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Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion., The metabolite methylthioadenosine (MTA) inhibits PRMT5. Therefore, MTA accumulation due to MTA phosphorylase (MTAP) deletion has been proposed as a vulnerability for PRMT5-targeted therapy in cancer. Here, the authors show that MTA does not accumulate in MTAP-deficient cancer cells but is secreted and metabolized by MTAP-intact cells in the tumour microenvironment.

The maintenance of skeletal muscle mass depends on the overall balance between the rates of protein synthesis and degradation. Thus, age-related muscle atrophy and function, commonly known as sarcopenia, may result from decreased protein synthesis, increased proteolysis, or simultaneous changes in both processes governed by complex multifactorial mechanisms. Growing evidence implicates oxidative stress and reactive oxygen species (ROS) as an essential regulator of proteolysis. Our previous studies have shown that genetic deletion of CuZn superoxide dismutase (CuZnSOD, Sod1) in mice leads to elevated oxidative stress, muscle atrophy and weakness, and an acceleration in age-related phenotypes associated with sarcopenia. The goal of this study is to determine whether oxidative stress directly influences the acceleration of proteolysis in skeletal muscle of Sod1(−/−) mice as a function of age. Compared to control, Sod1(−/−) muscle showed a significant elevation in protein carbonyls and 3-nitrotyrosine levels, suggesting high oxidative and nitrosative protein modifications were present. In addition, age-dependent muscle atrophy in Sod1(−/−) muscle was accompanied by an upregulation of the cysteine proteases, calpain, and caspase-3, which are known to play a key role in the initial breakdown of sarcomeres during atrophic conditions. Furthermore, an increase in oxidative stress-induced muscle atrophy was also strongly coupled with simultaneous activation of two major proteolytic systems, the ubiquitin-proteasome and lysosomal autophagy pathways. Collectively, our data suggest that chronic oxidative stress in Sod1(−/−) mice accelerates age-dependent muscle atrophy by enhancing coordinated activation of the proteolytic systems, thereby resulting in overall protein degradation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00200-5) contains supplementary material, which is available to authorized users.

A recently published article by Li et al. titled, "Key Metabolic Enzymes in Remdesivir Activation in Lung Cells," validates the canonical McGuigan enzymes (CES1, CTSA, HINT1) involved in remdesivir's (RDV's) bioactivation using bioinformatic and biochemical approaches (1).….

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes growth of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors. SIGNIFICANCE: Lung cancer is the leading cause of cancer deaths. The overall survival rate for lung cancer patients remains low despite recent therapeutic advances, and a majority of lung cancer patients lack a druggable target. Therefore, there is a great need for a better understanding of the molecular mechanisms driving lung cancer. Epigenetic modifiers are frequently lost in lung cancer, but how this provokes lung tumorigenesis remains unclear. We show that KMT2D, which is recurrently mutated in lung cancer, is a lung tumor suppressor. Our results uncover a tumor-promoting epigenetic mechanism by which KMT2D-inactivating mutations induce aberrant metabolic reprogramming via super-enhancer impairment in lung cancer. Our findings support a glycolysis-inhibitory approach as a therapeutic intervention strategy against KMT2D-mutant lung cancer.

Chemotherapy is central to oncology, perceived to operate only on prolific cancerous tissue. Yet, many non-neoplastic tissues are more prolific compared with typical tumors. Chemotherapies achieve sufficient therapeutic windows to exert antineoplastic activity because they are prodrugs that are bioactivated in cancer-specific environments. The advent of precision medicine has obscured this concept, favoring the development of high-potency kinase inhibitors. Inhibitors of essential mitotic kinases exemplify this paradigm shift, but intolerable on-target toxicities in more prolific normal tissues have led to repeated failures in the clinic. Proliferation rates alone cannot be used to achieve cancer specificity. Here, we discuss integrating the cancer specificity of prodrugs from classical chemotherapeutics and the potency of mitotic kinase inhibitors to generate a class of high-precision cancer therapeutics.

