Daciana Margineantu

HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition

The function of metabolic state in stemness is poorly understood. Mouse embryonicstem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent statesrepresenting the inner cell mass (ICM) and epiblast embryos. Human embryonic stemcells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic differencebetween these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalentin their energy production, dynamically switching from glycolysis to mitochondrialrespiration on demand. Despite having a more developed and expanding mitochondrialcontent, EpiSC/hESC have low mitochondrial respiratory capacity due to lowcytochrome c oxidase (COX) expression. Similarly, in vivo epiblastssuppress COX levels. These data reveal EpiSC/hESC functional similarity to theglycolytic phenotype in cancer (Warburg effect). We further show thathypoxia‐inducible factor 1α (HIF1α) is sufficient to drive ESC to aglycolytic Activin/Nodal‐dependent EpiSC‐like stage. This metabolic switch duringearly stem‐cell development may be deterministic.

Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells

Kaposi’s sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.

Hsp90 inhibition decreases mitochondrial protein turnover.

Background Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. Findings We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. Conclusions Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.

Bcl-2 family proteins as regulators of oxidative stress

The Bcl-2 family of proteins includes pro- and anti-apoptotic factors acting at mitochondrial and microsomal membranes. An impressive body of published studies, using genetic and physical reconstitution experiments in model organisms and cell lines, supports a view of Bcl-2 proteins as the critical arbiters of apoptotic cell death decisions in most circumstances (excepting CD95 death receptor signaling in Type I cells). Evasion of apoptosis is one of the hallmarks of cancer [Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70], relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins is observed in many cancers [Manion MK, Hockenbery DM. Targeting BCL-2-related proteins in cancer therapy. Cancer Biol Ther. 2003;2:S105-14; Olejniczak ET, Van Sant C, Anderson MG, Wang G, Tahir SK, Sauter G, et al. Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains. Mol Cancer Res. 2007;5:331-9]. The rekindled interest in aerobic glycolysis as a cancer trait raises interesting questions as to how metabolic changes in cancer cells are integrated with other essential alterations in cancer, e.g. promotion of angiogenesis and unbridled growth signals. Apoptosis induced by multiple different signals involves loss of mitochondrial homeostasis, in particular, outer mitochondrial membrane integrity, releasing cytochrome c and other proteins from the intermembrane space. This integrative process, controlled by Bcl-2 family proteins, is also influenced by the metabolic state of the cell. In this review, we consider the role of reactive oxygen species, a metabolic by-product, in the mitochondrial pathway of apoptosis, and the relationships between Bcl-2 functions and oxidative stress.

A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.

Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K–protein kinase B (PKB/AKT)–mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D–DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.

Mutant IDH1 regulates the tumor-associated immune system in gliomas

Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 (wtIDH1/2) tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutant IDH1 (muIDH1) and wtIDH1 gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared with wtIDH1. Also, we showed that IDH1 mutations caused down-regulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.

Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy

Significance Folliculin interacting protein-1 (Fnip1) is an intracellular protein known to interact with folliculin (a protein mutated in Birt Hogg Dube’ Syndrome) and the master metabolic sensor AMP kinase. However, the roles of Fnip1 in mammalian development and function are unclear. In this study, we used mice deficient in Fnip1 to show that Fnip1 regulates skeletal muscle fiber type specification. Mice deficient in Fnip1 were significantly enriched for highly oxidative skeletal muscle that is more resistant to fatigue than wild-type muscle. Loss of Fnip1 also decreased muscle damage in a mouse model of Duchenne muscular dystrophy. These results reveal a previously unidentified function for Fnip1 and suggest that pharmacological inhibition of Fnip1 may reduce muscle damage in patients with muscular dystrophy. Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I “red” slow twitch and type II “white” fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.

α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics.

Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma α-1-antitrypsin (AAT) levels in human donors and the development of acute GVHD in the recipients (n = 111; P = .0006). In murine models, treatment of transplant donors with human AAT resulted in an increase in interleukin-10 messenger RNA and CD8(+)CD11c(+)CD205(+) major histocompatibility complex class II(+) dendritic cells (DCs), and the prevention or attenuation of acute GVHD in the recipients. Ablation of DCs (in AAT-treated CD11c-DTR donors) decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells to one-third and abrogated the anti-GVHD effect. The graft-versus-leukemia (GVL) effect of donor cells (against A20 tumor cells) was maintained or even enhanced with AAT treatment of the donor, mediated by an expanded population of NK1.1(+), CD49B(+), CD122(+), CD335(+) NKG2D-expressing natural killer (NK) cells. Blockade of NKG2D significantly suppressed the GVL effect. Metabolic analysis showed a high glycolysis-high oxidative phosphorylation profile for NK1.1(+) cells, CD4(+)CD25(+)FoxP3(+) T cells, and CD11c(+) DCs but not for effector T cells, suggesting a cell type-specific effect of AAT. Thus, via altered metabolism, AAT exerts effective GVHD protection while enhancing GVL effects.

Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.