A gain of function mutation in AKT1 increases hexokinase 2 and diminishes oxidative stress in meningioma.

Increasing evidence suggests the oncogenic role of missense mutation (AKT1-E17K) of AKT1 gene in meningiomas. Upon investigating the connection between the pro-tumorigenic role of AKT1-E17K and cellular metabolic adaptations, elevated levels of glycolytic enzyme hexokinase 2 (HK2) was observed in meningioma patients with AKT1-E17K compared to patients harboring wild-type AKT1. In vitro experiments also suggested higher HK2 levels and its activity in AKT1-E17K cells. Treatment with the conventional drug of choice AZD5363 (a pan AKT inhibitor) enhanced cell death and diminished HK2 levels in AKT1 mutants. Given the role of AKT phosphorylation in eliciting inflammatory responses, we observed increased levels of inflammatory mediators (IL-1ß, IL6, IL8, and TLR4) in AKT1-E17K cells compared to AKT1-WT cells. Treatment with AKT or HK2 inhibitors dampened the heightened levels of inflammatory markers in AKT1-E17K cells. As AKT and HK2 regulates redox homeostasis, diminished ROS generation concomitant with increased levels of NF-E2- related factor 2 (Nrf2) and superoxide dismutase 1 (SOD1) were observed in AKT1-E17K cells. Increased sensitivity of AKT1-E17K cells to AZD5363 in the presence of HK2 inhibitor Lonidamine was reversed upon treatment with ROS inhibitor NAC. By affecting metabolism, inflammation, and redox homeostasis AKT1-E17K confers a survival advantage in meningioma cells. Our findings suggest that targeting AKT-HK2 cross-talk to induce ROS-dependent cell death could be exploited as novel therapeutic approach in meningiomas.

Dynamic modelling predicts lactate and IL-1ß as interventional targets in metabolic-inflammation-clock regulatory loop in glioma.

In an attempt to understand the role of dysregulated circadian rhythm in glioma, our recent findings highlighted the existence of a feed-forward loop between tumour metabolite lactate, pro-inflammatory cytokine IL-1ß and circadian CLOCK. To further elucidate the implication of this complex interplay, we developed a mathematical model that quantitatively describes this lactate dehydrogenase A (LDHA)-IL-1ß-CLOCK/BMAL1 circuit and predicts potential therapeutic targets. The model was calibrated on quantitative western blotting data in two glioma cell lines in response to either lactate inhibition or IL-1ß stimulation. The calibrated model described the experimental data well and most of the parameters were identifiable, thus the model was predictive. Sensitivity analysis identified IL-1ß and LDHA as potential intervention points. Mathematical models described here can be useful to understand the complex interrelationship between metabolism, inflammation and circadian rhythm, and in designing effective therapeutic strategies. Our findings underscore the importance of including the circadian clock when developing pharmacological approaches that target aberrant tumour metabolism and inflammation. Insight box  The complex interplay of metabolism-inflammation-circadian rhythm in tumours is not well understood. Our recent findings provided evidence of a feed-forward loop between tumour metabolite lactate, pro-inflammatory cytokine IL-1ß and circadian CLOCK/BMAL1 in glioma. To elucidate the implication of this complex interplay, we developed a mathematical model that quantitatively describes this LDHA-IL-1ß-CLOCK/BMAL1 circuit and integrates experimental data to predict potential therapeutic targets. The study employed a multi-start optimization strategy and profile likelihood estimations for parameter estimation and assessing identifiability. The simulations are in reasonable agreement with the experimental data. Sensitivity analysis found LDHA and IL-1ß as potential therapeutic points. Mathematical models described here can provide insights to understand the complex interrelationship between metabolism, inflammation and circadian rhythm, and in identifying effective therapeutic targets.

Reduced YAP1 and FOLR1 in gliomas predict better response to chemotherapeutics.

