Targeting Cell Metabolism Research Articles

Coagulation factors VII, IX and X are effective antibacterial proteins against drug-resistant Gram-negative bacteria

Infections caused by drug-resistant “superbugs” pose an urgent public health threat due to the lack of effective drugs; however, certain mammalian proteins with intrinsic antibacterial activity might be underappreciated. Here, we reveal an antibacterial property against Gram-negative bacteria for factors VII, IX and X, three proteins with well-established roles in initiation of the coagulation cascade. These factors exert antibacterial function via their light chains (LCs). Unlike many antibacterial agents that target cell metabolism or the cytoplasmic membrane, the LCs act by hydrolyzing the major components of bacterial outer membrane, lipopolysaccharides, which are crucial for the survival of Gram-negative bacteria. The LC of factor VII exhibits in vitro efficacy towards all Gram-negative bacteria tested, including extensively drug-resistant (XDR) pathogens, at nanomolar concentrations. It is also highly effective in combating XDR Pseudomonas aeruginosa and Acinetobacter baumannii infections in vivo. Through decoding a unique mechanism whereby factors VII, IX and X behave as antimicrobial proteins, this study advances our understanding of the coagulation system in host defense, and suggests that these factors may participate in the pathogenesis of coagulation disorder-related diseases such as sepsis via their dual functions in blood coagulation and resistance to infection. Furthermore, this study may offer new strategies for combating Gram-negative “superbugs”.

PF197 A NEW CLASSIFICATION OF ACUTE MYELOID LEUKEMIA BASED ON INTEGRATED GENOMICS AND METABOLOMICS

Background:An expanding body of evidence indicates that targeting cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML). Pyrimidine biosynthesis, oxidative phosphorylation, amino acid and arginine metabolism are among the most relevant actionable pathways (Sykes et al. Cell 2016; Molina et al. Nat Med 2018; Jones et al. Cancer Cell 2018; Mussai et al. Blood 2015). However, few studies identified specific metabolic vulnerabilites (Fenouille et al. Nat Med 2017; Gallipoli et al. Blood 2018) due to the lack of a comprehensive characterization of AML cell metabolism.Aims:The study aimed to stratify AML patients based on their metabolic landscape and integrate their genomic and metabolic profiles.Methods:Genomic data of AML patients were obtained by whole exome sequencing (n = 165) or targeted NGS (n = 18). Of them, 119 AML were evaluated for serum metabolites by nuclear magnetic resonance (NMR), along with 145 healthy donors. Intracellular metabolites of AML bone marrow cells (35 CD34+ and 15 CD33+CD14− samples) and controls (21 cord blood CD34+ and 21 peripheral blood CD33+ samples) were analyzed by mass spectrometry (Metabolon).Results:Unsupervised hierarchical clustering of AML based on the intracellular metabolic landscape clearly defined 3 clusters (C, Fig.A). C1 was characterized by high metabolite concentration, C2 by low metabolite levels and C3 by a more complex metabolic profile. By integrating genomic data into the metabolic classification we obtained a significant clusterization (p = 0.029). C1 was enriched for NPM1‐mutated (mut) AML (50%), C2 for cases with altered chromatin/splicing genes or inv(3) (37% and 13%, respectively), C3 for TP53‐mut/aneuploid AML (35%). Moreover, C3 included all core binding factor AML. By training a Gaussian Process classifier on NMR data, we obtained a significant separation of C1 and C3 also at serum level (Fig. B,C). In particular, NPM1‐mut AML displayed abundance of tricarboxylic cycle byproducts, while glutamine and creatinine were increased in NPM1‐wildtype (wt) cases (Fig.D,E). At intracellular level, NPM1‐mut AML were characterized by increased intermediates of purine and pyrimidine metabolism, in line with an active biosynthetic activity. Moreover, unsupervised NMR data analysis identified a clear separation (0:7 accuracy) between TP53‐wt and mut/deleted AML, the latter showing reduced threonine, alanine, glucose, lactate and glutamine serum concentration (Fig.F).At genomic level, 88% of patients carried at least one mutation in a metabolism‐related gene including enzymes (encoded by nuclear or mitchondrial DNA) and metabolic regulators, with lipid metabolism, cellular respiration (IDH1/2 mutations and oxidative phosphorylation with altered UQCRH, FASTKD3, ND and COX genes), metabolism of carbohydrates (e.g. glycosyltransferase GXYLT1, GDP‐mannose biosynthetic process involving PMM2), glucose (e.g. glycolysis‐related genes HK2, HK3, PKLR, BPGM) and nucleotides (e.g. AK2, AK5: nucleotide phosphate group transfer, NME7: synthesis of nucleoside triphosphates) being the pathways most frequently targeted by mutations.Summary/Conclusion:AML is characterized by a number of functional and genomic‐driven metabolic alterations. The integration of genomic and metabolic profiles provided a novel refined AML classification and suggested subtype‐specific metabolic targets, including nucleotide metabolic pathways and bioenergetics in NPM1‐mut and TP53‐mut/del AML, respectively.imageSupported by: EHA research fellowship award, AIRC, FP7‐NGS‐PTL, Fondazione del Monte.