Background Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett’s test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor–treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey’s test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

In BriefThe co-deletion ofMTAPin theCDKN2Alocus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitizeMTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTAin vitro. We further show that primary human glioblastoma tumors minimally accumulate MTAin vivo, which is likely explained by the metabolism of MTA byMTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associatedMTAPdeletions are present in primary human tumors, questioning their translational efficacy in the clinic.HighlightsMethylthioadenosine (MTA) is elevated inMTAP-deleted cancer cellsin vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitorsAccumulation of MTA inMTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.MTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism byMTAP-competent stromal cells.SUMMARYHomozygous deletion of theCDK2NAlocus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroboratedin vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show thatin vitroaccumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA fromMTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher inMTAP-deleted compared toMTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies betweenin vitromodels and primary human tumors and should thus be carefully considered in the development of the precision therapies targetingMTAP-homozygous deleted GBM.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization, thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel x-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

SUMMARY Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors., In Brief Through an in vivo epigenome-focused pooled RNAi screen, Maitituoheti et al. identify KMT2D as a tumor suppressor in melanoma. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways by reduction of H3K4me1-marked active enhancers, conferring sensitivity to glycolysis and IGFR inhibitors in melanoma with KMT2D-inactivating mutations., Graphical Abstract

Forkhead transcription factor family O (FoxO) maintains adult stem cell reserves by supporting their long-term proliferative potential. MicroRNAs (miRs) regulate neuronal stem/progenitor cell (NSPC) proliferation and differentiation during neural development by controlling the expression of a specific set of target genes. In the neurogenic subventricular zone, FoxO1 is specifically expressed in NSPCs and is no longer detected during the transition to neuroblast stage, forming an inverse correlation with miR-9 expression. The 3'-untranslated region of FoxO1 contains a conserved target sequence of miR-9 and FoxO1 expression is coordinated in concert with miR-9 during neuronal differentiation. Our study demonstrates that FoxO1 contributes to NSPC fate decision through its cooperation with the Notch signaling pathway.

Summary Neurodegeneration resulting in cognitive and motor impairment is an inevitable consequence of aging. Little is known about the genetic regulation of this process despite its overriding importance in normal aging. Here, we identify the Forkhead Box O (FOXO) transcription factor 1, 3, and 4 isoforms as a guardian of neuronal integrity by inhibiting age‐progressive axonal degeneration in mammals. FOXO expression progressively increased in aging human and mouse brains. The nervous system‐specific deletion of Foxo transcription factors in mice accelerates aging‐related axonal tract degeneration, which is followed by motor dysfunction. This accelerated neurodegeneration is accompanied by levels of white matter astrogliosis and microgliosis in middle‐aged Foxo knockout mice that are typically only observed in very old wild‐type mice and other aged mammals, including humans. Mechanistically, axonal degeneration in nerve‐specific Foxo knockout mice is associated with elevated mTORC1 activity and accompanying proteotoxic stress due to decreased Sestrin3 expression. Inhibition of mTORC1 by rapamycin treatment mimics FOXO action and prevented axonal degeneration in Foxo knockout mice with accelerated nervous system aging. Defining this central role for FOXO in neuroprotection during mammalian aging offers an invaluable window into the aging process itself.

Glioblastoma expression subtypes have been previously been associated with genomic abnormalities, treatment response, and differences in tumor microenvironment. We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical, a revision of the previously reported TCGA subtypes. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and the presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Comparison of matching primary and recurrent gliomas elucidated treatment-induced phenotypic tumor evolution, including expression subtype switching, in 45% of our cohort as well as associations between microenvironmental components and treatment response. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence was associated with CD8+ T cell enrichment. Frequency of M2 macrophage detection was associated with short-term relapse after radiation therapy. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild-type glioblastoma during treatment modulated tumor evolution. Characterization of the evolving glioblastoma transcriptome and tumor microenvironment aids in designing more effective immunotherapy trials.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ Tcell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

Malignant neoplasms evolve in response to changes in oncogenic signalling(1). Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance(2,3). Here we elucidate the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1–Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Elevated protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α–MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

SummaryWe leveraged IDH wild type glioblastomas and derivative neurospheres to define tumor-intrinsic transcription phenotypes. Transcriptomic multiplicity correlated with increased intratumoral heterogeneity and tumor microenvironment presence. In silico cell sorting demonstrated that M2 macrophages/microglia are the most frequent type of immune cells in the glioma microenvironment, followed by CD4 T lymphocytes and neutrophils. Hypermutation associated with CD8+ T cell enrichment. Longitudinal transcriptome analysis of 124 pairs of primary and recurrent gliomas showed expression subtype is retained in 53% of cases with no proneural to mesenchymal transition being apparent. Inference of the tumor microenvironment through gene signatures revealed a decrease in invading monocytes but a subtype dependent increase in M2 macrophages/microglia cells after disease recurrence. All expression datasets are accessible through http://recur.bioinfo.cnio.es/.SignificanceIDH wild type glioblastoma expression phenotypes have been related to tumor characteristics including genomic abnormalities and treatment response. We explored the intratumoral transcriptomic landscape, including a definition of tumor-intrinsic gene expression subtypes and how they relate to the different cellular components of the tumor immune environment. Comparison of matching primary and recurrent gliomas provided insights into the treatment-induced phenotypic tumor evolution. Proneural to mesenchymal transitions have long been suspected but were not apparent, while intratumoral heterogeneity was a predictor of subtype transition upon recurrence. Characterizing the evolving glioblastoma transcriptome en tumor microenvironment aids in designing more effective immunotherapy trials. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild type GBM during treatment modulated tumor evolution.HighlightsNext generation GBM-intrinsic transcriptional subtypes: proneural, classical, mesenchymalM2 macrophages, CD4+ T-lymphocytes and neutrophils dominate glioblastoma microenvironmentSensitivity to radiotherapy may associate with M2 macrophage presenceCD8+ T cells are enriched in hypermutated GBMs at diagnosis and recurrence

