Mutant IDH1 Promotes Glioma Formation In Vivo.

Isocitrate 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 proneural 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.

In vitro visualization and characterization of wild type and mutant IDH homo- and heterodimers using Bimolecular Fluorescence Complementation.

Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) were recently found in ~80% of WHO grade II-III gliomas and secondary glioblastomas. These mutations reduce the enzyme's ability to convert isocitrate to α-ketoglutarate and, instead, confer a novel gain-of-function resulting in the conversion of α-ketoglutarate to 2-hydroxglutarate (2-HG). However, IDH mutations exist in a heterozygous state such that a functional wild type allele is retained. Recent data suggest that the ability of mutant IDH1, but not mutant IDH2, to produce 2-HG is dependent on the activity of the retained wild type allele. In this study, we aimed to further our understanding of the interaction and function of wild type and mutant IDH heterodimers utilizing Bimolecular Fluorescence Complementation (BiFC). Dimerization of wild type and mutant IDH monomers conjugated to the N- and C-terminus of Venus protein, respectively, is directly proportional to the amount of fluorescence emitted and can be used as an approach to visualize and assess IDH dimerization. Thus, we utilized this method to visualize IDH homo- and heterodimers and to examine their cellular physiology based on subcellular localization, NADPH production, and 2-HG levels. Our results demonstrate that wild type and mutant IDH1 or IDH2 heterodimers display similar physiological characteristics to that of mutant IDH1 or IDH2 homodimers with the exception of their ability to generate NADPH. IDH1 heterodimers consistently generate NADPH whereas IDH2 heterodimers do not. However, the presence of mutant IDH1 or IDH2 in homo- or heterodimer configurations consistently generates equivalent levels of 2-HG. Our data suggest that the wild type protein is not required for the generation of 2-HG.

Akt signaling accelerates tumor recurrence following ras inhibition in the context of ink4a/arf loss.

Aberrant activation of the RAS signaling pathway contributes to nearly all human cancers, including gliomas. To determine the dependence of high-grade gliomas on this signaling pathway, we developed a doxycycline-regulated KRas glioma mouse model. Using this model we previously demonstrated that inhibition of KRas expression in gliomas induced by activated KRas and Akt results in complete tumor regression. We have also shown that, in the context of Ink4a/Arf loss, abrogation of KRas signaling is sufficient to decrease tumor burden but resistance ensues. In this study, we sought to determine the effect of activated Akt signaling in combination with activated KRas and loss of Ink4a/Arf on the growth and recurrence of brain tumors following suppression of KRas expression. We observed significant tumor formation in Ink4a/Arf(lox/lox) mice injected with retroviruses containing tetracycline responsive element (TRE)-KRas, Tet-off, Akt, and Cre. Abrogation of KRas signaling resulted in significant tumor regression; however, resistance developed after a relatively short latency. Tumor recurrence occurred more rapidly and the tumors were more aggressive in the presence of activated Akt signaling compared with loss of Ink4a/Arf alone suggesting that this pathway contributes to tumor progression in this context.

Glucose--a sweet way to die: metabolic switching modulates tumor cell death.

TRAIL, a putative anticancer cytokine, induces extrinsic cell death by activating the caspase cascade directly (Type I cells) via the death-inducing signaling complex (DISC) or indirectly (Type II cells) by caspase-8 cleavage of Bid and activation of the mitochondrial cell death pathway. Cancer cells are characterized by their dependence on aerobic glycolysis, which, although inefficient in terms of ATP production, facilitates tumor metabolism. Our studies show that TRAIL-induced cell death is significantly affected by the metabolic status of the cell. Inhibiting glycolysis with 2-deoxyglucose potentiates TRAIL-induced cell death, whereas glucose deprivation can paradoxically inhibit apoptosis. These conflicting responses to glycolysis inhibition are modulated by the balance between the Akt and AMPK pathways and their subsequent downstream regulation of mTORC1. This results in marked changes in protein translation, in which the equilibrium between anti- and pro-apoptotic Bcl-2 family member proteins is decided by their individual degradation rates. This regulates the mitochondrial cell death pathway and alters its sensitivity not only to TRAIL, but to ABT-737, a Bcl-2 inhibitor. Taken together, our studies show that the sensitivity of cancer cells to apoptosis can be modulated by targeting their unique metabolism in order to enhance sensitivity to apoptotic agents.

A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.

Formation 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.