Application of targeting cell metabolism in immune disease

Recent studies have shown that cell metabolism plays an important role in the regulation and function of immune cells. By targeting the regulation of immune cells in the metabolic pathway, the generation, effect and outcome of immune response can be affected, the occurrence and development of certain immune pathology could also be influenced. Further study of the metabolic characteristics of immune cells and the relevant regulatory mechanisms can offer more theoretical basis for targeting metabolism in the treatment of immune diseases and establish the foundation for the research of novel targeted medicine. The aim of this article is to introduce the progress of the application of targeting metabolism in the treatment of immune disorders, we summarize the metabolic characteristics of different immune cells and their regulatory mechanisms and introduce the application of targeting metabolism in bone marrow transplantation, solid organ transplantation, systemic lupus erythematosus (SLE) and sepsis. Key words: Targeting metabolism; Immune diseases

Hexosamine Biosynthetic Pathway Inhibition Leads to AML Cell Differentiation and Cell Death.

Treatment for acute myeloid leukemia (AML) has remained unchanged for past 40 years. Targeting cell metabolism is a promising avenue for future cancer therapy. We found that enzymes involved in metabolic hexosamine biosynthetic pathway (HBP) are increased in patients with AML. Inhibiting GFAT (the rate-limiting enzyme of HBP) induced differentiation and apoptosis in AML cells, sparing normal cells. UDP-GlcNAc, the end product of HBP, is the substrate for O-GlcNAcylation, a posttranslational modification. O-GlcNAc transferase (OGT) is the enzyme which transfers GlcNAc from UDP-GlcNAc to target proteins. Inhibition of O-GlcNAcylation, using OGT inhibitors as well as genetic knockdown of OGT, also led to cell differentiation and apoptosis of AML cells. Finally, HBP inhibition in vivo reduced the tumor growth in a subcutaneous AML xenograft model and tumor cells showed signs of differentiation in vivo A circulating AML xenograft model also showed clearance of tumor load in bone marrow, spleen, and blood, after HBP inhibition, with no signs of general toxicity. This study reveals an important role of HBP/O-GlcNAcylation in keeping AML cells in an undifferentiated state and sheds light into a new area of potential AML therapy by HBP/O-GlcNAc inhibition. Mol Cancer Ther; 17(10); 2226-37. ©2018 AACR.

Corrigendum: Zoledronate Triggers Vδ2 T Cells to Destroy and Kill Spheroids of Colon Carcinoma: Quantitative Image Analysis of Three-Dimensional Cultures.

[This corrects the article on p. 998 in vol. 9, PMID: 29867975.].

Zoledronate Triggers Vδ2 T Cells to Destroy and Kill Spheroids of Colon Carcinoma: Quantitative Image Analysis of Three-Dimensional Cultures.