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species, has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress-induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (60% of ad libitum fed diet). We have previously shown that the Sod1(-/-) mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1(-/-) mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation, and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2-isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress-induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.

Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2(fl/fl)). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ∼4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (∼75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2fl/fl mice). In the present study, we used TnIFastCreSod2fl/fl mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2fl/fl mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2fl/fl mice, when compared with control mice. Complex II activity is reduced by 47% in young and by ~90% in old TnIFastCreSod2fl/fl mice, associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2fl/fl mice. Complex II-linked mitochondrial ATP production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2fl/fl mice. Furthermore, in old TnIFastCreSod2fl/fl mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains greater than 2-fold elevated; and oxidative damage (measured as F2 isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2fl/fl mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo 1 . Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses 1 . Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2 1 neural progenitors, Dcx 1 newborn neurons, and Olig2 1 oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons 2 ,t his wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk 1,3 and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity. Accelerating structural and functional decline across diverse organ systems is observed in the aged 1,3,4 . The loss of genome integrity and associated DNA damage signalling and cellular checkpoint responses are well-established intrinsic instigators that drive tissue degeneration during ageing 5 . Of particular relevance to this study, age-progressive loss of telomere function in mice has been shown to provoke widespread p53 activation resulting in activation of cellular checkpoints of apoptosis, impaired proliferation and senescence, compromised tissue stem cell and progenitor function, marked tissue atrophy and physiological impairment in many organ systems 1,6 .

Cancer genomes possess many deletion events targeting tumor suppressor genes (TSG) and neighboring genes in these loci. These deletion patterns prompted us to consider a systematic approach, termed “collateral lethality”, designed to identify cancer-specific vulnerabilities resulting from the deletion of neighboring genes. These bystander genes do not appear to be involved in cancer pathogenesis, yet encode cell-essential functions and are members of multi-gene families that are functionally redundant and co-expressed. Homozygous deletion of SMAD4 is a frequent event in pancreas cancer and other cancer types, totaling >30,000 cases in US annually. SMAD4 deletion often results in co-deletion of the neighboring mitochondrial malic enzyme 2 (ME2) gene. In mammalian cells, two genes (ME2 and ME3) encode redundant cell-essential mitochondrial ME activity. Together, ME2 and ME3 function to generate pyruvate to fuel the TCA cycle, and NADPH to maintain ROS homeostasis. These observations prompted us to hypothesize that the genetic or pharmacological extinction of ME3 activity in a ME2 null cell would specifically compromise cancer cells yet be tolerated in normal host cells possessing ME2 activity. Inducible shRNA strategies were employed to genetically deplete ME3 in ME2-null versus ME2-intact cells followed by apoptosis measurements, integrated metabolomics, and molecular investigations. In collaboration with IONIS Pharmaceuticals, we tested and validated anti-sense oligonucleotide (ASO) to target ME3. Genetic depletion of ME3 in ME2 null, but not ME2 intact, cells resulted in apoptosis and blocked tumorigenic potential. Mechanistically, integrated metabolomic and molecular investigation of mitochondrial ME-deficient cells revealed diminished NADPH production and consequent high ROS, which activates AMP activated protein kinase (AMPK) and which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 (Branched chain amino acid transaminase 2) gene. We also determined that mitochondrial MEs regulate the utilization of branched chain amino acid (BCAA) via BCAT2, a transaminase required for BCAA catabolism. Notably, enforced expression of BCAT2 can restore tumorigenic potential of ME2/3 deficiency, and free nucleotides can restore proliferation in cell culture. Specific targeting of ME3 using ASOs could dramatically reduce tumor burden in a subQ tumor model of pancreatic cancer. Thus, a key mechanism driving cancer cell lethality involves BCAAs as crucial metabolites under the critical regulation of the mitochondrial MEs. These studies reveal a collateral lethal vulnerability in pancreas and other cancers that can be targeted pharmacologically in genotype-defined patient populations. We propose that highly specific ME3 inhibitors could provide an effective therapy across a substantial number of cancer patients. Citation Format: Prasenjit Dey, Joelle Baddour, Youngsoo Kim, Robert Macloed, Florian Muller, Chia Chin Wu, Huamin Wang, Andrea Viale, Haoqiang Ying, Giulio Draetta, Anirban Maitra, Alan Wang, Deepak Nagrath, Ronald DePinho. Malic enzyme 3 as a collateral lethality target in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 777.

