Inhibition Of Oxidative Phosphorylation Research Articles

The phenotype of peritoneal mouse macrophages depends on the mitochondria and ATP/ADP homeostasis

Different macrophage subtypes have different morphologies/shapes and functions. Naïve M0 macrophages are elongated. Pro-inflammatory M1 that produce the bactericidal molecule iNos are round. Anti-inflammatory M2 macrophages that produce the pro-healing enzyme Arg-1 are highly elongated. We showed previously that the morphologies of M0 and M2 but not M1 macrophages are RhoA-dependent. Macrophage-specific deletion of RhoA causes the extreme elongation (hummingbird phenotype) of M0 and M2 but not M1 macrophages. The M1 and M2 macrophages also differ in their metabolic status. Here, we studied the effect of the oxidative phosphorylation inhibitors, antimycin A and oligomycin A, at a suboptimal dose, which depolarizes mitochondria but does not eliminate mitochondrial functions, on the mitochondria/energy production and phenotype of wild-type and RhoA-deleted M0, M1 and M2 peritoneal mouse macrophages. We found that, while untreated M1 macrophages had the lowest and the M2 had the highest level of ATP the ATP/ADP ratio was nearly identical between M0, M1 and M2 macrophages. Inhibitor treatment resulted in approximately 60% increase in ATP level and ATP/ADP ratio in M0 and M2 macrophages, and decrease in the level of filamentous (F) actin, and these changes correlated with a drastic shortening/tail retraction of M0 and M2 macrophages, and decreased expression of Arg-1 in M2 macrophages. The treatment of M1 macrophages caused only a 30% increase in the ATP level and ATP/ADP ratio, and while it did not affect the shape of M1 macrophages, it increased the production of iNos. This indicates that the maintenance of mouse macrophage phenotypes depends on mitochondrial function and ATP/ADP homeostasis.

Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

Mitochondrial electron transport is essential for oxidative phosphorylation (OXPHOS). Electron transport chain (ETC) activity generates an electrochemical gradient that is used by the ATP synthase to make ATP. ATP synthase is organized into supramolecular units called synthasomes that increase the efficiency of ATP production, while within ATP synthase is the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (PTP). We investigated whether synthasomes are dynamic structures that respond to metabolic demands and whether CypD regulates this dynamic. Isolated heart mitochondria from wild-type (WT) and CypD knockout (KO) mice were treated to either stimulate OXPHOS or open the PTP. The presence and dynamics of mitochondrial synthasomes were investigated by native electrophoresis, immunoprecipitation, and sucrose density centrifugation. We show that stimulation of OXPHOS, inhibition of the PTP, or deletion of CypD increased high order synthasome assembly. In contrast, OXPHOS inhibition or PTP opening increased synthasome disassembly in WT, but not in CypD KO heart mitochondria. CypD activity also correlated with synthasome assembly in other tissues, such as liver and brain. We conclude that CypD not only regulates the PTP, but also regulates the dynamics of synthasome assembly depending on the bioenergetic state of the mitochondria.

Protective effect of casuarinin against glutamate-induced apoptosis in HT22 cells through inhibition of oxidative stress-mediated MAPK phosphorylation

Glutamate is the major excitatory neurotransmitter in the central nervous system and is involved in oxidative stress during neurodegeneration. In the present study, casuarinin prevented glutamate-induced HT22 murine hippocampal neuronal cell death by inhibiting intracellular reactive oxygen species (ROS) production. Moreover, casuarinin reduced chromatin condensation and annexin-V-positive cell production induced by glutamate. We also confirmed the underlying protective mechanism of casuarinin against glutamate-induced neurotoxicity. Glutamate markedly increased the phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38, which are crucial in oxidative stress-mediated neuronal cell death. Conversely, treatment with casuarinin diminished the phosphorylation of ERK1/2 and P38. In conclusion, the results of this study suggest that casuarinin, obtained from natural products, acts as potent neuroprotective agent by suppressing glutamate-mediated apoptosis through the inhibition of ROS production and activation of the mitogen activated protein kinase (MAPK) pathway. Thus, casuarinin can be a potential therapeutic agent in the treatment of neurodegenerative diseases.