Gliomas harbouring mutations in IDH1 (isocitrate dehydrogenase 1) are characterized by greater sensitivity to chemotherapeutics. These mutants also exhibit diminished levels of transcriptional coactivator YAP1 (yes-associated protein 1). Enhanced DNA damage in IDH1 mutant cells, as evidenced by γH2AX formation (phosphorylation of histone variant H2A.X) and ATM (serine/threonine kinase; ataxia telangiectasia mutated) phosphorylation, was accompanied by reduced FOLR1 (folate receptor 1) expression. Diminished FOLR1, concomitant with heightened γH2AX levels, was also observed in patient-derived IDH1 mutant glioma tissues. Chromatin immunoprecipitation, overexpression of mutant YAP1, and treatment with YAP1-TEAD (TEA domain transcription factors) complex inhibitor verteporfin demonstrated regulation of FOLR1 expression by YAP1 and its partner transcription factor TEAD2. TCGA (The Cancer Genome Atlas) data analysis demonstrated better patient survival with reduced FOLR1 expression. Depletion of FOLR1 rendered IDH1 wild-type gliomas more susceptible to temozolomide-mediated death. Despite heightened DNA damage, IDH1 mutants exhibited reduced levels of IL6 (interleukin 6) and IL8 (interleukin 8) - pro-inflammatory cytokines known to be associated with persistent DNA damage. While both FOLR1 and YAP1 influenced DNA damage, only YAP1 was involved in regulating IL6 and IL8. ESTIMATE and CIBERSORTx analyses revealed the association between YAP1 expression and immune cell infiltration in gliomas. By identifying the influence of YAP1-FOLR1 link in DNA damage, our findings suggest that simultaneous depletion of both could amplify the potency of DNA damaging agents, while concomitantly reducing the release of inflammatory mediators and potentially affecting immune modulation. This study also highlights the novel role of FOLR1 as a probable prognostic marker in gliomas, predicting responsiveness to temozolomide and other DNA damaging agents.

PRMT1 driven PTX3 regulates ferritinophagy in glioma.

Mutations in the Krebs cycle enzyme IDH1 (isocitrate dehydrogenase (NADP(+)) 1) are associated with better prognosis in gliomas. Though IDH1 mutant (IDH1R132H) tumors are characterized by their antiproliferative signatures maintained through hypermethylation of DNA and chromatin, mechanisms affecting cell death pathways in these tumors are not well elucidated. On investigating the crosstalk between the IDH1 mutant epigenome, ferritinophagy and inflammation, diminished expression of PRMT1 (protein arginine methyltransferase 1) and its associated asymmetric dimethyl epigenetic mark H4R3me2a was observed in IDH1R132H gliomas. Reduced expression of PRMT1 was concurrent with diminished levels of PTX3, a key secretory factor involved in cancer-related inflammation. Lack of PRMT1 H4R3me2a in IDH1 mutant glioma failed to epigenetically activate the expression of PTX3 with a reduction in YY1 (YY1 transcription factor) binding on its promoter. Transcriptional activation and subsequent secretion of PTX3 from cells was required for maintaining macroautophagic/autophagic balance as pharmacological or genetic ablation of PTX3 secretion in wild-type IDH1 significantly increased autophagic flux. Additionally, PTX3-deficient IDH1 mutant gliomas exhibited heightened autophagic signatures. Furthermore, we demonstrate that the PRMT1-PTX3 axis is important in regulating the levels of ferritin genes/iron storage and inhibition of this axis triggered ferritinophagic flux. This study highlights the conserved role of IDH1 mutants in augmenting ferritinophagic flux in gliomas irrespective of genetic landscape through inhibition of the PRMT1-PTX3 axis. This is the first study describing ferritinophagy in IDH1 mutant gliomas with mechanistic details. Of clinical importance, our study suggests that the PRMT1-PTX3 ferritinophagy regulatory circuit could be exploited for therapeutic gains.Abbreviations: 2-HG: D-2-hydroxyglutarate; BafA1: bafilomycin A1; ChIP: chromatin immunoprecipitation; FTH1: ferritin heavy chain 1; FTL: ferritin light chain; GBM: glioblastoma; HMOX1/HO-1: heme oxygenase 1; IHC: immunohistochemistry; IDH1: isocitrate dehydrogenase(NADP(+))1; MDC: monodansylcadaverine; NCOA4: nuclear receptor coactivator 4; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PTX3/TSG-14: pentraxin 3; PRMT: protein arginine methyltransferase; SLC40A1: solute carrier family 40 member 1; Tan IIA: tanshinone IIA; TCA: trichloroacetic acid; TEM: transmission electron microscopy; TNF: tumor necrosis factor.

