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Large-scale chromosomal alterations, particularly chromosomal deletions in cancer genomes confer functional advantages to cancer cells via the loss of tumor suppressor genes (TSGs). However, due to the nature of these focal and arm-level deletions, essential house-keeping genes in the neighborhood of TSGs are potentially lost. We explore the emergence of metabolic adaptations and vulnerabilities that arise due to the collateral loss of essential metabolic genes. In our previous work, we showed that genomic loss in the locus containing SMAD4 and ME2 in pancreatic ductal adenocarcinomas forces these cells to rely on ME3 to compensate for the collateral loss of ME2; thereby revealing a highly selective metabolic target in these cells. Cancer cells can not only exploit such genetic redundancies but also rely on redundancies built into their complex metabolic network to compensate for the loss of metabolic function. Importantly, there is an unexplored landscape of these genomic loss events beyond well-characterized TSGs. To address these challenges, we have developed a platform to identify patient-specific metabolic vulnerabilities emerging due to distinct patterns of genomic loss events across tumors. Our platform presents opportunities for precision-based therapeutic intervention by targeting metabolic vulnerabilities in cancer patients. It uses genomic and clinical data available in cancer patient databases to obtain candidate metabolic genes that are lost to genomic deletions in an unbiased manner. To delineate metabolic redundances and tackle the complexity of genome-scale metabolic models, we employ an innovative multi-objective metabolic flux analysis approach. The utility of this platform is demonstrated via the discovery of a novel metabolic target in a cohort of ovarian cancer patients. The predicted collateral lethal target is validated in vitro using RNA interference and small-molecule inhibitors. Furthermore, we verify the metabolic mechanism of vulnerability predicted by the algorithm using deuterium tracing experiments. The target is also validated in vivo with mice containing ovarian tumors derived from cancer cells with and without the genomic deletion. Surprisingly, the collateral lethal metabolic target was also found to exist in a subset of aggressive endometrial cancers. Finally, we developed a multi-layer machine learning model to predict occurrence of the particular genomic deletion in ovarian and endometrial cancer patients with minimal molecular information to remove the need for whole-genome sequencing data. The model was trained and tested using the publicly-available molecular data from TCGA and AACR GENIE datasets. Citation Format: Abhinav Achreja, Tao Yu, Anjali Mittal, Srinadh Choppara, Noah Meurs, Olamide Animasahun, Jin Heon Jeon, Aradhana Mohan, Anusha Jayaraman, Reva Kulkarni, Justin Reinhold, Michele Cusato, Analisa Difeo, Xiongbin Lu, Deepak Nagrath. Genomic loss in cancers enable discovery of metabolic targets for precision cancer therapy via multiobjective flux analysis and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 236.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis, and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 plays a role in the development of fatty liver in inbred-animals. The ability to perform similar studies using human tissue has been limited by the genetically variability in man. We generated human induced pluripotent stem (iPS)-cells with controllable expression of SIRT1. By differentiating edited-iPS cells into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1-knockdown iPS-derived hepatocytes and found that the human iPS-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically-edited human liver tissue may become an important tool for investigating human liver biology and disease.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin’s effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression., Advanced ovarian cancer usually spreads to the omentum. Here, the authors show that circulating intelectin-1 (ITLN1) has prognostic significance in patients with advanced ovarian cancer, and that mesothelial cell-derived ITLN1 in the omental tumor microenvironment suppresses ovarian cancer progression.