Transcriptomic data from two primary cell models stimulating human monocytes suggest inhibition of oxidative phosphorylation and mitochondrial function by N. meningitidis which is partially up-regulated by IL-10

BackgroundBiological interpretation of DNA microarray data may differ depending on underlying assumptions and statistical tests of bioinformatics tools used. We used Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to analyze previously generated DNA microarray data from human monocytes stimulated with N. meningitidis and IL-10 (“the model system”), and with meningococcal sepsis plasma before and after immunodepletion of IL-10 (“the patient plasma system”). The objectives were to compare if the two bioinformatics methods resulted in similar biological interpretation of the datasets, and to identify whether GSEA provided additional insight compared with IPA about the monocyte host response to meningococcal activation.ResultsIn both experimental models, GSEA and IPA identified genes associated with pro-inflammatory innate immune activation, including TNF-signaling, Toll-like receptor signaling, JAK-STAT-signaling, and type I and type II interferon signaling. GSEA identified genes regulated by the presence of IL-10 with similar gene sets in both the model system and the patient plasma system. In the model system, GSEA and IPA in sum identified 170 genes associated with oxidative phosphorylation/mitochondrial function to be down-regulated in monocytes stimulated with meningococci. In the patient plasma system, GSEA and IPA in sum identified 122 genes associated with oxidative phosphorylation/mitochondrial dysfunction to be down-regulated by meningococcal sepsis plasma depleted for IL-10. Using IPA, we identified IL-10 to up-regulate 18 genes associated with oxidative phosphorylation/mitochondrial function that were down-regulated by N. meningitidis.ConclusionsBiological processes associated with the gene expression changes in the model system of meningococcal sepsis were comparable with the results found in the patient plasma system. By combining GSEA with IPA, we discovered an inhibitory effect of N. meningitidis on genes associated with mitochondrial function and oxidative phosphorylation, and that IL-10 partially reverses this strong inhibitory effect, thereby identifying, to our knowledge, yet another group of genes where IL-10 regulates the effect of LPS. We suggest that relying on a single bioinformatics tool together with an arbitrarily chosen filtering criteria for data analysis may result in overlooking relevant biological processes and signaling pathways associated with genes differentially expressed between compared experimental conditions.

Drug resistance induces the upregulation of H2S-producing enzymes in HCT116 colon cancer cells

Hydrogen sulfide (H2S) production in colon cancer cells supports cellular bioenergetics and proliferation. The aim of the present study was to investigate the alterations in H2S homeostasis during the development of resistance to 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent. A 5-FU-resistant HCT116 human colon cancer cell line was established by serial passage in the presence of increasing 5-FU concentrations. The 5-FU-resistant cells also demonstrated a partial resistance to an unrelated chemotherapeutic agent, oxaliplatin. Compared to parental cells, the 5-FU-resistant cells rely more on oxidative phosphorylation than glycolysis for bioenergetic function. There was a significant increase in the expression of the drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP2A6 in 5-FU-resistant cells. The CYP450 inhibitor phenylpyrrole enhanced 5-FU-induced cytotoxicity in 5-FU-resistant cells. Two major H2S-generating enzymes, cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) were upregulated in the 5-FU-resistant cells. 5-FU-resistant cells exhibited decreased sensitivity to the CBS inhibitor aminooxyacetate (AOAA) in terms of suppression of cell viability, inhibition of cell proliferation and inhibition of oxidative phosphorylation. However, 5FU-resistant cells remained sensitive to the antiproliferative effect of benserazide (a recently identified, potentially repurposable CBS inhibitor). Taken together, the current data suggest that 5-FU resistance in HCT116 cells is associated with the upregulation of drug-metabolizing enzymes and an enhancement of endogenous H2S production. The anticancer effect of prototypical H2S biosynthesis inhibitor AOAA is impaired in 5-FU-resistant cells, but benserazide remains efficacious. Pharmacological approaches aimed at restoring the sensitivity of 5-FU-resistant cells to chemotherapeutic agents may be useful in the formulation of novel therapeutic strategies against colorectal cancer.