New successful anti-cancer strategies are based on the stimulation of immune reaction against tumors: however, preclinical testing of such treatments is still a challenge. To improve the screening of anti-cancer drugs, three-dimensional (3D) culture systems, including spheroids, have been validated as preclinical models. We propose the spheroid 3D system to test anti-tumor drug-induced immune responses. We show that colorectal carcinoma (CRC) spheroids, generated with the epithelial growth factor (EGF), can be co-cultured with Vδ2 T cells to evaluate the anti-tumor activity of these effector lymphocytes. By computerized image analysis, the precise and unbiased measure of perimeters and areas of tumor spheroids is achievable, beside the calculation of their volume. CRC spheroid size is related to ATP content and cell number, as parameters for cell metabolism and proliferation; in turn, crystal violet staining can check the viability of cells inside the spheroids to detect tumor killing by Vδ2 T cells. In this 3D cultures, we tested (a) zoledronate that is known to activate Vδ2 T cells and (b) the therapeutic anti-EGF receptor humanized antibody cetuximab that can elicit the antibody-dependent cytotoxicity of tumor cells by effector lymphocytes. Zoledronate triggers Vδ2 T cells to kill and degrade CRC spheroids; we detected the T-cell receptor dependency of zoledronate effect, conceivably due to the recognition of phosphoantigens produced as a drug effect on target cell metabolism. In addition, cetuximab triggered Vδ2 T lymphocytes to exert the antibody-dependent cellular cytotoxicity of CRC spheroids. Finally, the system reveals differences in the sensitivity of CRC cell lines to the action of Vδ2 T lymphocytes and in the efficiency of anti-tumor effectors from distinct donors. A limitation of this model is the absence of cells, including fibroblasts, that compose tumor microenvironment and influence drug response. Nevertheless, the system can be improved by setting mixed spheroids, made of stromal and cancer cells. We conclude that this type of spheroid 3D culture is a feasible and reliable system to evaluate and measure anti-tumor drug-induced immune responses beside direct anti-cancer drug effect.

Impact of Aldehyde Dehydrogenase Activity on Gliomas

High-grade gliomas are primary brain tumors with a highly aggressive, invasive phenotype and dismal outcome. Targeting cell metabolism and cancer bioenergetics has emerged as a potential effective therapeutic approach. Selective aldehyde dehydrogenase isoenzyme activity regulates glioma tumor growth, invasiveness, and resistance to current treatments, and has potential as a therapeutic target.

TLR-mediated metabolic reprogramming in the tumor microenvironment: potential novel strategies for cancer immunotherapy

Cellular energy metabolism not only promotes tumor cell growth and metastasis but also directs immune cell survival, proliferation and the ability to perform specific and functional immune responses within the tumor microenvironment. A better understanding of the molecular regulation of metabolism in different cell components in the tumor-suppressive microenvironment is critical for the development of effective strategies for human cancer treatments. Toll-like receptors (TLRs) have recently been recognized as critical factors involved in tumor pathogenesis, regulating both tumor cells and tumor-infiltrating innate and adaptive immune cells. However, little is known about the molecular crosstalk between TLR signaling and tumor or/and immune cell metabolism, although there is abundant expression of TLRs in these cells. In this review, we explore the functional role of TLR signaling in reprogramming cell metabolism in the tumor microenvironment. In particular, we discuss how malignant tumors regulate metabolism to support their growth and survival, summarize more recently identified metabolic profiles of different immune cell subsets and TLR-mediated regulation of cellular metabolism in both tumor and immune cells, and further explore potential strategies targeting cell metabolism for TLR-based cancer therapy. An improved understanding of these issues should open new avenues for the development of novel strategies via TLR-mediated metabolic reprogramming of the tumor microenvironment for cancer immunotherapy.

Metformin and temozolomide, a synergic option to overcome resistance in glioblastoma multiforme models.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration.

Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles

ABSTRACTExtracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

Abstract 5212: Metabolic reprogramming of radiation fibrosis using adipose derived stromal cells

Abstract Background: Soft tissue radiation fibrosis (RF) affects up to 70% of cancer survivors post-radiotherapy. RF is an irreversible and progressive side effect of radiotherapy characterized by poor tissue elasticity and increased ECM deposition, clinically translating to increased morbidity due to hardening, distortion, and pain. Recent evidence has highlighted the potential role of metabolic alterations in the onset and progression of fibrosis. Adipose derived stromal cells (ADSCs) have been used effectively for enhancing complex wound repair in a number of clinical trials. The therapeutic effect of ADSCs has been attributed to its secretion of paracrine factors, which can regulate the metabolism of target cells. Even so, the metabolic effects of ADSCs on target cells is not well described. Methods and Results: Transcriptomic profiling and targeted metabolomics of human radiated tissue and a murine model of RF revealed that suppression of fatty acid oxidation (FAO) is hallmark of RF. TGF-B, a master regulator of fibrosis, has a large effect in suppressing FAO in primary human fibroblasts in vitro through downregulating genes in the PPAR pathway, a major regulatory pathway for FAO, and through inhibiting oxidation of the long chain fatty acid, palmitic acid (p<0.05, t-test). Treatment of TGF-B stimulated fibroblasts with ADSC conditioned media resulted in an improvement in FAO and a reduction in fibronectin, collagen-1, and PAI-1 protein levels, three of major contributors to fibrosis. Transplantation of ADSCs into murine RF resulted in a metabolic shift back to FAO and a significant reduction in RF both functionally (percentage leg contracture 36% vs 26%, 2-way ANOVA < 0.05) and histologically through image analysis of trichrome staining (p<0.01, t-test). Pathway analysis of significant genes altered by ADSCs revealed an upregulation of the PPAR pathway was the most significant effect of ADSC transplantation. Untargeted metabolomics confirmed that FAO metabolites were significantly upregulated with ADSC transplantation. To confirm the importance of metabolic regulation by ADSCs, we utilized a pharmacogenomics strategy to uncover a compound that mimicked the effect of ADSCs. Drug A ranked highest and replicated the anti-fibrotic and pro-FAO effects of ADSCs both on TGF-B stimulated primary fibroblasts and in murine RF. Etomoxir, a FAO inhibitor, inhibited the effect of ADSCs and Drug A in regulating protein levels of fibronectin and collagen, revealing that ADSCs and Drug A require an intact FAO pathway to exert their anti-fibrotic effects. Conclusions: Inhibition of FAO is a novel hallmark of RF. ADSC-mediated metabolic reprogramming resulted in an enhancement of FAO and a reduction in RF through upregulating the PPAR pathway. ADSC-directed pharmacogenomics uncovered Drug A, which mimicked the pro-FAO and anti-fibrotic effects of ADSCs. Our research has highlighted the importance of reversing metabolic aberrations to reduce RF. Citation Format: Xiao Zhao, Laleh Soltan Ghoraie, Pamela Psarianos, Kenneth Yip, Laurie Ailles, Benjamin Haibe-Kains, Fei-Fei Liu. Metabolic reprogramming of radiation fibrosis using adipose derived stromal cells [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 5212. doi:10.1158/1538-7445.AM2017-5212

3-bromopyruvate ameliorate autoimmune arthritis by modulating Th17/Treg cell differentiation and suppressing dendritic cell activation

Recent studies have shown that cellular metabolism plays an important role in regulating immune cell functions. In immune cell differentiation, both interleukin-17-producing T (Th17) cells and dendritic cells (DCs) exhibit increased glycolysis through the upregulation of glycolytic enzymes, such as hexokinase-2 (HK2). Blocking glycolysis with 2-deoxyglucose was recently shown to inhibit Th17 cell differentiation while promoting regulatory T (Treg) cell generation. However, 2-DG inhibits all isoforms of HK. Thus, it is unclear which isoform has a critical role in Th17 cell differentiation and in rheumatoid arthritis (RA) pathogenesis. Here we demonstrated that 3-bromopyruvate (BrPA), a specific HK2 inhibitor, significantly decreased the arthritis scores and the histological scores in SKG mice, with a significant increase in Treg cells, decrease in Th17 cells, and decrease in activated DCs in the spleen. In vitro, BrPA facilitated the differentiation of Treg cells, suppressed Th17 cells, and inhibited the activation of DCs. These results suggested that BrPA may be a therapeutic target of murine arthritis. Although the role of IL-17 is not clarified in the treatment of RA, targeting cell metabolism to alter the immune cell functions might lead to a new therapeutic strategy for RA.