Branched chain amino acids (BCAAs) in cancer serve as requisite precursors for protein synthesis, maintaining metabolite pools in the tricarboxylic acid (TCA) cycle, and sustaining production of nucleotides and lipids. However, the role of stromal cancer associated fibroblasts (CAFs) in support of BCAA metabolism in tumors is still poorly understood. Since most studies in pancreatic cancers have focused on systemic or cancer cell autonomous BCAA metabolism, understanding cancer-stromal ecosystem requires insight into the intersection of cancer-associated transformations in the stroma with reprogramming of their BCAA metabolism. Deciphering the precise role of various cellular components in BCAA metabolism of tumors is complicated by conflicting evidence from past studies and the challenging nature of the intricate tumor microenvironment (TME). Neither systemic in vivo BCAA metabolism nor cancer cells’ BCAA metabolism alone is sufficient to dissect the stromal role. The difficulty in understanding BCAA metabolism in the tumor milieu is exacerbated by nutrient-scarcity, exchange reactions, and metabolite sharing between cancer and stromal cells. Both, the fibrotic environment and nutrient scarcity are difficult to mimic in aggressive murine PDAC models. The metabolic fates of the BCAAs, leucine, valine, and isoleucine, are cell- and tissue-dependent. BCAA transaminases (BCAT1/2), first deaminate BCAAs to branched chain a-ketoacids (BCKAs). The second step in BCAA metabolism involves irreversible BCKA oxidation catalyzed by the mitochondrial BCKA dehydrogenase (BCKDH) complex. Further, oxidation of BCKAs results in succinyl-CoA and acetyl-CoA that act as anaplerotic or ketogenic sources for the TCA cycle. Our recent study revealed differential BCAA metabolism in cancer and stromal compartments of PDAC tumors. We identified a strikingly higher BCAA catabolic flux in CAFs but increased BCKA oxidative flux in cancer cells. Further, CAF-secreted BCKAs were used for maintaining protein synthesis, augmenting TCA cycle metabolite pools, and increasing oxidative phosphorylation in cancer cells. To corroborate the mechanistic underpinnings discovered in our human CAF and cancer cell-line model, we employed two patient-derived models: circulating tumor cells (CTCs) and tumor slice cultures. Collectively, we elucidated an undiscovered metabolic-signaling crosstalk between PDAC and stromal cells and demonstrated that targeting BCAA metabolism in PDAC tumors could mitigate PDAC aggression. Ziwen Zhu, Abhinav Achreja, Noah Meurs, Olamide Animasahun, Sarah Owen, Anjali Mittal, Pooja Parikh, TingWen Lo, Janusz Franco-Barraza, Jiaqi Shi, Mara Sherman, Edna Cuikerman, Andrew Pickering, Anirban Maitra, Vaibhav Sahai, Meredith Morgan, Sunitha Nagrath, Thedore Lawrence, Deepak Nagrath, "Tumor Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors", Nature Metabolism, 2 (8), 2020. Citation Format: Deepak Nagrath. Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT009.

Omental adipose stromal cells (O-ASC) are a multipotent population of mesenchymal stem cells contained in the omentum tissue that promote endometrial and ovarian tumor proliferation, migration, and drug resistance. The mechanistic underpinnings of O-ASCs' role in tumor progression and growth are unclear. Here, we propose a novel nitric oxide (NO)–mediated metabolic coupling between O-ASCs and gynecologic cancer cells in which O-ASCs support NO homeostasis in malignant cells. NO is synthesized endogenously by the conversion of l-arginine into citrulline through nitric oxide synthase (NOS). Through arginine depletion in the media using l-arginase and NOS inhibition in cancer cells using NG-nitro-l-arginine methyl ester (l-NAME), we demonstrate that patient-derived O-ASCs increase NO levels in ovarian and endometrial cancer cells and promote proliferation in these cells. O-ASCs and cancer cell cocultures revealed that cancer cells use O-ASC–secreted arginine and in turn secrete citrulline in the microenvironment. Interestingly, citrulline increased adipogenesis potential of the O-ASCs. Furthermore, we found that O-ASCs increased NO synthesis in cancer cells, leading to decrease in mitochondrial respiration in these cells. Our findings suggest that O-ASCs upregulate glycolysis and reduce oxidative stress in cancer cells by increasing NO levels through paracrine metabolite secretion. Significantly, we found that O-ASC–mediated chemoresistance in cancer cells can be deregulated by altering NO homeostasis. A combined approach of targeting secreted arginine through l-arginase, along with targeting microenvironment-secreted factors using l-NAME, may be a viable therapeutic approach for targeting ovarian and endometrial cancers. Cancer Res; 75(2); 456–71. ©2014 AACR.

Nitric oxide (NO) is a signaling molecule with pleiotropic physiological roles in normal cells and pathophysiological roles in cancer. NO synthetase expression and NO synthesis are linked to altered metabolism, neoplasticity, invasiveness, chemoresistance, immune evasion, and ultimately to poor prognosis of cancer patients. Exogenous NO in the microenvironment facilitates paracrine signaling, mediates immune responses, and triggers angiogenesis. NO regulates posttranslational protein modifications, S-nitrosation, and genome-wide epigenetic modifications that can have both tumor-promoting and tumor-suppressing effects. We review mechanisms that link NO to cancer hallmarks, with a perspective of co-targeting NO metabolism with first-line therapies for improved outcome. We highlight the need for quantitative flux analysis to study NO in tumors.