Repurposing Methotrexate in Dampening SARS-CoV2-S1-Mediated IL6 Expression: Lessons Learnt from Lung Cancer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) is associated with uncontrolled inflammatory responses. Loss of pulmonary angiotensin-converting enzyme 2 (ACE2) function has been associated with SARS-CoV-2 infection. The aberrant signalling and dysregulated inflammation characteristic of lung cancer have marked similarities with SARS-CoV-2 infection. Spearman's correlation analysis of The Cancer Genome Atlas (TCGA) datasets indicated an inverse correlation between ACE2 and IL6 in lung adenocarcinoma. qRT-PCR analysis revealed CoV-2-SRBD-mediated diminished ACE2 expression in lung cancer cells that was concomitant with increased IL6 expression. Western blot and qRT-PCR analysis suggested that treatment with methotrexate (MTx) dampened CoV-2-SRBD-mediated increase in JAK1/STAT3 phosphorylation, gp130, IL6, and folate-binding protein (FBP) expressions. MTx also rescued the diminished expression of ACE2 in CoV-2-SRBD transfected cells. As lung tissue injury in severely affected COVID-19 patients is characterised by aberrant inflammatory response, repurposing MTx as an effective therapy against critical regulators of inflammation in SARS-CoV-2 infection warrants investigation.

Pyruvate dehydrogenase kinase 1 promotes neuronal apoptosis upon Japanese encephalitis virus infection.

Infection by Japanese Encephalitis Virus (JEV) in humans is primarily characterized by signs and symptoms including non-specific febrile illness, arthralgia, myalgia etc. followed by its resolution due to joint action of host innate and adaptive immunity. However, in selective cases, complications arise owing to invasion of central nervous system (CNS) by JEV. Patients being unable to control peripheral viral replication owing to differences in host genetics and immunity experience JEV-associated neurological complications manifested in the form of headache, nausea, meningoencephalitis, coma and eventual death. Entry of JEV into CNS activates complex cascade of events resulting in loss of neuronal physiology and thus CNS tissue integrity. In present study, we have demonstrated role played by JEV in modulation of neuronal pyruvate dehydrogenase kinase 1 (PDK1) abundance and its effect upon neuronal health. Infection of neuron by JEV culminates into upregulation of PDK1 abundance. Albeit inhibition of JEV-induced PDK1-upregulation was accompanied by enhanced JEV propagation in neurons, abrogation of PDK1-upregulation was demonstrated to ameliorate neuronal apoptosis. PDK1 inhibition-associated reduction in neuronal death was observed to be associated with reduced generation of reactive oxygen species (ROS) in neurons. Our study hence provides a possible therapeutic target which upon modulation might help combat JEV infection-associated neuronal apoptosis via restoration of JEV-associated ROS generation.

YAP1-mediated regulation of mitochondrial dynamics in IDH1 mutant gliomas.

Mutation of the isocitrate dehydrogenase 1 (IDH1) gene leads to the production of oncometabolite D-2-hydroxyglutarate (2-HG) from α-ketoglutarate and is associated with better prognosis in glioma. As Yes-associated protein 1 (YAP1) is an important regulator of tumor progression, its role in glioma expressing IDH1 with an R132H mutation was investigated. Diminished nuclear levels of YAP1 in IDH1 mutant glioma tissues and cell lines were accompanied by decreased levels of mitochondrial transcription factor A (TFAM). Luciferase reporter assays and chromatin immunoprecipitation were used to investigate the functionality of the TEAD2-binding site on the TFAM promoter in mediating its YAP1-dependent expression. YAP1-dependent mitochondrial fragmentation and ROS generation were accompanied by decreased telomerase reverse transcriptase (TERT) levels and increased mitochondrial TERT localization in IDH1 R132H cells. Treatment with the Src kinase inhibitor bosutinib, which prevents extranuclear shuttling of TERT, further elevated ROS in IDH1 R132H cells and triggered apoptosis. Importantly, bosutinib treatment also increased ROS levels and induced apoptosis in IDH1 wild-type cells when YAP1 was concurrently depleted. These findings highlight the involvement of YAP1 in coupling mitochondrial dysfunction with mitochondrial shuttling of TERT to constitute an essential non-canonical function of YAP1 in the regulation of redox homeostasis. This article has an associated First Person interview with the first author of the paper.

