Epigenetic therapy a good BET for glioblastoma? A systems biology approach to identify a rational combination therapy.

Glioblastoma (GBM) is the most aggressive primary brain tumour in adults with a median overall survival of only 15 months. Despite multiple attempts, single agent therapies have failed in clinical trials and new strategies for combination treatments are urgently needed. Here, our aim was to predict rational combination therapies with BET inhibi- tors (BETi) that target bromodomain and extra-terminal tail (BET) proteins. BET pro- teins are readers of lysine acetylation on histone tails, therefore promoting gene transcription. BETi are currently being evaluated as anti-cancer drugs. First, we have assessed the biological activity of the tool drug JQ1, a small molecule inhibitor of BET, using glioblastoma derived sphere lines (GS-lines). The results suggested intermediate sensitivity of GS-lines to JQ1. Importantly, we ob- served that JQ1 impaired the self-renewal capacity of all 4 tested GS-lines. With the view to identify BETi-induced vulnerabilities in cancer relevant path- ways that may be targeted with a second drug, we obtained differential expression profiles of glioma sphere lines treated with the BETi JQ1. Gene set enrichment anal- ysis using MSigDB collections revealed several significantly disturbed pathways. They included IFN-α response genes and signatures of response to histone deacety- lase inhibitors (HDACi). In order to validate the observed down regulation of the interferon response genes, we primed our GS-lines with IFN-α and confirmed that interferon-stimulated genes (ISGs), such as MX1, OAS1, and CD274 are down regulated after a 4-hour exposure to JQ1. Importantly, the levels of pSTAT1 in the nucleus remained un- changed upon JQ1 treatment, suggesting that JQ1 was acting directly on the tran- scriptional level of ISGs and not on the IFN-induced JAK-STAT signalling. Similar results were obtained in adherent GBM cell lines that constitutively express ISGs. Moreover, in U87MG orthotopic xenografts in mice, a single i.p. injection of JQ1 down regulated OAS1 and CD274 expression. Finally, we showed that HDACi and JQ1 synergize to reduce cell viability of GS-lines in vitro. Further experiments are necessary to test HDACi and BETi drug combinations in mouse orthotopic xenografts of GS-lines. -- Le glioblastome (GBM) est la tumeur cérébrale la plus agressive avec une survie globale médiane de seulement 15 mois. Malgré de multiples tentatives, les traitements en monothérapie ont échoué dans les essais cliniques et de nouvelles stratégies pour les traitements combinés sont nécessaires. Notre objectif était de prédire les combinaisons thérapeutiques rationnelles avec les inhibiteurs BET (BETi) qui ciblent la bromodomaine et les protéines de la queue extra-terminale (BET), et qui sont actuellement évaluées comme médicaments anticancéreux. Premièrement, nous avons évalué l'activité biologique du médicament JQ1, une petite molécule inhibant BET, en utilisant des lignées de sphères dérivées du glioblastome (lignées-GS). Nos résultats suggèrent une sensibilité intermédiaire des lignées-GS à JQ1. Fait important, nous avons observé que JQ1 a réduit la capacité d’auto-renouvellement des quatre lignées-GS testées. Afin d'identifier les vulnérabilités induites par JQ1 dans les voies pathologiques cancéreuses susceptibles d'être ciblées par un second médicament, nous avons obtenu des profils d'expression différentielle des lignées-GS traitées avec JQ1. L'analyse de l'enrichissement des ensembles de gènes a révélé plusieurs voies significativement perturbées qui comprenaient des gènes de réponse à IFN-α et des signatures de réponse aux inhibiteurs de l'histone désacétylase (HDACi). Afin de valider la régulation négative observée de la signature du gène de réponse à l'interféron, nous avons exposés les lignées-GS à IFN-α et confirmé que l’expression des gènes stimulés par interféron (ISGs), tels que MX1, OAS1 et CD274, est réduite par JQ1. Notamment, les niveaux de pSTAT1 dans le noyau sont inchangés lors du traitement JQ1, suggérant que JQ1 agissait directement au niveau transcriptionnel des ISG et non sur la voie JAK-STAT. Des résultats similaires ont été obtenus dans des lignées cellulaires GBM adhérentes qui expriment constitutivement des ISG. De plus, dans les xénogreffes orthotopiques U87MG chez la souris, une seule injection i.p. de JQ1 a diminué l'expression OAS1 et CD274. Enfin, nous avons montré que HDACi et JQ1 synergisent pour réduire la viabilité cellulaire des lignées-GS in vitro. D'autres expériences sont nécessaires pour tester les combinaisons de médicaments HDACi et BETi dans des xénogreffes orthotopiques murines des lignées-GS

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

Here, we report that nuclear envelope proteins are subjected to autophagic proteolysis in human cells undergoing oncogene-induced senescence. This degradation occurs in parallel with autophagy and lysosomal activity induction that accompanies the establishment of the senescence respons

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

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