Abstract B12: Analysis of Lipid Metabolism and Gene Expression in Hepatocellular Carcinoma Reveals Mitochondrial Function as a Potential Target for Combinatorial Treatment

Abstract Background: Hepatocellular carcinoma (HCC), the most common malignant liver tumor, is a heterogeneous disease with little efficacious chemotherapy. Lipid metabolic dysfunction is integral to the pathogenesis of HCC, but the underlying mechanisms remain poorly understood. HCC cell lines and patient tumors can be classified into hepatocyte and hepatoblast subtypes based on gene expression patterns, with the hepatoblast type being more aggressive. Methods: We characterized the RNA expression profiles of 20 HCC cell lines (13 hepatoblast and 7 hepatocyte type) and 1 normal immortalized liver cell line. We also characterized the steady state concentrations of hundred of metabolites of these cells lines. In addition, to find the specific small molecules to inhibit their growth we tested a panel of metabolic pathway inhibitors for activity against these cell lines. Results: There are significant differences between the transcriptional profiles of the two subtypes of HCC, as well as between HCC cell lines and the normal liver cell line. The most important determinants of these differences are in lipid metabolic pathways. Analysis of the microarray data, corroborated by real time quantitative PCR analysis showed significant difference between transcript levels of ACOX1, ACOX2, FASN, SCD1, and SCAP, as well as in fatty acid transport proteins (FATPs, SLC27As) and elongation enzymes (ELOVL). Metabolomic studies show that not only are there differences between the steady state amounts of long chain and very long chain fatty acids among the groups, but there are also differences in the levels of acyl-carnitines. These differences indicate that there is variability in their reliance on mitochondrial oxidative respiration. Accordingly, there are significant differences between the sensitivity of the HCC cell lines to oligomycin compared with a normal liver cell line. Conclusion: These data point to the importance of lipid metabolic and mitochondrial oxidative dysfunction in the pathogenesis of hepatocellular carcinoma. We anticipate combining the inhibition of oxidative phosphorylation with the standard of care treatment consisting of sorafenib to allow for a much lower and better tolerated dose of both drugs while maintaining anti-tumor efficacy. Citation Format: Ali Zarrinpar, Tiziana Palumbo, Dinesh Barupal. Analysis of Lipid Metabolism and Gene Expression in Hepatocellular Carcinoma Reveals Mitochondrial Function as a Potential Target for Combinatorial Treatment [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 B12.

Metabolic heterogeneity and plasticity of glioma stem cells in a mouse glioblastoma model.

Glioblastomas have been shown to rely on glycolysis as an energy source. However, recent evidence suggests that at least a subset of glioma cells with stem cell-like properties can thrive on oxidative phosphorylation. It remains unclear whether both metabolic phenotypes support tumor propagation, if they are independent, and how stable they are. The present study investigated these questions with the use of isogenic murine glioma stem cells (GSCs). GSCs were established from tumors formed by Ink4a/Arf-null, H-RasV12-expressing glioma-initiating cells that differed in extracellular acidification potential. Metabolic characteristics of GSCs were determined by measurement of glucose, oxygen, and glutamine uptake, ATP content, and lactate production. Effects of metabolic inhibitors and changes in oxygen or nutrient availability on lactate production and tumorsphere growth were also determined. GSCs were found either to consume more glucose and produce more lactate or to consume more oxygen and maintain a higher ATP content depending on the metabolic characteristics of the tumor cells of origin. The latter, mitochondrial-type GSCs increased lactate production after treatment with the oxidative phosphorylation inhibitor oligomycin or phenformin. Exposure to hypoxia also increased lactate production and expression of glycolysis-related enzymes and metabolites in mitochondrial-type GSCs in a reversible manner. Both glycolytic and mitochondrial-type energy production can sustain tumor propagation by isogenic GSCs. Whereas both phenotypes can be independent and stable, cells that rely on oxidative phosphorylation can also switch to a more glycolytic phenotype in response to metabolic stress, suggesting that plasticity is a further characteristic of GSC metabolism.