Brg1 mutation alters oxidative stress responses in glioblastoma.

Increasing evidences suggest that the SWI/SNF chromatin remodeling complex involved in the organization of chromatin architecture via ATP hydrolysis, plays an important role in human cancer. As TCGA gene expression analyses revealed signature of enhanced oxidative stress in GBMs harbouring Brg1mutations, we examined the involvement of ATPase subunit of BRG1 in regulating oxidative stress responses in glioma. BRG1-MUT overexpressing glioma cells exhibit intrinsically higher reactive oxygen species (ROS) levels as compared to BRG1-WT. Elevated ROS generation was concomitant with decreased expression of NF-E2- related factor 2 (NRF2), superoxide dismutases (SOD-1,2) and thioredoxins (TrX-1,2). A similar change in redox regulatory genes and ROS production was observed upon siRNA-mediated knockdown of Brg1. Increased sensitivity to temozolomide was observed upon loss of BRG1-ATPase catalytic domain. These findings highlight the role of ATPase domain of BRG1 in regulating redox homeostasis and sensitivity to oxidative stressors in glioma cells. BRG1 mutation created vulnerability to elevated ROS levels can be therapeutically exploited, with ROS stressors as a promising therapeutic target for the treatment of BRG1-mutant cancers.

Rewiring of Lactate-Interleukin-1ß Autoregulatory Loop with Clock-Bmal1: a Feed-Forward Circuit in Glioma.

A desynchronized circadian rhythm in tumors is coincident with aberrant inflammation and dysregulated metabolism. As their interrelationship in cancer etiology is largely unknown, we investigated the link among the three in glioma. The tumor metabolite lactate-mediated increase in the proinflammatory cytokine interleukin-1ß (IL-1ß) was concomitant with elevated levels of the core circadian regulators Clock and Bmal1. Small interfering RNA (siRNA)-mediated knockdown of Bmal1 and Clock decreased (i) lactate dehydrogenase A (LDHA) and IL-1ß levels and (ii) the release of lactate and proinflammatory cytokines. Lactate-mediated deacetylation of Bmal1 and its interaction with Clock regulate IL-1ß levels and vice versa. Site-directed mutagenesis and luciferase reporter assays indicated the functionality of E-box sites on LDHA and IL-1ß promoters. Sequential chromatin immunoprecipitation (ChIP-re-ChIP) revealed that lactate-IL-1ß cross talk positively affects the corecruitment of Clock-Bmal1 to these E-box sites. Clock-Bmal1 enrichment was accompanied by decreased H3K9me3 and increased H3K9ac and RNA polymerase II (Pol II) occupancy. The lactate-IL-1ß-Clock (LIC) loop positively regulated the expression of genes associated with the cell cycle, DNA damage, and cytoskeletal organization involved in glioma progression. TCGA (The Cancer Genome Atlas) data analysis suggested the presence of lactate-IL-1ß cross talk in other cancers. The responsiveness of stomach and cervical cancer cells to lactate inhibition followed the same trend as that exhibited by glioma cells. In addition, components of the LIC loop were found to be correlated with (i) patient survival, (ii) clinically actionable genes, and (iii) anticancer drug sensitivity. Our findings provide evidence for potential cancer-specific axis wiring of IL-1ß and LDHA through Clock-Bmal1, the outcome of which is to fuel an IL-1ß-lactate autocrine loop that drives proinflammatory and oncogenic signals.

Glycyrrhizin prevents SARS-CoV-2 S1 and Orf3a induced high mobility group box 1 (HMGB1) release and inhibits viral replication.