SummaryThe PI3K-AKT-FoxO pathway is integral to lifespan regulation in lower organisms and essential for the stability of long-lived cells in mammals. Here, we report the impact of combined FoxO1, 3, and 4 deficiencies on mammalian brain physiology with a particular emphasis on the study of the neural stem/progenitor cell (NSC) pool. We show that the FoxO family plays a prominent role in NSC proliferation and renewal. FoxO-deficient mice show initial increased brain size and proliferation of neural progenitor cells during early postnatal life, followed by precocious significant decline in the NSC pool and accompanying neurogenesis in adult brains. Mechanistically, integrated transcriptomic, promoter, and functional analyses of FoxO-deficient NSC cultures identified direct gene targets with known links to the regulation of human brain size and the control of cellular proliferation, differentiation, and oxidative defense. Thus, the FoxO family coordinately regulates diverse genes and pathways to govern key aspects of NSC homeostasis in the mammalian brain.

Previously, we demonstrated that mitochondria from denervated muscle exhibited dramatically higher Amplex Red dependent fluorescence (thought to be highly specific for hydrogen peroxide) compared with control muscle mitochondria. We now demonstrate that catalase only partially inhibits the Amplex Red signal in mitochondria from denervated muscle. In contrast, ebselen (a glutathione peroxidase mimetic and inhibitor of fatty acid hydroperoxides) significantly inhibits the Amplex Red signal. This suggests that the majority of the Amplex Red signal in mitochondria from denervated muscle is not derived from hydrogen peroxide. Because Amplex Red cannot react with substrates in the lipid environment, we hypothesize that lipid hydroperoxides formed within the mitochondrial lipid bilayer are released as fatty acid hydroperoxides and react with the Amplex Red probe. We also suggest that the release of fatty acid hydroperoxides from denervated muscle mitochondria may be an important determinant of muscle atrophy. In support of this, muscle atrophy and the Amplex Red signal are inhibited in caloric restricted mice and in transgenic mice that overexpress the lipid hydroperoxide-detoxifying enzyme glutathione peroxidase 4. Finally, we propose that cytosolic phospholipase A2 may be a potential source of these hydroperoxides.

It is proposed that conformational changes induced in proteins by oxidation can lead to loss of activity or protein aggregation through exposure of hydrophobic residues and alteration in surface hydrophobicity. Because increased oxidative stress and protein aggregation are consistently observed in amyotrophic lateral sclerosis (ALS), we used a 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (BisANS) photolabeling approach to monitor changes in protein unfolding in vivo in skeletal muscle proteins in ALS mice. We find two major proteins, creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), conformationally affected in the ALS G93A mouse model concordant with a 43% and 41% reduction in enzyme activity, respectively. This correlated with changes in conformation and activity that were detected in CK and GAPDH with in vitro oxidation. Interestingly, we found that GAPDH, but not CK, is conformationally and functionally affected in a longer-lived ALS model (H46R/H48Q), exhibiting a 22% reduction in enzyme activity. We proposed a reaction mechanism for BisANS with nucleophilic amino acids such as lysine, serine, threonine, and tyrosine, and BisANS was found to be primarily incorporated to lysine residues in GAPDH. We identified the specific BisANS incorporation sites on GAPDH in nontransgenic (NTg), G93A, and H46R/H48Q mice using liquid chromatography–tandem mass spectrometry analysis. Four BisANS-containing sites (K52, K104, K212, and K248) were found in NTg GAPDH, while three out of four of these sites were lost in either G93A or H46R/H48Q GAPDH. Conversely, eight new sites (K2, K63, K69, K114, K183, K251, S330, and K331) were found on GAPDH for G93A, including one common site (K114) for H46R/H48Q, which is not found on GAPDH from NTg mice. These data show that GAPDH is differentially affected structurally and functionally in vivo in accordance with the degree of oxidative stress associated with these two models of ALS.