Insult dependent oxidative‐induced cell death

SummaryMitochondrial dysfunction caused by inhibition of oxidative phosphorylation results both in reduced ATP production and oxidative stress because of a significant elevation of ROS. In the reported studies oxidative stress to cultures of cells (RGC‐5 cells) was initiated by inhibition of mitochondrial phosphorylation (blue light, sodium azide) or by use of hydrogen peroxide known to induce superoxide through the activation of cellular NADPH oxidase. While all insults resulted in cell death and attenuated by the antioxidant EGCG differences occurred in various measured biochemical parameters implicated in cell death. For example, blue light‐induced apoptosis was attenuated by the presence of the mitochondrial the uncoupler M3778 but potentiated by the presence of cobalt. In contrast, hydrogen‐peroxide‐induced apoptosis was unaffected by M3778 but attenuated by cobalt. Also, blue light‐induced cell death occurs via necroptosis, in that it was inhibited by necrostatin‐1 and was caspase‐independent. This was not the case for sodium azide, where the death process was caspase‐dependent, occurred via apoptosis and was unaffected by necrostatin‐1.

Exploring acute myeloid leukaemia bioenergetics and drug sensitivity by rendering acute myeloid leukaemia cell lines impaired for either glycolysis or oxidative phosphorylation

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy that results in the overproduction of immature myeloid cells. While chemotherapy is the standard treatment, majority of patients relapse or are refractory to treatment, highlighting the need to develop new therapeutic options. Since cancer cells are known to undergo severe metabolic reprogramming, targeting AML aberrant bioenergetics could be a more effective therapeutic approach. The two main processes for energy production are glycolysis and oxidative phosphorylation. Therefore, AML cell lines made deficient in one or the other of these metabolic pathways can be used to characterise AML metabolic reliance and establish a screen for drugs targeting each metabolic dependency. Two AML cell lines have been treated with 2DG (a glycolytic inhibitor) or oligomycin (an inhibitor of oxidative phosphorylation), in order to obtain drug resistant cells that are either glycolysis- or oxidative phosphorylation-deficient, respectively. These cells have been tested for their acquired drug resistance and their metabolic phenotype. Drug resistance was confirmed by running the cells in survival assays where increased survival was observed when the cells were treated with 2DG (for 2DG-resistant cells) or oligomycin (for oligomycin-resistant cells) compared to parental cells. To assess the metabolic phenotypes, the set of resistant and parental cells have been run in a number of metabolic assays that have established the impaired glycolysis and oxidative phosphorylation profile for the 2DG-resistant and oligomycin-resistant cells, respectively. These cells can now be used in drug screening where the glycolytic deficient cells would be sensitive to drugs that target oxidative phosphorylation while the oxidative phosphorylation deficient cells would be sensitive to glycolysis targeting drugs. The discovery of such drugs targeting both metabolic pathways can then be tested further in combination studies. This study offers a new way to treat AML by uncovering new drug combinations able to target malignant metabolism bioenergetics.

Disrupting mitochondrial Ca2+ homeostasis causes tumor-selective TRAIL sensitization through mitochondrial network abnormalities

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycinA and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers withminimally compromising the tumor-selectivity.

Roles of autophagy and metabolism in pancreatic cancer cell adaptation to environmental challenges.

