SummaryFormation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.

TRAIL (TNF (tumour necrosis factor)-related apoptosis-inducing ligand) a putative anti-cancer cytokine induces apoptosis through DISC (death-inducing signalling complex)-mediated activation of caspase-8 and/or cleavage of Bid. TRAIL is relatively specific for tumour cells but primary chronic lymphocytic leukaemia and mantle cell lymphoma (MCL) cells are resistant. Herein, we show that cellular metabolism influences cell death and that MCL cells (Z138 cell line) can survive/proliferate in glucose-free media by switching from aerobic glycolysis to 'coupled' oxidative phosphorylation. Extracellular flux analysis and mitochondrial inhibitors reveal that in the absence of glycolysis, Z138 cells have enhanced respiratory capacity coupled to ATP synthesis, similar to 'classical' state 3 mitochondria. Conversely, 2-deoxyglucose (2DG) blocked glycolysis and partially inhibited glycolyticdependent oxidative phosphorylation, resulting in a 50% reduction in cellular ATP levels. Also, 2DG sensitised Z138 cells to TRAIL and induced a marked decrease in caspase-8, -3, cFLIP S , Bid and Mcl-1 expression but Bak remained unchanged, altering the Mcl-1/Bak ratio, facilitating cytochrome c release and cell death. Conversely, under glucose-free conditions, Z138 cells were less sensitive to TRAIL with reduced TRAIL-R1/R2 surface receptor expression and impaired DISC formation. Anti-apoptotic proteins Bcl-2 and XIAP were up-regulated while pro-apoptotic BAX was down-regulated. Additionally, mitochondria had higher levels of cytochrome c and ultrastucturally exhibited a condensed configuration with enhanced intracristal spaces. Thus, metabolic switching was accompanied by mitochondrial proteome and ultrastructural remodelling enabling enhanced respiration activity. Cytochrome c release was decreased in glucose-free cells, suggesting that either pore formation was inhibited or that cytochrome c was more tightly bound. Glucose-free Z138 cells were also resistant to intrinsic cell death stimuli (ABT-737 and ionising radiation). In summary, in MCL cells, the anti-glycolytic effects of 2DG and glucose restriction produced opposite effects on TRAIL-induced cell death, demonstrating that mitochondrial metabolism directly modulates sensitivity of tumour cells to apoptosis.

SUMMARYIsocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II–III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled pro-neural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.