Glioblastoma Mesenchymal Transition and Invasion are Dependent on a NF-κB/BRD2 Chromatin Complex.

Glioblastoma (GBM) represents the most aggressive subtype of glioma, noted for its profound invasiveness and molecular heterogeneity. The mesenchymal (MES) transcriptomic subtype is frequently associated with therapy resistance, rapid recurrence, and increased tumor-associated macrophages. Notably, activation of the NF-κB pathway and alterations in the PTEN gene are both associated with this malignant transition. Although PTEN aberrations have been shown to be associated with enhanced NF-κB signaling, the relationships between PTEN, NF-κB and MES transition are poorly understood in GBM. Here, we show that PTEN regulates the chromatin binding of bromodomain and extraterminal (BET) family proteins, BRD2 and BRD4, mediated by p65/RelA localization to the chromatin. By utilizing patient-derived glioblastoma stem cells and CRISPR gene editing of the RELA gene, we demonstrate a crucial role for RelA lysine 310 acetylation in recruiting BET proteins to chromatin for MES gene expression and GBM cell invasion upon PTEN loss. Remarkably, we found that BRD2 is dependent on chromatin associated acetylated RelA for its recruitment to MES gene promoters and their expression. Furthermore, loss of BRD2 results in the loss of MES signature, accompanied by an enrichment of proneural signature and enhanced therapy responsiveness. Finally, we demonstrate that disrupting the NFκB/BRD2 interaction with a brain penetrant BET-BD2 inhibitor reduces mesenchymal gene expression, GBM invasion, and therapy resistance in GBM models. This study uncovers the role of hitherto unexplored PTEN-NF-κB-BRD2 pathway in promoting MES transition and suggests inhibiting this complex with BET-BD2 specific inhibitors as a therapeutic approach to target the MES phenotype in GBM.

TERT promoter C228T mutation in neural progenitors confers growth advantage following telomere shortening in vivo.

BACKGROUND: Heterozygous TERT (telomerase reverse transcriptase) promoter mutations (TPMs) facilitate TERT expression and are the most frequent mutation in glioblastoma (GBM). A recent analysis revealed this mutation is one of the earliest events in gliomagenesis. However, no appropriate human models have been engineered to study the role of this mutation in the initiation of these tumors. METHOD: We established GBM models by introducing the heterozygous TPM in human induced pluripotent stem cells (hiPSCs) using a two-step targeting approach in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, and EGFRvIII overexpression. The impact of the mutation was evaluated through the in vivo passage and in vitro experiment and analysis. RESULTS: Orthotopic injection of neuronal precursor cells (NPCs) derived from hiPSCs with the TPM into immunodeficient mice did not enhance tumorigenesis compared to TERT promoter wild type NPCs at initial in vivo passage presumably due to relatively long telomeres. However, the mutation recruited GA-Binding Protein and engendered low-level TERT expression resulting in enhanced tumorigenesis and maintenance of short telomeres upon secondary passage as observed in human GBM. These results provide the first insights regarding increased tumorigenesis upon introducing a TPM compared to isogenic controls without TPMs. CONCLUSION: Our novel GBM models presented the growth advantage of heterozygous TPMs for the first time in the context of GBM driver mutations relative to isogenic controls, thereby allowing for the identification and validation of TERT promoter-specific vulnerabilities in a genetically accurate background.

PTEN deficiency leads to proteasome addiction: a novel vulnerability in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months; therefore, more effective treatment options for GBM are required. To identify new drugs targeting GBMs, we performed a high-throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype. METHODS: High-throughput drug screening was performed on GSCs followed by a dose-response assay of the 5 identified original "hits." A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome Inhibition Response Signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using three-dimensional (3D) GBM-organoids and preclinical orthotopic models. RESULTS: We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D GBM-organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition. CONCLUSIONS: Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.

Cellular environment controls the dynamics of histone H3 lysine 56 acetylation in response to DNA damage in mammalian cells.