Pancreatic ductal adenocarcinoma (PDAC) displays extensive and poorly vascularized desmoplastic stromal reaction, and therefore, pancreatic cancer (PaCa) cells are confronted with nutrient deprivation and hypoxia. Here, we investigate the roles of autophagy and metabolism in PaCa cell adaptation to environmental stresses, amino acid (AA) depletion, and hypoxia. It is known that in healthy cells, basal autophagy is at a low level, but it is greatly activated by environmental stresses. By contrast, we find that in PaCa cells, basal autophagic activity is relatively high, but AA depletion and hypoxia activate autophagy only weakly or not at all, due to their failure to inhibit mechanistic target of rapamycin. Basal, but not stress-induced, autophagy is necessary for PaCa cell proliferation, and AA supply is even more critical to maintain PaCa cell growth. To gain insight into the underlying mechanisms, we analyzed the effects of autophagy inhibition and AA depletion on PaCa cell metabolism. PaCa cells display mixed oxidative/glycolytic metabolism, with oxidative phosphorylation (OXPHOS) predominant. Both autophagy inhibition and AA depletion dramatically decreased OXPHOS; furthermore, pharmacologic inhibitors of OXPHOS suppressed PaCa cell proliferation. The data indicate that the maintenance of OXPHOS is a key mechanism through which autophagy and AA supply support PaCa cell growth. We find that the expression of oncogenic activation mutation in GTPase Kras markedly promotes basal autophagy and stimulates OXPHOS through an autophagy-dependent mechanism. The results suggest that approaches aimed to suppress OXPHOS, particularly through limiting AA supply, could be beneficial in treating PDAC.NEW & NOTEWORTHY Cancer cells in the highly desmoplastic pancreatic ductal adenocarcinoma confront nutrient [i.e., amino acids (AA)] deprivation and hypoxia, but how pancreatic cancer (PaCa) cells adapt to these conditions is poorly understood. This study provides evidence that the maintenance of mitochondrial function, in particular, oxidative phosphorylation (OXPHOS), is a key mechanism that supports PaCa cell growth, both in normal conditions and under the environmental stresses. OXPHOS in PaCa cells critically depends on autophagy and AA supply. Furthermore, the oncogenic activation mutation in GTPase Kras upregulates OXPHOS through an autophagy-dependent mechanism.

Abstract 4990: High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities

Abstract Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Mårten Fryknäs. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Abstract 3702: Quantitative imaging of metabolic changes in macrophage subsets

Abstract Macrophages play an important role in tumor therapeutic response. However, current methods cannot image heterogeneities in macrophage activity on a single-cell level over time within intact, living samples. Macrophage metabolism is closely linked to macrophage function, as M1-like macrophages rely on aerobic glycolysis and M2-like macrophages rely on fatty acid oxidation and oxidative phosphorylation. Thus, we propose to develop optical metabolic imaging (OMI) using two-photon microscopy to monitor cell-level changes in macrophage polarization and functional activity. OMI exploits the auto-fluorescence intensities and lifetimes of the metabolic co-enzymes NAD(P)H and FAD, and has been previously used to dynamically image in vivo tumors with cell-level resolution. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) provides a global measure of redox balance within individual cells. The fluorescence lifetimes of NAD(P)H and FAD provide information in protein-binding activities in metabolic reactions. OMI was used to monitor metabolic changes in macrophages on a single-cell level during polarization and after metabolic perturbations. RAW 264.7 macrophages were stimulated towards an M1- or M2-like phenotype, and imaged with OMI at 24, 48, and 72 hours post-polarization. M1- and M2-like macrophages exhibited significant differences (p<0.05) in redox ratio and FAD fluorescence lifetime values at 48 and 72 hours post-polarization. The M1-like and M2-like character of the macrophages were validated with quantitative polymerase chain reaction (qPCR) analysis. Population density modeling of single-cell metabolism established differences in intra-sample heterogeneity across subsets over time. M1- and M2-like macrophages were also treated with 2-deoxy-glucose (glycolysis inhibitor), etomoxir (fatty acid oxidation inhibitor), and cyanide (oxidative phosphorylation inhibitor. Treatment with etomoxir caused a significant decrease in redox ratio in M2-like macrophages (p<0.05) with no significant effect on M1-like macrophages. In contrast, the redox ratio decreased in M1-like macrophages upon inhibition of glycolysis (p<0.05). Comparable to previously published data, inhibition of oxidative phosphorylation in both groups yielded a decreased NAD(P)H mean lifetimes (p<0.05) and increased FAD mean lifetimes (p<0.05). These results establish OMI as a method to image heterogeneity in macrophage populations within intact, living samples. This method could be used to understand macrophage interactions in the microenvironment of intact in vivo tumors. Citation Format: Tiffany M. Heaster, David L. Elion, Rebecca S. Cook, Melissa C. Skala. Quantitative imaging of metabolic changes in macrophage subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3702. doi:10.1158/1538-7445.AM2017-3702

