Mitochondrial Metabolism Regulation of T Cell-Mediated Immunity.

Recent evidence supports the notion that mitochondrial metabolism is necessary for T cell activation, proliferation, and function. Mitochondrial metabolism supports T cell anabolism by providing key metabolites for macromolecule synthesis and generating metabolites for T cell function. In this review, we focus on how mitochondrial metabolism controls conventional and regulatory T cell fates and function.

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation.

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

NSD2 drives t(4;14) myeloma cell dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome.

Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase NSD2. A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased NADP(H) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine (SAM), compromising synthesis of creatine from its precursor guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum consistent with impaired utilization of mitochondrial ATP. Accordingly, AK2 suppression increased sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

SIRT1 and AMPK pathways are essential for the proliferation and survival of primary effusion lymphoma cells.

Primary effusion lymphoma (PEL) is a rare and aggressive B-cell lymphoma with a dismal prognosis caused by infection of Kaposi's sarcoma-associated herpesvirus. Despite the findings that numerous viral genes and cellular pathways are essential for the proliferation and survival of PEL cells, there is currently no effective therapeutic treatment for PEL. Here, we report that the metabolic sensor SIRT1 is functionally required for sustaining the proliferation and survival of PEL cells. Knockdown of SIRT1 with specific shRNAs or inhibition of SIRT1 with an inhibitor (tenovin-6) induced cell cycle arrest and apoptosis in PEL cells. We detected high levels of AMPK activation in PEL cells, reflected in AMPKα1 phosphorylation at T174. Knockdown or inhibition of SIRT1 reduced AMPK activation, indicating that SIRT1 was required for AMPK activation. Interestingly, knockdown of AMPK with specific shRNAs or inhibition of AMPK with the inhibitor compound C recapitulated the phenotype of SIRT1, and induced cell cycle arrest and apoptosis, whereas overexpression of a constitutively active AMPK construct rescued the cytotoxic effect of SIRT1 knockdown. Remarkably, treatment with tenovin-6 effectively inhibited the initiation and progression of PEL, and significantly extended the survival of mice in a murine PEL model. Taken together, these results illustrate that the SIRT1-AMPK axis is essential for maintaining the proliferation and survival of PEL and identify SIRT1 and AMPK as potential therapeutic targets, and tenovin-6 as a candidate therapeutic agent for PEL patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) demonstrates a global down-regulation of miR-15a and miR-16 and a selective silencing of the related miR-29b in aggressive disease. Deletions in chromosome 13 [del(13q14)] partially account for the loss of expression of miR-15a and miR-16, but the mechanisms by which miR-29b becomes silenced is unknown. In the present study, we show that the histone deacetylases (HDACs) are overexpressed in CLL and mediate the epigenetic silencing of miR-15a, miR-16, and miR-29b. HDAC inhibition triggered the accumulation of the transcriptionally activating chromatin modification H3K4me2 and restored the expression of miR-15a, miR-16, and miR-29b in approximately 35% of samples. Ectopic expression of miR-15a and miR-16 and HDAC inhibition-induced expression of miR-15a, miR-16, or miR-29b in primary CLL cells was associated with declines in the levels of Mcl-1, but not Bcl-2, mitochondrial dysfunction, and induction of cell death. Therefore, our results show that HDACs aberrantly silence the expression of the critical tumor suppressors miR-15a, miR-16, and miR-29b in CLL. Deacetylase inhibition may be a therapeutic strategy that restores the expression of these miRs to antagonize Mcl-1, an important survival protein in these cells. Consequently, CLL patients who exhibit such epigenetic silencing may benefit from HDAC inhibitor-based therapy.

Pre-illness data reveals differences in multiple metabolites and metabolic pathways in those who do and do not recover from infectious mononucleosis.