Cell Connections by Tunneling Nanotubes: Effects of Mitochondrial Trafficking on Target Cell Metabolism, Homeostasis, and Response to Therapy.

Intercellular communications play a major role in tissue homeostasis and responses to external cues. Novel structures for this communication have recently been described. These tunneling nanotubes (TNTs) consist of thin-extended membrane protrusions that connect cells together. TNTs allow the cell-to-cell transfer of various cellular components, including proteins, RNAs, viruses, and organelles, such as mitochondria. Mesenchymal stem cells (MSCs) are both naturally present and recruited to many different tissues where their interaction with resident cells via secreted factors has been largely documented. Their immunosuppressive and repairing capacities constitute the basis for many current clinical trials. MSCs recruited to the tumor microenvironment also play an important role in tumor progression and resistance to therapy. MSCs are now the focus of intense scrutiny due to their capacity to form TNTs and transfer mitochondria to target cells, either in normal physiological or in pathological conditions, leading to changes in cell energy metabolism and functions, as described in this review.

History of endocrinology in the world and in Poland

Endocrinology is a branch of medicine dealing with the functioning of endocrine glands. Theain role of the endocrine glands is to control various functions and to adopt internal environment to changes in the external environment. Glands manage to do this by regulating the metabolism of target cells by hormones. One of the earliest connections with endocrinology is found in theHippocratic humoral hypothesis dating about 400 years BC. The author of the first endocrinological observations was Aristotle, who noticed changes in behavior and appearance in castrated roosters. In the Middle Ages winners of battles ate their enemies organs, such as the heart, brain, or gonads, believing that they were a source of special power.However, the last two centuries were a time of intensive development of the research in this field. Certainly, we can now look to the future awaiting the next breakthroughs that will revolutionize today's perception of endocrinology.

Abstract 45: Preventing and treating hepatic metastatic colon and pancreatic cancers by targeting cell metabolism

Abstract One fundamental change in cancer cells in oncogenesis is the alteration of glucose metabolism. With few exceptions, cancer cells exhibit aerobic glycolysis. A majority of pyruvate derived from glycolysis is converted to lactate instead of entering mitochondria for oxidative phosphorylation. This feature of cancer cells is also referred as “the Warburg effect”. The functional significance of the inefficient metabolic mode to cancer cells is becoming clear. In essence, reduction in the flux of pyruvate into mitochondria prevents complete oxidation of glucose. This increases production of NADPH and metabolic intermediates required for biosynthesis of macromolecules essential for cell proliferation. Pyruvate flux into mitochondria can be dramatically enhanced through mitochondrial uncoupling, a process that allows protons cross the mitochondrial inner membrane without producing ATP. As a result, mitochondrial uncoupler promotes “futile” oxidation of acetyl-CoA, leading to complete glucose oxidation. Therefore, safe mitochondrial uncouplers could be potent anti-cancer agents by antagonizing the function of the Warburg effect. Here we tested this novel cancer chemotherapeutic strategy with niclosamide ethanolamine (NEN), a well-characterized mitochondrial uncoupler with an excellent safety profile, and one of its derivative (OXY-1) in culture cell models and a mouse model. Mouse colon cancer cells (MC38) and mouse pancreatic cells (Panc02) were treated with NEN and OXY-1. At effective mitochondrial uncoupling concentrations, NEN and OXY-1 reduced cells viability, induced cell cycle arrest, and reduced clonegenicity. Moreover, NEN and OXY-1 also inhibited cell migration. These effects were associated with the activation of AMPK and reduction of NADPH/NADP ratio. We further examined the anti- cancer effect of NEN and OXY-1 on a metastatic cancer mouse model. First, we tested if the uncouplers are effective in inhibiting cancer growth. The MC38 or Panc02 cells were injected into the liver of NOD mice. Oral treatment of OXY-1 (800 ppm in food) significantly reduced tumor size and tumor incidence in mouse liver. We then tested if these drugs could inhibit tumor metastasis. MC38 or Panc02 cells were intrasplenically injected into the mice and the tumor metastasis to liver was analyzed. Oral NEN (2000 ppm in food) or OXY-1 (800 ppm in food) either totally prevented tumor metastasis to liver or drastically reduced metastatic tumor numbers and tumor volume. Taken together, our results strongly suggest that the safe and mild mitochondrial uncouplers NEN and OXY-1 could be effective in preventing and treating hepatic metastasis of colon and pancreatic cancers. Our results may provide a new and attractive way for preventing and treating these incurable tumors. Citation Format: AMER H. ALASADI, Jingjing Guo, Hanlin Tao, Shengkan Jin. Preventing and treating hepatic metastatic colon and pancreatic cancers by targeting cell metabolism. [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 45.