Abstract 1487: ONC201 kills breast cancer cells by inhibiting mitochondrial respiration

Abstract Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. Methods: ONC201 was obtained from Oncoceutics, Inc. Recombinant GST-TRAIL was prepared in the laboratory. Cell viability was tested with MTS assay and CellTiter-Glo luminescent cell viability assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNAseq and western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal microscopy and electron microscopy analysis were performed to study mitochondrial morphology. Results: We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases and live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggest that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces the stress pathway proteins ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate but did not inhibit glycolysis as measured by the extracellular acidification rate. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. Supplementing glucose to cells grown in galactose medium partially prevented ONC201-dependent ATP depletion, induction of phospho-AMPK, ATF4 and CHOP, and cell death. These data are consistent with an inhibition of oxidative phosphorylation (OxPhos) by ONC201. RNAseq revealed ONC201 inhibits expression of multiple mitochondrial genes involved in OxPhos and other mitochondrial functions, and western blot confirmed those findings. Confocal and electron microscopic evaluation revealed abnormal mitochondrial morphology. Conclusion: Our data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving disruption of mitochondrial morphology and inhibition of mitochondrial respiration. Citation Format: Yoshimi Greer, Samuel Gilbert, Celia Islam, Yun Ji, Luca Gattinoni, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Xiantao Wang, Markus Hafner, Kunio Nagashima, Donna Voeller, Stanley Lipkowitz. ONC201 kills breast cancer cells by inhibiting mitochondrial respiration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2017-1487

Abstract 1021: Mutations in SWI/SNF chromatin remodeling complex sensitize tumors to OXPHOS inhibition

Abstract Recent large-scale cancer genomic studies have established a framework in which biological functions and genetic interactions of established and novel cancer genes can be explored. One of the major findings of these studies has been the discovery of frequent genetic alterations in chromatin and epigenetic regulators including inactivating mutations in components of the SWI/SNF chromatin remodeling complex. In lung adenocarcinoma in particular, a quarter of tumors have inactivating mutations in the SWI/SNF components Smarca4, ARID1A or the histone modifier SETD2. With the aim of understanding the mechanism of tumor development and identifying potential vulnerabilities of SWI/SNF mutant tumor, we developed a genetically engineered mouse model (GEMM) of lung adenocarcinoma by selectively ablating Smarca4 in the respiratory epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss. Cross species integrative gene expression analyses revealed signatures of enhanced oxidative phosphorylation and reactive oxygen species (ROS) response in Smarca4 mutant murine and human lung tumors. We further show that Smarca4 mutant cells have increased oxygen consumption, enhanced basal and maximal respiratory capacity and production of ROS. This is primarily driven by activation of the mitochondrial master regulator, PGC1alpha. Finally, we show that Smarca4 and other SWI/SNF mutant lung cancer cell lines have exquisite sensitivity to inhibition of oxidative phosphorylation using a novel small molecule that is under development. These findings provide a mechanistic basis for further development of OXPHOS inhibitors as therapies in SWI/SNF mutant tumors. Citation Format: Yonathan Lissanu Deribe. Mutations in SWI/SNF chromatin remodeling complex sensitize tumors to OXPHOS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1021. doi:10.1158/1538-7445.AM2017-1021

P 019 - Alterations of cultured myotubes and fasting plasma metabolite profiles related to mitochondrial dysfunction in Type 2 diabetes subjects