Insulin resistance is a characteristic feature of type 2 diabetes and obesity. Insulin-resistant individuals manifest multiple disturbances in free fatty acid (FFA) metabolism and have excessive lipid accumulation in insulin target tissues. Although much evidence supports a causal role for altered FFA metabolism in the development of insulin resistance, i.e., "lipotoxicity", the intracellular mechanisms by which elevated plasma FFA levels cause insulin resistance have yet to be completely elucidated. Recent studies have implicated a possible role for mitochondrial dysfunction in the pathogenesis of insulin resistance in skeletal muscle. We examined the effect of FFA metabolites [palmitoyl carnitine (PC), palmitoyl-coenzyme A (CoA), and oleoyl-CoA] on ATP synthesis in mitochondria isolated from mouse and human skeletal muscle. At concentrations ranging from 0.5 to 2 microM, these FFA metabolites stimulated ATP synthesis; however, above 5 microM, there was a dose-response inhibition of ATP synthesis. Furthermore, 10 microM PC inhibits ATP synthesis from pyruvate. Elevated PC concentrations (or =10 microM) inhibit electron transport chain activity and decrease the mitochondrial inner membrane potential. These acquired mitochondrial defects, caused by a physiological increase in the concentration of FFA metabolites, provide a mechanistic link between lipotoxicity, mitochondrial dysfunction, and muscle insulin resistance.

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase ( Sod1) and glutathione peroxidase-1 ( Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3–6 h in wild-type mice without any lethality. In contrast, treatment of Sod1−/−or Gpx1−/−mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1−/−mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1−/−, and Sod1−/−mice and in untreated Sod1−/−mice is an upregulation of p53 target genes ( p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H2O2) with glutamate+succinate (approximately 1100 pmol of H2O2 x min(-1) x mg(-1)) were unexpectedly much higher than with succinate (approximately 400 pmol of H2O2 x min(-1) x mg(-1)) or glutamate (approximately 80 pmol of H2O2 x min(-1) x mg(-1)) alone. Superoxide production with glutamate+succinate remained high even at low substrate concentrations (

Genomic deletion of tumor suppressor genes (TSGs) is a rite of passage for virtually all human cancers. The synthetic lethal paradigm has provided a framework for the development of molecular targeted therapeutics that are functionally linked to the loss of specific TSG functions. In the course of genomic events that delete TSGs, a large number of genes with no apparent direct role in tumor promotion also sustain deletion as a result of chromosomal proximity to the target TSG. In this perspective, we review the novel concept of ‘collateral lethality', which has served to identify cancer-specific therapeutic vulnerabilities resulting from codeletion of passenger genes neighboring TSGs. The large number of collaterally deleted genes, playing diverse functions in cell homeostasis, offers a rich repertoire of pharmacologically targetable vulnerabilities presenting novel opportunities for the development of personalized antineoplastic therapies.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity.

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.

The cytochrome (cyt) bc(1) complex is central to energy transduction in many species. Most investigators now accept a modified Q-cycle as the catalytic mechanism of this enzyme. Several thermodynamically favorable side reactions must be minimized for efficient functioning of the Q-cycle. Among these, reduction of oxygen by the Q(o) site semiquinone to produce superoxide is of special pathobiological interest. These superoxide-producing bypass reactions are most notably observed as the antimycin A- or myxothiazol-resistant reduction of cyt c. In this work, we demonstrate that these inhibitor-resistant cyt c reductase activities are largely unaffected by removal of O(2) in the isolated yeast cyt bc(1) complex. Further, increasing O(2) tension 5-fold stimulated the antimycin A-resistant reduction by a small amount ( approximately 25%), while leaving the myxothiazol-resistant reduction unchanged. This most likely indicates that the rate-limiting step in superoxide production is the formation of a reactive species (probably a semiquinone), capable of rapid O(2) reduction, and that in the absence of O(2) this species can reduce cyt c by some other pathway. We suggest as one possibility that a semiquinone escapes from the Q(o) site and reduces either O(2) or cyt c directly. The small increase in antimycin A-resistant cyt c reduction rate at high O(2) can be explained by the accumulation of a low concentration of a semiquinone inside the Q(o) site. Under aerobic conditions, addition of saturating levels of superoxide dismutase (SOD) inhibited 50% of cyt c reduction in the presence of myxothiazol, implying that essentially all bypass reactions occur with the production of superoxide. However, SOD inhibited only 35% of antimycin A-resistant cyt c reduction, suggesting the presence of a second, slower bypass reaction that does not reduce O(2). Given that myxothiazol blocks cyt b reduction whereas antimycin A promotes it, we propose that this second bypass occurs by reduction of the Q(o) site semiquinone by prereduced cyt b(L).

Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

To evaluate evolutionary patterns of GBM recurrence, we analyzed whole genome sequencing (WGS) and multi-sector exome sequencing data from pairs of primary and posttreatment GBM. WGS on ten primary-recurrent pairs detected a median number of 12,214 mutations which we utilized to uncover clonal structures, by analyzing the distribution of mutation cellular frequencies (the fraction of tumor cells harboring a mutation). On average, 41 % of the mutations were shared by primary and recurrence. The majority of shared mutations were clonal in both primary and recurrence, but we also observed many clonal mutations that were uniquely detected in either the primary or the recurrence. This raises the intriguing possibility that major tumor clones in the primary tumor and disease relapse both evolved from a shared ancestral tumor cell population. At least one subclone was identified in the majority of WGS samples, and we observed groups of mutations that were at low cancer cell fractions in both primary and recurrence, suggesting that both subclones evolved from the same ancestral tumor cells separate from the major clone ancestral cells. To address the possibility that the lack of overlap between subsequent tumors was due to intratumoral heterogeneity, we analyzed exome sequencing from a second tumor sector of seven primary and six recurrent tumors. We found that the majority of "second biopsy" mutations were not conserved between time points, suggesting that intratumoral heterogeneity did not explain the large number of mutations uniquely detected in primary and recurrence. The limited overlap of mutations in primary and recurrence provides evidence for ancestral tumor cell populations that could not be eradicated by therapy, while offspring cell populations contained unique mutations, were selectively killed by treatment and could therefore no longer be detected after disease relapse. This study has provided new insights into patterns and dynamics of tumor evolution.

Arising from E.-Z. Shen et al. , 128–132 (2014); doi:10.1038/nature1301210.1038/nature13012 Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial; several studies suggest that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al.1 provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of three-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of mitoflashes as ‘bursts of superoxide radicals’ assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide2, but this interpretation has been criticized because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide3,4,5,6,7. Here we show that purified cpYFP is completely unresponsive to superoxide, and that mitoflashes do not reflect superoxide generation or provide a link between mitochondrial free radical dynamics and lifespan. There is a Reply to this Brief Communication Arising by Cheng, H. Nature 514, http://dx.doi.org/10.1038/nature13859 (2014).

Meeting abstracts The ability of the immune system to treat established tumors is very well recognized. Immunotherapeutic approaches such as Adoptive T cell therapy and anti-CTLA-4 antibody treatment can result in long-term responses in some patients with metastatic melanoma. However, the majority

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non- toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.

Large scale genomic characterization efforts such as TCGA have painted an unprecedentedly detailed picture of the genetic alterations that underlie tumorigenesis. Yet, the majority of genetic alterations are passenger rather than driver events and are considered unactionable. We have previously proposed that passenger or collateral deletions could serve as pharmacologically targetable vulnerabilities Collateral Lethality, in case passenger genes are homozygously deleted and member of a paralogous gene family carrying out an essential housekeeping function. We have presented proof-of-principal, whereby passenger deletion of the glycolytic gene Enolase 1 (ENO1) as part of the 1p36-tumor suppressor locus, renders glioma cells harboring such deletions highly sensitive to ablation of its redundant paralogue, ENO2. While our original analysis identified ENO1-homozygous deletions in Glioblastoma (GBM), recent bioinformatics analyzes, backed by immunohistochemistry, show that ENO1-homozygous deletions also occur in Hepatocellular Carcinoma and Cholangiocarcinoma. In GBM, multisector analysis of primary and recurrent tumors, indicate that ENO1 deletion is an early event which is homogenously distributed through the primary tumor and persist during recurrence. To pharmacologically exploit ENO1-deletion, we have pursued two approaches. First, we have synthesized cell-permeable prodrug derivatives of the natural Enolase inhibitor SF2312. The lead compound, POMHEX, shows potent killing of ENO1-deleted glioma cells in the low nM range while ENO1-restored isogenic or normal cells can tolerate μM doses. POMHEX has a short half-life yet can eradicate intracranial xenografted ENO1-deleted tumors, provided extensive breakdown of the blood-brain barrier. Our second approach to targeting ENO1-deletion consisted of chemical biology screening of drug libraries for the ability to kill ENO1-deleted but not isogenic rescued cells. We find that ENO1-deleted cells show a dramatic sensitization to inhibitors of the mitochondrial electron transport chain. These include tool compounds such as rotenone as well as compounds not previously associated with mitochondria, such as Mubritinib and an experimental anti-neoplastic agent previously described as a HIF1-inhibitor, now known to inhibit mitochondrial Complex I. The latter agent shows potent activity against ENO1-deleted intracranial xenografts. The likely cause for this sensitivity is the inability of ENO1-deleted cells to compensatory upregulate glycolysis in response mitochondrial inhibition, the typical response of ENO1-intact glioma cells and normal cells. Together, these data indicate that passenger deletion of ENO1 is an encouraging drug-target and provide support for collateral lethality as a viable therapeutic strategy, which, given the large number of passenger deletions in the cancer genome, may be broadly applicable. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Wang, Ronald A. DePinho, Florian L. Muller. Passenger deletion of ENO1 as a collateral lethality target in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3837.