Efforts to understand host factors critical for COVID-19 pathogenesis have identified high mobility group box 1 (HMGB1) to be crucial for regulating susceptibility to SARS-CoV-2. COVID-19 disease severity is correlated with heightened inflammatory responses, and HMGB1 is an important extracellular mediator in inflammation processes.In this study, we evaluated the effect of HMGB1 inhibitor Glycyrrhizin on the cellular perturbations in lung cells expressing SARS-CoV-2 viral proteins. Pyroptosis in lung cells transfected with SARS-CoV-2 S-RBD and Orf3a, was accompanied by elevation of IL-1ß and extracellular HMGB1 levels. Glycyrrhizin mitigated viral proteins-induced lung cell pyroptosis and activation of macrophages. Heightened release of proinflammatory cytokines IL-1ß, IL-6 and IL-8, as well as ferritin from macrophages cultured in conditioned media from lung cells expressing SARS-CoV-2 S-RBD and Orf3a was attenuated by glycyrrhizin. Importantly, Glycyrrhizin inhibited SARS-CoV-2 replication in Vero E6 cells without exhibiting cytotoxicity at high doses. The dual ability of Glycyrrhizin to concomitantly halt virus replication and dampen proinflammatory mediators might constitute a viable therapeutic option in patients with SARS-CoV-2 infection.

p53 affects epigenetic signature on SOCS1 promoter in response to TLR4 inhibition.

Suppressor of cytokine signaling (SOCS1) functions as a negative regulator of toll-like receptor (TLR) induced inflammatory signaling. As silencing of SOCS1 is concomitant with elevated TLR4 levels in glioblastoma, we investigated the effect of TLR4 inhibition on SOCS1 expression. Pharmacological inhibition of TLR4 signaling by TAK242 or its siRNA-mediated knockdown in p53 mutant or wild-type glioma cells resulted in either increased or decreased SOCS1 expression and promoter activity, respectively. Genetic manipulation of p53 indicated that SOCS1 expression upon TLR4 inhibition is dependent on p53 mutational status. Increased SOCS1 level was concomitant with diminished nucleosomal occupancy around p53-binding site on SOCS1 promoter. This altered nucleosomal landscape was accompanied by (i) diminished nuclear H3K9me3 and (ii) increased JMJD2A and Brg1 levels. JMJD2A inhibition or ectopic expression of ATPase-deficient BRG1 prevented TAK242 mediated increase in SOCS1 expression. Recruitment of Brg1-p53-JMJD2A complex on p53 binding sites of SOCS1 promoter upon TLR4 inhibition was concomitant with increased SOCS1 expression in p53-mutant cells. The Cancer Genome Atlas (TCGA) dataset indicated an inverse correlation between TLR4 and SOCS1 levels in p53 mutant but not in p53WT GBM. Taken together, p53 mutational status regulates transcriptional plasticity of SOCS1 promoter through differential recruitment of chromatin remodelers and epigenetic regulators in response to TLR4 inhibition.

Mutant Isocitrate Dehydrogenase 1 Disrupts PKM2-ß-Catenin-BRG1 Transcriptional Network-Driven CD47 Expression.

A gain-of-function mutation in isocitrate dehydrogenase 1 (IDH1) affects immune surveillance in gliomas. As elevated CD47 levels are associated with immune evasion in cancers, its status in gliomas harboring mutant IDH1 (IDH1-MT cells) was investigated. Decreased CD47 expression in IDH1-R132H-overexpressing cells was accompanied by diminished nuclear ß-catenin, pyruvate kinase isoform M2 (PKM2), and TCF4 levels compared to those in cells harboring wild-type IDH1 (IDH1-WT cells). The inhibition of ß-catenin in IDH1-WT cells abrogated CD47 expression, ß-catenin-TCF4 interaction, and the transactivational activity of ß-catenin/TCF4. The reverse effect was observed in IDH1-MT cells upon the pharmacological elevation of nuclear ß-catenin levels. Genetic and pharmacological manipulation of nuclear PKM2 levels in IDH1-WT and IDH1-MT cells suggested that PKM2 is a positive regulator of the ß-catenin-TCF4 interaction. The Cancer Genome Atlas (TCGA) data sets indicated diminished CD47, PKM2, and ß-catenin levels in IDH1-MT gliomas compared to IDH1-WT gliomas. Also, elevated BRG1 levels with mutations in the ATP-dependent chromatin-remodeling site were observed in IDH1-MT glioma. The ectopic expression of ATPase-deficient BRG1 diminished CD47 expression as well as TCF4 occupancy on its promoter. Sequential chromatin immunoprecipitation (ChIP-re-ChIP) revealed the recruitment of the PKM2-ß-catenin-BRG1-TCF4 complex to the TCF4 site on the CD47 promoter. This occupancy translated into CD47 transcription, as a diminished recruitment of this complex was observed in glioma cells bearing IDH1-R132H. In addition to its involvement in CD47 transcriptional regulation, PKM2-ß-catenin-BRG1 cross talk affected the phagocytosis of IDH1-MT cells by microglia.

