed the APLN-dependent vasogenic edema assessed by comparison of T2w-MRI to HE tumor volumes. To follow vasogenic edema formation in vivo, T1w 3D FLASH images were acquired every second over a 360s time course after gadolinium-based MR-contrast agent injection and demonstrated a delayed washout of the contrast in APLN-deficient GBM. CONCLUSION Together, our study shows that DCE-MRI can document APLN-dependent intratumoral vascular normalization and allows inspecting vasogenic edema formation in vivo. In addition to its anti-angiogenic / anti-invasive effect, Apelin-F13A can potently reduce vasogenic edema and might thus serve as a multimodal therapy for the treatment of human GBM in the future.