Cancer cells display metabolic properties distinct from normal cells and are therefore believed to be dependent on key metabolic enzymes. The effectiveness of targeting metabolism for cancer therapy has been largely restricted due to metabolic adaptability. We postulate that tumors with metabolic vulnerabilities will be limited in such adaptation, thus providing a unique opportunity for therapeutic intervention. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is a metabolically vulnerable cancer, due to mutations in a key TCA cycle enzyme - fumarate hydratase (FH), thus rendering them deficient in oxidative phosphorylation (OXPHOS). We performed a loss-of-function genetic screen in a FH-null HLRCC model (UOK262 cells) and identified 6-phosphogluconate dehydrogenase (6PGD) - a rate-limiting enzyme in the non-oxidative arm of pentose phosphate pathway - as a critical regulator of cell growth. Inhibition of 6PGD using either dox-inducible shRNA or a small molecule inhibitor dramatically blocks the growth of UOK262 cells, and in contrast, has no effect on 6PGD-deficient NB1 cells. The growth inhibition from knockdown is fully rescued by expressing shRNA-resistant 6PGD. Conversely, ectopic expression of 6PGD in NB1 cells results in a gain of function phenotype. Importantly, in vivo, 6PGD knockdown causes robust regression of established UOK262 xenografts. Mechanistically, 6PGD inhibition causes the accumulation of 6-phosphogluconate, blocks glycolysis, and induces ROS production. Furthermore, in tumors with intact TCA cycle, 6PGD inhibition shows synergism with OXPHOS Complex I inhibitor IACS-10759. Together, our data suggest that 6PGD is a bona fide oncology target and provide a strong rationale for developing small molecule inhibitors that could be used to treat OXPHOS-deficient tumors as single agent, or combined with inhibitors of OXPHOS. Citation Format: Yuting Sun, Madhavi Bandi, Timothy Lofton, Melinda Smith, Christopher Bristow, Norma Rogers, Chang Edward, Mary Geck Do, Yongying Jiang, Pietro Morlacchi, Florian Muller, Faika Mseeh, Barbara Czako, Wylie Palmer, Carlo Toniatti, Philip Jones, Giulio Draetta, Timothy Heffernan, Joseph Marszalek. Functional genomics reveals dependency on 6-phosphogluconate dehydrogenase in OXPHOS-deficient tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1023.

Mitochondrial free radicals and redox poise are central to metabolism and cell fate. Their measurement in living cells remains a major challenge and their in vivo dynamics are poorly understood. Reports of 'superoxide flashes' in single mitochondria have therefore been perceived as a major breakthrough: single mitochondria expressing the genetically encoded sensor circularly permuted yellow fluorescent protein (cpYFP) display spontaneous flashes of fluorescence that are responsive to metabolic changes and stressors. We critically review the evidence that underpins the interpretation of mitochondrial cpYFP flashes as bursts of superoxide production and conclude that flashes do not represent superoxide bursts but instead are caused by transient alkalinisation of the mitochondrial matrix. We provide a revised framework that will help to clarify the interpretation of mitochondrial flashes. © 2012 Elsevier Ltd.