Hexokinase 2 and nuclear factor erythroid 2-related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells.

A dynamic network of metabolic adaptations, inflammatory responses, and redox homeostasis is known to drive tumor progression. A considerable overlap among these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1ß in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1ß-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1ß-induced JNK had no effect on cell viability under glucose deprivation, it mediated nuclear translocation of hexokinase 2 (HK2). This event was accompanied by increases in the levels of sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), and xanthine oxidoreductase (XOR). SIRT6 not only induced ROS-mediated cell death but also facilitated nuclear Nrf2-HK2 interaction. Recruitment of the Nrf2-HK2 complex to the ARE site on XOR promoter regulated its expression. Importantly, HK2 served as transcriptional coactivator of Nrf2 to regulate XOR expression, indicated by decreased XOR levels in siRNA-mediated Nrf2 and HK2 knockdown experiments. Our results highlight a non-metabolic role of HK2 as transcriptional coactivator of Nrf2 to regulate XOR expression under conditions of proinflammatory and metabolic stresses. Our insights also underscore the importance of nuclear activities of HK2 in the regulation of genes involved in redox homeostasis.

Reciprocal role of SIRT6 and Hexokinase 2 in the regulation of autophagy driven monocyte differentiation.

Emerging evidences suggest the impact of autophagy on differentiation but the underlying molecular links between metabolic restructuring and autophagy during monocyte differentiation remain elusive. An increase in PPARγ, HK2 and SIRT6 expression was observed upon PMA induced monocyte differentiation. While PPARγ positively regulated HK2 and SIRT6 expression, the latter served as a negative regulator of HK2. Changes in expression of these metabolic modelers were accompanied by decreased glucose uptake and increase in Chibby, a potent antagonist of ß-catenin/Wnt pathway. Knockdown of Chibby abrogated PMA induced differentiation. While inhibition of HK2 either by Lonidamine or siRNA further elevated PMA induced Chibby, mitochondrial ROS, TIGAR and LC3II levels; siRNA mediated knock-down of SIRT6 exhibited contradictory effects as compared to HK2. Notably, inhibition of autophagy increased HK2, diminished Chibby level and CD33 expression. In addition, PMA induced expression of cytoskeletal architectural proteins, CXCR4, phagocytosis, acquisition of macrophage phenotypes and release of pro-inflammatory mediators was found to be HK2 dependent. Collectively, our findings highlight the previously unknown reciprocal influence of SIRT6 and HK2 in regulating autophagy driven monocyte differentiation.

miR-217-casein kinase-2 cross talk regulates ERK activation in ganglioglioma.

Gangliogliomas (GGs) are the most commonly diagnosed long-term epilepsy-associated tumors (LEATs). Although molecular characterizations of brain tumors have identified few novel biomarkers among the LEATs, mechanisms of pathogenesis remain poorly understood. In this study, global microarray-based microRNA (miRNA) expression profile on a set of 9 GGs indicated 66 miRNAs to be differentially expressed in GG as compared to normal brain. The differences validated by qRT-PCR indicated microRNA-217 to be the most downregulated. Through insilico analysis, ERK1/2 and casein kinase (CK-2α) were predicted to be miR-217 regulated. As decreased miR-217 expression was concomitant with upregulated ERK1/2 and CK-2α levels in GG; the interplay between these molecules was investigated in primary human neural precursor cells to mimic the glioneuronal characteristics of these tumors. miR-217 over-expression-mediated decrease in pERK, CK-2α, and mGluR1 levels was accompanied with increase in glycogen accumulation. Importantly, increase in miR-217 levels upon CK-2α inhibition indicated inverse correlation between the two. Inhibition of CK-2α also decreased ERK and mGluR1 levels. By demonstrating, for the first time, the existence of miR-217-CK-2 cross talk and its effects on known epileptogenic factors, these findings provide a unique insight into the pathogenesis of ganglioglioma. By highlighting the role of CK-2 in affecting miR-217/ERK/mGluR1 interplay, this study suggests that targeting CK-2 may afford a novel strategy aimed at LEATs. KEY MESSAGES: Global microarray of ganglioglioma indicates downregulation of miR-217. Decreased miR-217 expression is concomitant with elevated CK-2α and Erk levels. Inverse correlation between miR-217 and CK-2α in primary human neural precursors. miR-217 agomir or CK-2α inhibition decreases pERK and mGluR1 levels. CK-2α affects miR-217/ERK/mGluR1 interplay in long-term epilepsy-associated tumors.

