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.

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.