Metabolic pathways related to energy production, amino acids, nucleotides, nitrogen, lipids, and neurotransmitters in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) may contribute to the pathophysiology of ME/CFS. 4501 Northwestern University college students were enrolled in a prospective, longitudinal study. We collected data before illness, during infectious mononucleosis (IM), and at a 6 month follow-up for those who recovered (N = 18) versus those who went on to develop ME/CFS 6 months later (N = 18). Examining pre-illness blood samples, we found significant detectable metabolite differences between participants fated to develop severe ME/CFS following IM versus recovered controls. We identified glutathione metabolism, nucleotide metabolism, and the TCA cycle (among others) as potentially dysregulated pathways. The pathways that differed between cases and controls are essential for proliferating cells, particularly during a pro-inflammatory immune response. Performing a series of binary logistic regressions using a leave-one-out cross-validation (LOOCV), our models correctly classified the severe ME/CFS group and recovered controls with an accuracy of 97.2%, sensitivity of 94.4%, and specificity of 100.0%. These changes are consistent with the elevations in pro-inflammatory cytokines that we have reported for patients fated to develop severe ME/CFS 6 months after IM.

Genes Involved in Maintaining Mitochondrial Membrane Potential Upon Electron Transport Chain Disruption.

Mitochondria are biosynthetic, bioenergetic, and signaling organelles with a critical role in cellular physiology. Dysfunctional mitochondria are associated with aging and underlie the cause of a wide range of diseases, from neurodegeneration to cancer. Through signaling, mitochondria regulate diverse biological outcomes. The maintenance of the mitochondrial membrane potential, for instance, is essential for proliferation, the release of mitochondrial reactive oxygen species, and oxygen sensing. The loss of mitochondrial membrane potential triggers pathways to clear damaged mitochondria and often results in cell death. In this study, we conducted a genome-wide positive selection CRISPR screen using a combination of mitochondrial inhibitors to uncover genes involved in sustaining a mitochondrial membrane potential, and therefore avoid cell death when the electron transport chain is impaired. Our screen identified genes involved in mitochondrial protein translation and ATP synthesis as essential for the induction of cell death when cells lose their mitochondrial membrane potential. This report intends to provide potential targets for the treatment of diseases associated with mitochondrial dysfunction.

Reduced expression of mitochondrial complex I subunit Ndufs2 does not impact healthspan in mice.

Aging in mammals leads to reduction in genes encoding the 45-subunit mitochondrial electron transport chain complex I. It has been hypothesized that normal aging and age-related diseases such as Parkinson's disease are in part due to modest decrease in expression of mitochondrial complex I subunits. By contrast, diminishing expression of mitochondrial complex I genes in lower organisms increases lifespan. Furthermore, metformin, a putative complex I inhibitor, increases healthspan in mice and humans. In the present study, we investigated whether loss of one allele of Ndufs2, the catalytic subunit of mitochondrial complex I, impacts healthspan and lifespan in mice. Our results indicate that Ndufs2 hemizygous mice (Ndufs2+/-) show no overt impairment in aging-related motor function, learning, tissue histology, organismal metabolism, or sensitivity to metformin in a C57BL6/J background. Despite a significant reduction of Ndufs2 mRNA, the mice do not demonstrate a significant decrease in complex I function. However, there are detectable transcriptomic changes in individual cell types and tissues due to loss of one allele of Ndufs2. Our data indicate that a 50% decline in mRNA of the core mitochondrial complex I subunit Ndufs2 is neither beneficial nor detrimental to healthspan.

Mitochondrial Metabolism as a Target for Cancer Therapy.

Recent evidence in humans and mice supports the notion that mitochondrial metabolism is active and necessary for tumor growth. Mitochondrial metabolism supports tumor anabolism by providing key metabolites for macromolecule synthesis and generating oncometabolites to maintain the cancer phenotype. Moreover, there are multiple clinical trials testing the efficacy of inhibiting mitochondrial metabolism as a new cancer therapeutic treatment. In this review, we discuss the rationale of using these anti-cancer agents in clinical trials and highlight how to effectively utilize them in different tumor contexts.