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with in vitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma.

SIRT6 regulated nucleosomal occupancy affects Hexokinase 2 expression.

To understand the molecular association between inflammation and dysregulated metabolism in glioblastoma, the effect of IL-1ß on Hexokinase 2 (HK2) expression was investigated. IL-1ß induced HK2 expression was accompanied by heightened SIRT6 and MZF1 levels. IL-1ß mediated overall decrease in chromatin compactness on HK2 promoter involved diminished nucleosomal occupancy around the most labile region bearing MZF1 sites. Importantly, SIRT6 and MZF1 served as negative regulators of HK2. Ectopic SIRT6 induced formation and recruitment of MZF1-SIRT6 complex to MZF1 site was concomitant with increased nucleosomal occupancy. The function of SIRT6 as co-repressor of MZF1 was inconspicuous in cells treated with IL-1ß alone, as IL-1ß-induced HIF-1α prevented SIRT6 availability for interaction with MZF1. Taken together, SIRT6 over-expression establishes a condition whereby reconfiguration of the HK2 promoter chromatin structure makes it receptive to interaction with MZF1/SIRT6 complex, thereby favouring a regulatory state conducive to diminished transcription.

Concerted action of histone methyltransferases G9a and PRMT-1 regulates PGC-1α-RIG-I axis in IFNγ treated glioma cells.

IFNγ induced de-differentiation markers are negatively regulated by retinoic acid inducible gene (RIG-I) in glioma cells. In addition to RIG-I, IFNγ treatment increased H3K9me2; histone methyltransferases (HMTs) G9a and protein arginine methyltransferase-1 (PRMT-1) levels. While G9a inhibition further increased IFNγ induced RIG-I, PRMT-1 inhibition abrogated IFNγ elevated RIG-I levels. IFNγ induced Sp1 and NFκB served as negative regulators of RIG-I, with decreased occupancy of Sp1 and NFκB observed on the RIG-I promoter. A diminished H3K9Me2 enrichment was observed at the NFκB but not at Sp-1 binding site. IFNγ induced PPAR gamma coactivator-1 alpha (PGC-1α) positively regulated RIG-I; with PRMT-1 and G9a affecting PGC-1α in a counter-regulatory manner. These findings demonstrate how concerted action of HMTs aid PGC-1α driven RIG-I for the sustenance of glioma cells in a de-differentiated state.

CK2 induced RIG-I drives metabolic adaptations in IFNγ-treated glioma cells.

Given the known anti-tumorigenic properties of IFNγ, its effect on glioma cell survival was investigated. Though IFNγ had no effect on glioma cell viability, it induced cell cycle arrest. This was accompanied by increased expression of p53 and retinoic acid inducible gene (RIG-I). While RIG-I had no effect on glioma cell survival, it increased expression of p53 and its downstream target TP53 induced glycolysis and apoptosis regulator (TIGAR). IFNγ induced mitochondrial co-localization of RIG-I was concomitant with its ability to regulate ROS generation, oxidative phosphorylation (OXPHOS) and key enzymes involved in glycolysis and pentose phosphate pathway. Importantly, metabolic gene profiling indicated a suppressed glycolytic pathway in glioma cells upon IFNγ treatment. In addition, IFNγ mediated increase in casein kinase 2 (CK2) expression positively regulated RIG-I expression. These findings demonstrate how IFNγ induced CK2 regulates RIG-I to drive a complex program of metabolic adaptation and redox homeostasis, crucial for determining glioma cell fate.