A plethora of metabolic profiling studies have identified several metabolic signatures in the patient’s biofluids and tissues to be correlated with type 2 diabetes (T2D) and its comorbidities. However, the association between specific metabolite-based markers and occurrence of mitochondrial dysfunction among T2D subjects has not been adequately addressed. Therefore, the current study sought to determine whether circulating metabolite signatures in cultured myotubes and fasting plasma of T2D subjects are associated with mitochondrial dysfunction. A cellular disease model was first established using human myotubes treated with antimycin A, an oxidative phosphorylation inhibitor. The cultured sample of these intracellular-defined metabolites on treated myotubes was analyzed using mass spectrometry based approaches. Further, a targeted metabolic profiling of fasting blood plasma from normal (n=83) and T2D (n=92) subjects in a cross sectional study was validated. Multivariable-adjusted conditional logical regression analysis was computed to estimate odds ratios (ORs) and 95% confidence intervals for verifying differentiating metabolites correlated with T2D. Several metabolites were considerably altered in cultured myotubes with reduced mitochondrial function. Targeted metabolic fingerprinting of fasting blood plasma adjusted for age, sex and body mass index (BMI) revealed 20 significant robust metabolites (P

Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation

Triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Qd-binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues.

Modulation of the energy metabolism inhibits plasmacytoid dendritic cell activation and delays onset of autoantibody production in murine models of systemic lupus erythematosus

Abstract Despite the heterogeneity of systemic lupus erythematosus (SLE) clinical manifestations, an increased expression of type I interferon (IFN) regulated genes, termed the interferon signature, has been reported in most SLE patients. Long recognized to facilitate the progression of SLE, Type I IFNs can be produced in small amounts by most cell types, but the plasmacytoid dendritic cells (pDCs) are the primary producers. Since murine models of SLE have shown that depletion of pDCs stop the onset and progression of autoimmunity, we speculated that it is important to suppress pDC activities that go beyond the production of type I IFNs. We have confirmed that changes in fatty acid metabolism are essential for pDC activation. Using inhibitors of fatty acid oxidation, oxidative phosphorylation, and glycolysis, we have found that metabolic modulation prevents pDC activation upon stimulation with TLR ligands CpG and R848. This metabolic inhibition did not affect pDC viability. Moreover, in vivo treatment of mice with metabolic inhibitors delayed the onset of autoantibody production in murine models of SLE. Indeed, the inhibition of energy metabolism strongly reduced the production of anti-dsDNA and antichromatin autoanti-bodies in i) a model of lupus induced by Graft versus Host Disease (GvHD) and ii) in lupus-prone mice accelerated by TLR stimulation. Therefore, our findings suggest that the in vivo suppression of the major producers of type I IFN through inhibition of energy metabolism may therapeutically downregulate the production of autoantibodies in murine lupus. Our data raises the possibility of manipulating cellular metabolic pathways to regulate the detrimental type I IFN effects in SLE.

The Symbiotic Bacterium Fuels the Energy Metabolism of the Host Trypanosomatid Strigomonas culicis

The mutualistic relationship between trypanosomatids and their respective endosymbiotic bacteria represents an excellent model for studying metabolic co-evolution since the symbiont completes essential biosynthetic routes of the host cell. In this work, we investigated the influence of the endosymbiont on the energy metabolism of Strigomonas culicis by comparing the wild strain with aposymbiotic protists. The bacterium maintains a frequent and close association with glycosomes, which are distributed around the prokaryote. Furthermore, 3D reconstructions revealed that the shape and distribution of glycosomes are different in symbiont-bearing protists compared to symbiont-free cells. Results of bioenergetic assays showed that the presence of the symbiont enhances the O2 consumption of the host cell. When the quantity of intracellular or released glycerol was evaluated, the aposymbiotic strain presented higher values when compared to symbiont-containing cells. Furthermore, inhibition of oxidative phosphorylation by potassium cyanide increased the rate of glycerol release and slightly diminished the ATP content in cells without the symbiont, indicating that the host trypanosomatid enhances its fermentative activity when the bacterium is lost.