BET protein inhibition sensitizes glioblastoma cells to temozolomide treatment by attenuating MGMT expression.

Bromodomain and extra-terminal tail (BET) proteins have been identified as potential epigenetic targets in cancer, including glioblastoma. These epigenetic modifiers link the histone code to gene transcription that can be disrupted with small molecule BET inhibitors (BETi). With the aim of developing rational combination treatments for glioblastoma, we analyzed BETi-induced differential gene expression in glioblastoma derived-spheres, and identified 6 distinct response patterns. To uncover emerging actionable vulnerabilities that can be targeted with a second drug, we extracted the 169 significantly disturbed DNA Damage Response genes and inspected their response pattern. The most prominent candidate with consistent downregulation, was the O-6-methylguanine-DNA methyltransferase (MGMT) gene, a known resistance factor for alkylating agent therapy in glioblastoma. BETi not only reduced MGMT expression in GBM cells, but also inhibited its induction, typically observed upon temozolomide treatment. To determine the potential clinical relevance, we evaluated the specificity of the effect on MGMT expression and MGMT mediated treatment resistance to temozolomide. BETi-mediated attenuation of MGMT expression was associated with reduction of BRD4- and Pol II-binding at the MGMT promoter. On the functional level, we demonstrated that ectopic expression of MGMT under an unrelated promoter was not affected by BETi, while under the same conditions, pharmacologic inhibition of MGMT restored the sensitivity to temozolomide, reflected in an increased level of γ-H2AX, a proxy for DNA double-strand breaks. Importantly, expression of MSH6 and MSH2, which are required for sensitivity to unrepaired O6-methylguanine-lesions, was only briefly affected by BETi. Taken together, the addition of BET-inhibitors to the current standard of care, comprising temozolomide treatment, may sensitize the 50% of patients whose glioblastoma exert an unmethylated MGMT promoter.

Metabolic and transcriptomic profiles of glioblastoma invasion revealed by comparisons between patients and corresponding orthotopic xenografts in mice.

The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients' tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H Magnetic Resonance Spectroscopy (1H MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients' tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma.

BET inhibitors repress expression of interferon-stimulated genes and synergize with HDAC inhibitors in glioblastoma.

BACKGROUND: The development of rational combination therapies is key to overcome inherent treatment resistance of glioblastoma (GBM). We aim at identifying new druggable targets by disturbing GBM cells with inhibitors of bromodomain and extra-terminal motif (BET) proteins to reveal cancer-relevant vulnerabilities that may sensitize to a second drug. BET proteins are epigenetic modulators and have been associated with proto-oncogene overexpression in cancer. METHODS: A GBM-derived sphere-line was treated with the BET inhibitor (BETi) JQ1 over a time-course of 48 hours, followed by RNA-sequencing. Four chromatin marks were investigated by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Signatures of interest were functionally validated in vitro and in orthotopic xenografts. Combination therapies were evaluated for synergistic effects. RESULTS: Cancer-relevant pathways significantly modulated by JQ1 comprised interferon alpha (IFN-α) response genes and response signatures to histone deacetylase inhibitors (HDACi). The IFN-signature was reminiscent of a GBM-derived IFN-signature comprising CD274 (PD-L1). Functional pathway analysis suggested that JQ1 was acting directly on the transcriptional level of IFN-response genes and not via the canonical JAK/STAT pathway. This was in line with JQ1 modulated expression and BRD4 and Pol II occupancy at IFN-signature genes, supporting a direct mechanistic interaction. Finally, we showed that combining HDACi with JQ1 acts synergistically in reducing cell viability of GS-lines. CONCLUSIONS: Our approach identified BETi-induced vulnerabilities in cancer-relevant pathways, potentially amenable to synergistic combinatorial therapy, such as combination with HDACi. The direct inhibitory effect of BETi on IFN-responsive genes in GBM cells, including CD274, indicates modulation of the tumor immune landscape and warrants further studies.

Hyperpolarized 13C-glucose magnetic resonance highlights reduced aerobic glycolysis in vivo in infiltrative glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted "Warburg effect". However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.

Glioma epigenetics: From subclassification to novel treatment options.

Gliomas are the most common malignant primary brain tumors, of which glioblastoma is the most malignant form (WHO grade IV), and notorious for treatment resistance. Over the last decade mutations in epigenetic regulator genes have been identified as key drivers of subtypes of gliomas with distinct clinical features. Most characteristic are mutations in IDH1 or IDH2 in lower grade gliomas, and histone 3 mutations in pediatric high grade gliomas that are also associated with characteristic DNA methylation patterns. Furthermore, in adult glioblastoma patients epigenetic silencing of the DNA repair gene MGMT by promoter methylation is predictive for benefit from alkylating agent therapy. These epigenetic alterations are used as biomarkers and play a central role for classification of gliomas (WHO 2016) and treatment decisions. Here we review the pivotal role of epigenetic alterations in the etiology and biology of gliomas. We summarize the complex interactions between "driver" mutations, DNA methylation, histone post-translational modifications, and overall chromatin organization, and how they inform current efforts of testing epigenetic compounds and combinations in preclinical and clinical studies.

Autophagy-mediated degradation of nuclear envelope proteins during oncogene-induced senescence.

Cellular senescence is a largely irreversible form of cell cycle arrest triggered by various types of damage and stress, including oncogene expression (termed oncogene-induced senescence or OIS). We and others have previously demonstrated that OIS occurs in human benign lesions, acting as a potent tumor suppressor mechanism. Numerous phenotypic changes occur during OIS, both in the cytoplasm and in the nucleus. These include the activation of autophagy, a catabolic process operating in the cytoplasm and downregulation of lamin B1, a component of the nuclear lamina. However, it is unknown whether these changes relate to each other. We discovered that cells entering BRAF(V600E)- or H-RAS(G12V)-induced senescence downregulate not only lamin B1 but also lamin A, as well as several other nuclear envelope (NE) proteins, resulting in an altered NE morphology. Depletion of LMNB1 or LMNA/C was sufficient to recapitulate some OIS features, including cell cycle exit and downregulation of NE proteins. We further found that the global loss of NE proteins is a consequence of their degradation by the autophagy machinery, which occurs concomitantly with autophagy induction and increased lysosomal content and activity. Our study therefore reveals a previously unknown connection between autophagy and the disruption of NE integrity during OIS.