Abstract 1001: Expression of cytochrome C oxidase 4 (COX4) in thyroid cancer cells

Abstract Background: Targeting cell metabolism has emerged as a therapeutic strategy for the treatment of cancer. Metabolically active thyroid cancers are resistant to treatment with radioactive iodine. Aberrant expression of genes controlling glycolysis was demonstrated in thyroid cancer cells, but little is known regarding the role of mitochondrial proteins in thyroid carcinogenesis. Cytochrome c oxidase 4 (COX4) plays pivotal roles in oxidative phosphorylation and the cellular response to oxidative stress. COX4 may thus represent a promising therapeutic target. Objectives: We examined expression of COX4 in human thyroid tumors and performed functional studies using thyroid cancer cell lines. Material and Methods Expression of COX4 was examined by immunostaining in 25 follicular adenomas (FAs), 22 follicular cancers (FTCs), 90 papillary cancers (PTCs) and in 48 samples from normal thyroid tissue. FTC-derived (FTC133) and PTC-derived (BCPAP) cell lines were used to create COX4-deficient cells by lentiviral transfection. The efficiency of COX4 inhibition was examined. Mitochondrial membrane potential was examined by JC-1 staining. DNA-damage signaling was examined after cell exposure to γ radiation (6-18 Gy). We also determined the effects of 2-deoxyglucose (2DG) on viability of thyroid cancer cells with compromised COX4. Caspase-3 and PARP cleavage assays were performed to measure apoptosis. Results Positive immunostaining with anti-COX4 was detected in 6/48 (12.5%) normal tissue, 5/20 (25%) FAs, 7/22 (31%) FTCs, and 51/90 (56%) PTCs. The intensity of COX4 immunostaining was significantly higher in thyroid cancers than in either normal thyroid (p = 0.0001) or benign FAs (0.001). COX4 expression was more frequently detected in PTCs than in FTCs (p = 0.03). The mRNA level of COX4 was higher in BCPAP and FTC133 cells compared to normal thyroid. In both cell lines, silencing of COX4 altered intra-cellular distribution of JC-1 staining. In control cells, JC-1 staining was perinuclear, but in COX4-deficient cells it became diffusely cytoplasmic. COX4 silencing affected cell growth and response to γradiation in a cell type specific manner. In BCPAP cells, downregulation of COX4 was associated with inhibition of cell growth, block in G1 phase and inhibition of Cyclin D1. In BCPAP cells, COX4 silencing activated DNA-damage signaling and increased sensitivity to γ radiation. Inhibitor of glycolysis (2DG) was more efficient against COX4-deficient than COX4-expressing BCPAP cells. In FTC133 cells, silencing of COX4 increased the rate of growth and induced expression of Cyclin D1. COX4 silencing did not increase FTC133 cell sensitivity to γradiation nor to treatment with 2DG. Conclusion COX4 is implicated in regulation of thyroid cancer cell growth and response to DNA damaging or metabolic treatments. These data suggest that evaluation of COX4 in thyroid cancer could serve as a biomarker of response to treatment with metabolic agents. Citation Format: Vasyl V. Vasko, Athanasios Bikas, Aneeta Patel, John Costello, Rok Tkavc, Kenneth D. Burman, Kirk Jensen. Expression of cytochrome C oxidase 4 (COX4) in thyroid cancer cells. [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 1001.