Glutamine is the most abundant circulating amino acid in blood and muscle and is critical for many fundamental cell functions in cancer cells, including synthesis of metabolites that maintain mitochondrial metabolism; generation of antioxidants to remove reactive oxygen species; synthesis of nonessential amino acids (NEAAs), purines, pyrimidines, and fatty acids for cellular replication; and activation of cell signaling. In light of the pleiotropic role of glutamine in cancer cells, a comprehensive understanding of glutamine metabolism is essential for the development of metabolic therapeutic strategies for targeting cancer cells. In this article, we review oncogene-, tumor suppressor–, and tumor microenvironment–mediated regulation of glutamine metabolism in cancer cells. We describe the mechanism of glutamine's regulation of tumor proliferation, metastasis, and global methylation. Furthermore, we highlight the therapeutic potential of glutamine metabolism and emphasize that clinical application of in vivo assessment of glutamine metabolism is critical for identifying new ways to treat patients through glutamine-based metabolic therapy.

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC., Kras activation and p16 inactivation cooperatively promote pancreatic cancer progression. Here, the authors show that such cooperation depends upon an increased expression of the NAD(P)H oxidase NOX4 achieved through transcription factors independently regulated by the two oncogenic genetic alterations.

Dissecting the pleiotropic roles of tumor micro-environment (TME) on cancer progression has been brought to the foreground of research on cancer pathology. Extracellular vesicles such as exosomes, transport proteins, lipids, and nucleic acids, to mediate intercellular communication between TME components and have emerged as candidates for anti-cancer therapy. We previously reported that cancer-associated fibroblast (CAF) derived exosomes (CDEs) contain metabolites in their cargo that are utilized by cancer cells for central carbon metabolism and promote cancer growth. However, the metabolic fluxes involved in donor cells towards packaging of metabolites in extracellular vesicles and exosomes-mediated metabolite flux upregulation in recipient cells are still not known. Here, we have developed a novel empirical and computational technique, exosome-mediated metabolic flux analysis (Exo-MFA) to quantify flow of cargo from source cells to recipient cells via vesicular transport. Our algorithm, which based on (13)C metabolic flux analysis, successfully predicts packaging fluxes to metabolite cargo in CAFs, dynamic changes in rate of exosome internalization by cancer cells and flux of cargo release over time. We find that cancer cells internalize exosomes rapidly leading to depletion of extracellular exosomes within 24 hours. However, metabolite cargo significantly alters intracellular metabolism over the course of 24 hours by regulating glycolysis pathway fluxes via lactate supply. Furthermore, it can supply up to 35% of the TCA cycle fluxes by providing TCA intermediates and glutamine. Our algorithm will help gain insight into (i) metabolic interactions in multicellular systems (ii) biogenesis of extracellular vesicles and their differential packaging of cargo under changing environments, and (iii) regulation of cancer cell metabolism by its microenvironment.

The development of a sensitive sensor for detecting nitric oxide (NO) emissions from biological samples is reported. The sensor is based on tunable diode laser absorption spectroscopy (TDLAS) using a continuous wave, thermoelectrically cooled quantum cascade laser (QCL) and a 100-m astigmatic Herriot cell. A 2f-wavelength modulation spectroscopy technique was used to obtain QCL-based TDLAS NO emission measurements with an optimum signal-to-noise ratio. An absorption line at 1,900.076 cm−1 was targeted to measure NO with a minimum detection limit of 124 ppt. Positive control measurements with the NO donor DETA NONOate were performed to determine and optimize the sensor performance for measurements of biological samples. Our measurements with NO donor show the potential suitability of the sensor for monitoring NO emission from cancer cells for biological investigations.

Metabolic programs are rewired in tumors to support growth, progression, and immune evasion. A wealth of work in the past decade has delineated how these metabolic rearrangements are facilitated by signaling pathways downstream of oncogene activation and tumor suppressor loss. More recently, this field has expanded to include metabolic interactions among the diverse cell types that exist within a tumor and how this impacts the immune system. In this special issue, 17 review articles discuss these phenomena, and, alongside four original research manuscripts, the vulnerabilities associated with deregulated metabolic programming are highlighted and examined.