Epigenetic changes play a crucial role in sensing signals and responding to fluctuations in the extracellular environment. How the cellular micro-environment affects DNA damage response signalling in chromatin context is not extensively studied. Histone acetylation is dynamic and very sensitive to changes in the extracellular environment. Existing literature on H3 lysine 56 acetylation (H3K56ac) levels upon DNA damage in mammals presents a conflicting picture. The occurrence of both increased and decreased H3K56ac upon DNA damage in our experiments led us to investigate the role of the micro-environment on H3K56ac. Here, we show that the global levels of H3K56ac increase as cells grow from low density to high density while SIRT1 and SIRT6 expression decrease. Additionally, rising lactic acid levels increase H3K56ac. Our results show that cell density and accumulation of metabolites affect dynamics of H3K56ac in response to DNA damage. Upon DNA damage, H3K56ac increases in low density cells with low initial acetylation, while acetylation decreases in high cell density cells. These results highlight that H3K56ac levels upon DNA damage are dependent on the metabolites in the extracellular milieu which impact chromatin structure by regulating chromatin modifying enzymes. Accumulation of lactic acid at high cell density reflects conditions similar to the tumour micro-environment. As H3K56ac increases in tumours, lactic acid and low pH might alter H3K56ac in tumours, leading to deregulated gene expression, contributing to tumour progression.

Mammalian target of rapamycin complex 2 (mTORC2) controls glycolytic gene expression by regulating Histone H3 Lysine 56 acetylation.

Metabolic reprogramming is a hallmark of cancer cells, but the mechanisms are not well understood. The mammalian target of rapamycin complex 2 (mTORC2) controls cell growth and proliferation and plays a critical role in metabolic reprogramming in glioma. mTORC2 regulates cellular processes such as cell survival, metabolism, and proliferation by phosphorylation of AGC kinases. Components of mTORC2 are shown to localize to the nucleus, but whether mTORC2 modulates epigenetic modifications to regulate gene expression is not known. Here, we identified histone H3 lysine 56 acetylation (H3K56Ac) is regulated by mTORC2 and show that global H3K56Ac levels were downregulated on mTORC2 knockdown but not on mTORC1 knockdown. mTORC2 promotes H3K56Ac in a tuberous sclerosis complex 1/2 (TSC1/2) mediated signaling pathway. We show that knockdown of sirtuin6 (SIRT6) prevented H3K56 deacetylation in mTORC2 depleted cells. Using glioma model consisting of U87EGFRvIII cells, we established that mTORC2 promotes H3K56Ac in glioma. Finally, we show that mTORC2 regulates the expression of glycolytic genes by regulating H3K56Ac levels at the promoters of these genes in glioma cells and depletion of mTOR leads to increased recruitment of SIRT6 to these promoters. Collectively, these results identify mTORC2 signaling pathway positively promotes H3K56Ac through which it may mediate metabolic reprogramming in glioma.

Fission Yeast Sirtuin Hst4 Functions in Preserving Genomic Integrity by Regulating Replisome Component Mcl1.

The Schizosaccharomyces pombe sirtuin Hst4, functions in the maintenance of genome stability by regulating histone H3 lysine56 acetylation (H3K56ac) and promoting cell survival during replicative stress. However, its molecular function in DNA damage survival is unclear. Here, we show that hst4 deficiency in the fission yeast causes S phase delay and DNA synthesis defects. We identified a novel functional link between hst4 and the replisome component mcl1 in a suppressor screen aimed to identify genes that could restore the slow growth and Methyl methanesulphonate (MMS) sensitivity phenotypes of the hst4Δ mutant. Expression of the replisome component Mcl1 rescues hst4Δ phenotypes. Interestingly, hst4 and mcl1 show an epistatic interaction and suppression of hst4Δ phenotypes by mcl1 is H3K56 acetylation dependent. Furthermore, Hst4 was found to regulate the expression of mcl1. Finally, we show that hSIRT2 depletion results in decreased levels of And-1 (human orthologue of Mcl1), establishing the conservation of this mechanism. Moreover, on induction of replication stress (MMS treatment), Mcl1 levels decrease upon Hst4 down regulation. Our results identify a novel function of Hst4 in regulation of DNA replication that is dependent on H3K56 acetylation. Both SIRT2 and And-1 are deregulated in cancers. Therefore, these findings could be of therapeutic importance in future.