The discovery of potent inhibitors of the BRAF proto-oncogene has revolutionized therapy for melanoma harboring mutations in BRAF, yet NRAS-mutant melanoma remains without an effective therapy. Because direct pharmacological inhibition of the RAS proto-oncogene has thus far been unsuccessful, we explored systems biology approaches to identify synergistic drug combination(s) that can mimic RAS inhibition. Here, leveraging an inducible mouse model of NRAS-mutant melanoma, we show that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activates apoptosis but not cell-cycle arrest, which is in contrast to complete genetic neuroblastoma RAS homolog (NRAS) extinction, which triggers both of these effects. Network modeling pinpointed cyclin-dependent kinase 4 (CDK4) as a key driver of this differential phenotype. Accordingly, combined pharmacological inhibition of MEK and CDK4 in vivo led to substantial synergy in therapeutic efficacy. We suggest a gradient model of oncogenic NRAS signaling in which the output is gated, resulting in the decoupling of discrete downstream biological phenotypes as a result of incomplete inhibition. Such a gated signaling model offers a new framework to identify nonobvious coextinction target(s) for combined pharmacological inhibition in NRAS-mutant melanomas.

Dietary restriction (DR) and rapamycin (Rapa) have been shown to increase the lifespan of a variety of organisms leading to the speculation that these interventions increase lifespan through related mechanisms. However, both these interventions have a detrimental effect in the G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). Our previous work indicated that different ALS SOD1 mutant mouse models differ in disease pathogenesis; therefore in this study we measured the effect of DR and Rapa in a second ALS mutant mouse model (the H46R/H48Q mutant). Interestingly, in mice expressing this mutant SOD1 protein, DR significantly delays disease onset and extends lifespan, while Rapa has no effect. These findings suggest that: (1) the effect of DR in ALS is not mediated through pathways common with Rapa, (2) the deleterious effect of DR and Rapa in the G93A ALS mouse model may not be universal to disease caused by all SOD1 mutations, and (3) the results reinforce our previous conclusions that the pathogenic mechanisms in G93A and H46R/H48Q mice are distinct.

Publisher Summary This chapter presents a review of the literature that suggests that increasing the antioxidant concentration, whether exogenously through the diet or through use of transgenic mouse models, is not sufficient to extend the life span of mice bred under optimal animal husbandry conditions. It suggests that the first premise of the free radical theory of aging, i.e., that an increase in antioxidants should extend life span, appears to be true only when short lived animal models are used, whether it is because they are genetically short-lived or because of suboptimal husbandry conditions. With regard to the second premise of the free radical theory of aging, that an increase in oxidants should shorten life span, the data accumulated so far leave little doubt that uncontrolled levels of reactive oxygen species are incompatible with mammalian life. The deleterious phenotypes of Sod2–/– and Gpx4–/– mice make this point very clearly. In addition, the increased levels of oxidative damage brought about by the absence of Sod1 are clearly sufficient to result in an increase in oxidative damage and a significant reduction in life span as well as to accelerate the course of several age-related pathologies. However, the effect of high levels of oxidative damage on aging is difficult to interpret because Sod1–/– mice also have a significant increase in cancer, which may contribute to the reduction in life span.

Oxidative stress has been implicated in the etiology of age-related muscle loss (sarcopenia). However, the underlying mechanisms by which oxidative stress contributes to sarcopenia have not been thoroughly investigated. To directly examine the role of chronic oxidative stress in vivo, we used a mouse model that lacks the antioxidant enzyme CuZnSOD (Sod1). Sod1−/− mice are characterized by high levels of oxidative damage and an acceleration of sarcopenia. In the present study, we demonstrate that muscle atrophy in Sod1−/− mice is accompanied by a progressive decline in mitochondrial bioenergetic function and an elevation of mitochondrial generation of reactive oxygen species. In addition, Sod1−/− muscle exhibits a more rapid induction of mitochondrial-mediated apoptosis and loss of myonuclei. Furthermore, aged Sod1−/− mice show a striking increase in muscle mitochondrial content near the neuromuscular junctions (NMJs). Despite the increase in content, the function of mitochondria is significantly impaired, with increased denervated NMJs and fragmentation of acetylcholine receptors. As a consequence, contractile force in aged Sod1−/− muscles is greatly diminished. Collectively, we show that Sod1−/− mice display characteristics of normal aging muscle in an accelerated manner and propose that the superoxide-induced NMJ degeneration and mitochondrial dysfunction are potential mechanisms of sarcopenia.—Jang, Y. C., Lustgarten, M. S., Liu, Y., Muller, F. L., Bhattacharya, A., Liang, H., Salmon, A. B., Brooks, S. V., Larkin, L., Hayworth, C. R., Richardson, A., and Van Remmen, H. Increased superoxide in vivo accelerates age-associated muscle atrophy through mitochondrial dysfunction and neuromuscular junction degeneration.