Reactive oxygen species (ROS) are important signaling molecules in cancer. The level of ROS will determine physiological effects. While high levels of ROS can cause damage to tissues and cell death, low levels of ROS can have a proliferative effect. ROS are produced by tumor cells but also cellular components that make up the tumor microenvironment (TME). In this review, we discuss the mechanisms by which ROS can affect the TME with particular emphasis on tumor-infiltrating leukocytes. Greater insight into ROS biology in this setting may allow for therapeutic manipulation of ROS levels in order to remodel the tumor microenvironment and increase anti-tumor activity.

Circulating tumor cells (CTCs) has an important role in the spread of cancers and metastasis. CTCs are believed to be the most promising model to understand metastatic progression in patients with cancer. Glutamine, an alternative carbon source to glucose, has recently been shown to be important for the cancer cell survival, growth and progression. The role of glutamine metabolism in CTCs is not known. MicroRNAs (miRNAs) are a group of highly conserved noncoding RNAs and approximately 22 nucleotides in length. Here, we postulate that CTCs have unique microRNAs (miRNAs) expression compared to resident tumor cells. And miRNAs control CTC glutamine metabolism for the metastasis. In this study, we used 3 primary CTC cultures to study how miRNA controlling glutamine metabolism. We identified several miRNAs have high expressions in CTCs and these miRNAs are regulating glutamine metabolism in CTCs. Our results demonstrate the metabolic miRNA targets of CTCs on cancer progression. These insights will present a unique opportunity for diagnosing and treating cancer through CTCs. Note: This abstract was not presented at the meeting. Citation Format: Ziwen Zhu, Sarah N. Owen, Abhinav Achreja, Sunitha Nagrath, Deepak Nagrath. Role of microRNAs in circulating tumor cells glutamine metabolism pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1850.

Multiple myeloma (MM) is the second most common hematologic malignancy. In 2017, MM accounted for approximately 30,770 new diagnoses and 12770 deaths in the US. Although, advancements in treatment options have increased survival rates and life expectancy, MM remains incurable due to development of resistance. Venetoclax is a highly selective, potent BCL-2 antagonist, currently in phase I/II trials for MM and FDA approved for the treatment of CLL patients exhibiting 17p deletion. Venetoclax is effective in eliciting cell death as a single agent in a subset of MM with the (11;14) translocation (which we henceforth designate “sensitive”) in contrast to the majority of MM that is resistant (which we henceforth designate “resistant”). We previously reported that glutamine deprivation increases BIM binding to BCL-2 thereby sensitizing MM to venetoclax, while alpha-ketoglutarate supplementation reversed this sensitivity. We were therefore interested to explore if there was a metabolic basis for t(11;14)-myeloma sensitivity to single agent venetoclax to aid in 1) identifying venetoclax sensitive MM and; 2) inform us of metabolic targets that could be inhibited to sensitize resistant MM to venetoclax. We first queried the CoMMpass MM patient RNAseq trial data and cell lines for electron transport chain (ETC) and TCA cycle gene expression differences in t(11;14) vs non-t(11;14) patients and cell lines. All sensitive cells exhibited varied suppression of TCA/ETC genes, and reduced TCA cycle metabolites and oxygen consumption rates (OCR) compared to resistant cells. Examination of TCA cycle activities connected to the ETC and OCR showed significant decrease in Complex II Succinate ubiquinone reductase (SQR) activity in sensitive cell lines and purified CD38+ve MM patient cells and elevated SQR activity in resistant cells. Furthermore, inhibition of SQR with thenoyltrifluoroacetone (TTFA) sensitized resistant cells to venetoclax. Consistent with SQR inhibition leading to ABT-199 sensitivity, overexpression of an SQR mutant (SDHCR72C) in SDHC-knockout resistant MM, increased venetoclax sensitivity, identifying a unique role for SQR in regulating BCL-2 dependence. In interrogating the mechanistic underpinnings of SQR inhibition-induced sensitivity to venetoclax, we identified increased expression of ATF4 and BIM upon SQR inhibition. ATF4KD or BIMKO restored viability in TTFA and venetoclax co-treated MM further confirming the role of SQR inhibition-induced ATF4 and BIM in venetoclax sensitivity. In testing translationally relevant compounds, we determined that the histone deacetylase inhibitor, panobinostat reduced SQR activity in a dose dependent manner and sensitized MM cells to venetoclax. Our study thus identifies SQR as a novel target and predictive marker to aid in identifying ABT-199-responsive MM patients in a functional biomarker informed manner. Citation Format: Richa Bajpai, Abhinav Achreja, Changyong Wei, Arusha Siddiqa, Shannon M. Matulis, Vikas Gupta, Samuel K. McBrayer, Anjali Mittal, Manali Rupji, Hsiao-Rong Chen, Jeanne Kowalski, Sagar Lonial, Ajay K. Nooka, Lawrence H. Boise, Deepak Nagrath, Mala Shanmugam. Deciphering a metabolic basis for single-agent venetoclax efficacy in t(11;14) multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2715.