Repurposing Cytarabine for Treating Primary Effusion Lymphoma by Targeting Kaposi's Sarcoma-Associated Herpesvirus Latent and Lytic Replications.

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically linked to primary effusion lymphoma (PEL), an aggressive and nontreatable malignancy commonly found in AIDS patients. In this study, we performed a high-throughput screening of 3,731 characterized compounds and identified cytarabine, approved by the FDA for treating numerous types of cancer, as a potent inhibitor of KSHV-induced PEL. We showed the high efficacy of cytarabine in the growth inhibition of various PEL cells by inducing cell cycle arrest and apoptosis. Cytarabine inhibited host DNA and RNA syntheses and therefore induced cellular cytotoxicity. Furthermore, cytarabine inhibited viral DNA and RNA syntheses and induced the rapid degradation of KSHV major latent protein LANA (latency-associated nuclear antigen), leading to the suppression of KSHV latent replication. Importantly, cytarabine effectively inhibited active KSHV replication and virion production in PEL cells. Finally, cytarabine treatments not only effectively inhibited the initiation and progression of PEL tumors but also induced regression of grown PEL tumors in a xenograft mouse model. Altogether, our study has identified cytarabine as a novel therapeutic agent for treating PEL as well as eliminating KSHV persistent infection.IMPORTANCE Primary effusion lymphoma is an aggressive malignancy caused by Kaposi's sarcoma-associated herpesvirus. The outcome of primary effusion lymphoma is dismal without specific treatment. Through a high-throughput screening of characterized compounds, we identified an FDA-approved compound, cytarabine, as a potent inhibitor of primary effusion lymphoma. We showed that cytarabine induced regression of PEL tumors in a xenograft mouse model. Cytarabine inhibited host and viral DNA and RNA syntheses, resulting in the induction of cytotoxicity. Of interest, cytarabine induced the degradation of KSHV major latent protein LANA, hence suppressing KSHV latent replication, which is required for PEL cell survival. Furthermore, cytarabine inhibited KSHV lytic replication program, preventing virion production. Our findings identified cytarabine as a novel therapeutic agent for treating PEL as well as for eliminating KSHV persistent infection. Since cytarabine is already approved by the FDA, it might be an ideal candidate for repurposing for PEL therapy and for further evaluation in advanced clinical trials.

TLR4-Mediated Inflammation Promotes KSHV-Induced Cellular Transformation and Tumorigenesis by Activating the STAT3 Pathway.

Toll-like receptors (TLR) are conserved immune sensors mediating antimicrobial and antitumoral responses, but recent evidence implicates them in promoting carcinogenesis in certain cancers. Kaposi sarcoma is caused by infection of Kaposi sarcoma-associated herpesvirus (KSHV) and is characterized by uncontrolled neoangiogenesis and inflammation. Here, we show that TLR4 is upregulated in KSHV-infected spindle tumor cells in human Kaposi sarcoma lesions. In a model of KSHV-induced cellular transformation, KSHV upregulated expression of TLR4, its adaptor MyD88, and coreceptors CD14 and MD2. KSHV induction of TLR4 was mediated by multiple viral miRNAs. Importantly, the TLR4 pathway was activated constitutively in KSHV-transformed cells, resulting in chronic induction of IL6, IL1ß, and IL18. Accordingly, IL6 mediated constitutive activation of the STAT3 pathway, an essential event for uncontrolled cellular proliferation and transformation. TLR4 stimulation with lipopolysaccharides or live bacteria enhanced tumorigenesis while TLR4 antagonist CLI095 inhibited it. These results highlight an essential role of the TLR4 pathway and chronic inflammation in KSHV-induced tumorigenesis, which helps explain why HIV-infected patients, who frequently suffer from opportunistic bacterial infections and metabolic complications, frequently develop Kaposi sarcoma. Cancer Res; 77(24); 7094-108. ©2017 AACR.