642 - Targeting cell metabolism to improve prostate cancer therapeutics

642 - Targeting cell metabolism to improve prostate cancer therapeutics

In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state.

NADH and its oxidized form NAD(+) have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD(+)/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as SoNar (sensor of NAD(H) redox). The chimeric SoNar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein from Thermus aquaticus (T-Rex). It functions by binding to either NAD(+) or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of SoNar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing SoNar in mice. Typically, the approximate time frame for fluorescence imaging of SoNar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism.

Fluorescent and quantitative mitochondrial redox imaging of tumor targeted by Octa-RGD probe

Integrins, over-expressed in a broad range of cancer diseases, are widely utilized as a tumor biomarker. Metabolism investigation also plays important roles in tumor theranostics. Developing simple integrin-targetting probe and monitoring tumor metabolism will give opportunities to find ways for cancer treatment, however, the investigation of tumor metabolism with integrin receptor based probes has been rarely reported so far. Here, we developed an octavalent fluorescent probe Octa-RGD by convenient genetic method, based on one tetrameric far-red fluorescent protein (fRFP) linked with RGD peptides. We validated its intergin targeting by confocal imaging in vitro. Then we screened a variety of tumor cells, and differentiated their binding affinity based on the fluorescence of the probe via flow cytometry. Among these cells, CNE-2 cells had the highest uptake of the probe, while B16 cells had the lowest, corresponding with their intergin expression levels. Next, the fluorescent and metabolic imaging was performed in HT1080 (intergin postive) tumor, where nicotinamide adenine dinucleotide hydrogen (NADH), flavoprotein (Fp) and fRFP fluorescent signals were collected. The tumor from mice intravenously injected with Octa-RGD probe displayed obviously higher NADH redox ratio NADH/(Fp+NADH) and fRFP signal, than those with fRFP protein. It suggested that integrin targeting may have influence on the target cell metabolism, and further demonstrated Octa-RGD probe facilitated its uptake in the targeted tumor in vivo. This paper developed a useful probe, which can bind integrins specifically and efficiently in tumor cells, and together with tumor metabolic information, it may provide new insight for RGD targeting-based cancer therapeutics.

Abstract IA05: Oncogenic signaling pathways and resistance to therapy in T-ALL

Abstract Aberrant NOTCH1 signaling plays a major role in the pathogenesis of over 60% of T-ALLs harboring activating mutations in the NOTCH1 gene. Most notably, oncogenic NOTCH1 has been proposed as a therapeutic target in NOTCH1-mutant leukemias and small molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH signaling. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by the limited and delayed therapeutic response to these drugs, making the identification of highly effective and synergistic drug combinations capable of delivering strong antileukemic responses a top priority in the field. Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of clinical response to anti-NOTCH1 therapies. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, both inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapies. Moreover, Pten loss and consequent activation of the PI3K pathway induces increased glycolysis and consequently rescues leukemic cell metabolism abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL. These results highlight the fundamental importance of NOTCH1 signaling in the control of leukemia cell metabolism, extend our understanding of the prominent role of PTEN and the PI3K pathway in the control of oncogenic cell growth and provide the basis for the design of new therapeutic strategies targeting cell metabolism for the treatment of T-ALL. Citation Format: Adolfo Ferrando. Oncogenic signaling pathways and resistance to therapy in T-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA05.