The roles of long non-coding RNAs in cancer metabolism remain largely unexplored. Here we identify FILNC1 (FoxO-induced long non-coding RNA 1) as an energy stress-induced long non-coding RNA by FoxO transcription factors. FILNC1 deficiency in renal cancer cells alleviates energy stress-induced apoptosis and markedly promotes renal tumor development. We show that FILNC1 deficiency leads to enhanced glucose uptake and lactate production through upregulation of c-Myc. Upon energy stress, FILNC1 interacts with AUF1, a c-Myc mRNA-binding protein, and sequesters AUF1 from binding c-Myc mRNA, leading to downregulation of c-Myc protein. FILNC1 is specifically expressed in kidney, and is downregulated in renal cell carcinoma; also, its low expression correlates with poor clinical outcomes in renal cell carcinoma. Together, our study not only identifies FILNC1 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control energy metabolism and tumor development., FoxO are commonly down-regulated transcription factors and tumor suppressors in renal cell cancer (RCC). Here, the authors show that upon energy stress FoxOs induce the expression of the long non-coding RNA FILNC1, which inhibits survival of RCC by downregulating c-Myc and c-Myc-dependent metabolic rewiring.

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.

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001, eLife digest Cancer cells behave differently from healthy cells in many ways. Healthy cells rely on structures called mitochondria to provide them with energy via a process that requires oxygen. However cancer cells don’t rely on this process, and instead release energy by breaking down sugars outside of the mitochondria. This may explain why cancer cells are able to thrive even when little oxygen is available. Cancer cells also interact with neighboring cells called fibroblasts, which are a major part of a tumor’s microenvironment, and recruit them into the tumors. The fibroblasts communicate with cancer cells, in part, by releasing chemical messengers packaged into tiny bubble-like structures called exosomes. Recent studies have suggested that these exosomes may help cancer cells to thrive, but there are many questions remaining about how they might do this. Now, Zhao et al. show that the fibroblasts smuggle essential nutrients to cancer cells via the exosomes and disable oxygen-based energy production in cancer cells. First, exosomes released by cancer-associated fibroblasts from people with prostate cancer were collected and marked with a green dye. Next, the green-labeled exosomes were mixed with prostate cancer cells, and shown to be absorbed by the cells. Oxygen-based energy release was dramatically reduced in the exosome-absorbing cells, and sugar-based energy release increased. Next, Zhao et al examined the contents of the exosomes, and found that they contain the building blocks of proteins, fats, and other important molecules. Next, the experiments revealed that both prostate cancer and pancreatic cancer cells deprived of nutrients can use these smuggled resources to continue to grow. Importantly, this process did not involve the protein Kras, which previous studies had show helps cancer cells absorb nutrients. These findings suggest that preventing exosomes from smuggling resources to starving cancer cells might be an effective strategy to treat cancers. DOI: http://dx.doi.org/10.7554/eLife.10250.002

A general rate model was employed in concert with a preferential interaction quadratic adsorption isotherm for the characterization of HIC resins and the prediction of solute behavior in these separation systems. The results indicate that both pore and surface diffusion play an important role in protein transport in HIC resins. The simulated and experimental solute profiles were compared for two model proteins, lysozyme and lectin, for both displacement and gradient modes of chromatography. Our results indicate that a modeling approach using the generate rate model and preferential interaction isotherm can accurately predict the shock layer response in both gradient and displacement chromatography in HIC systems. While pore and surface diffusion played a major role and were limiting steps for proteins, surface diffusion was seen to play less of a role for the displacer. The results demonstrate that this modeling approach can be employed to describe the behavior of these non-linear HIC systems, which may have implications for the development of more efficient preparative HIC separations.

The current state of the art for linear optimization in Flux Balance Analysis has been limited to single objective functions. Since mammalian systems perform various functions, a multiobjective approach is needed when seeking optimal flux distributions in these systems. In most of the available multiobjective optimization methods, there is a lack of understanding of when to use a particular objective, and how to combine and/or prioritize mutually competing objectives to achieve a truly optimal solution. To address these limitations we developed a soft constraints based linear physical programming-based flux balance analysis (LPPFBA) framework to obtain a multiobjective optimal solutions. The developed framework was first applied to compute a set of multiobjective optimal solutions for various pairs of objectives relevant to hepatocyte function (urea secretion, albumin, NADPH, and glutathione syntheses) in bioartificial liver systems. Next, simultaneous analysis of the optimal solutions for three objectives was carried out. Further, this framework was utilized to obtain true optimal conditions to improve the hepatic functions in a simulated bioartificial liver system. The combined quantitative and visualization framework of LPPFBA is applicable to any large-scale metabolic network system, including those derived by genomic analyses.

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.

Our recent studies determined molecular interactions between genes in the ubiquitin-proteasome pathways and cancer cell metabolism. Ubiquitin-specific peptidase 13 (USP13) specifically deubiquitinates and thus upregulates ATP citrate lyase and oxoglutarate dehydrogenase that drive ovarian cancer metabolism. These findings may lead to the development of USP13 inhibitors and new-targeted therapies in ovarian cancers.

An optimization framework based on the use of hybrid models is presented for preparative chromatographic processes. The first step in the hybrid model strategy involves the experimental determination of the parameters of the physical model, which consists of the full general rate model coupled with the kinetic form of the steric mass action isotherm. These parameters are then used to carry out a set of simulations with the physical model to obtain data on the functional relationship between various objective functions and decision variables. The resulting data is then used to estimate the parameters for neural-network-based empirical models. These empirical models are developed in order to enable the exploration of a wide variety of different design scenarios without any additional computational requirements. The resulting empirical models are then used with a sequential quadratic programming optimization algorithm to maximize the objective function, production rate times yield (in the presence of solubility and purity constraints), for binary and tertiary model protein systems. The use of hybrid empirical models to represent complex preparative chromatographic systems significantly reduces the computational time required for simulation and optimization. In addition, it allows both multivariable optimization and rapid exploration of different scenarios for optimal design.

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

Ovarian cancers are one of the most lethal gynecological cancers because they are usually diagnosed in advanced stages when metastasis has already occurred in the peritoneal cavity. Interestingly, ovarian cancers frequently invade the fatty pad of adipose tissue along the stomach called the omentum [1]. Recent studies have shown that omentum contains a population of stem cell-like cells such as adipose stromal cells (ASCs) that engraft in tumors and encourage cancer progression [2]. Omentum-derived ASCs (O-ASCs) have been demonstrated to contribute to the formation of a hospitable environment for the development of ovarian cancer metastasis [2]. Previous studies have shown that O-ASCs enhance proliferation and migration of ovarian cancer cells while reducing their response to chemotherapy and radiation [2]. However, the mechanistic underpinnings of O-ASCs’ role in tumor progression and growth are still unclear. Recently we demonstrated that nitric oxide (NO)-mediated metabolic coupling between O-ASCs and ovarian cancer cells contributes to ovarian cancers’ growth and increased chemosensitivity [3]. Our study revealed that O-ASCs secreted arginine, which was used by cancer cells for NO generation. NO is generated endogenously by an enzymatic conversion of arginine into citrulline through nitric oxide synthase (NOS). NOS has been found to be differentially expressed in obese and non-obese individuals and has been shown to be overexpressed in aggressive ovarian tumors [4]. We revealed that O-ASCs secreted arginine could rescue reduced proliferation rates of ovarian cancers caused by arginine deprivation [3]. Notably, overweight O-ASCs were able to rescue the reduced proliferation of cancer cells more than their lean counterparts. However, more studies are required to shed light on the role of obesity in ovarian cancer development and progression. Several studies indicate that NO is a double-edged molecule. It is a tumor promoter at lower concentrations (less than 500 nM), but damages DNA and induces apoptosis at higher concentrations (millimolar range) [5]. We found that ovarian cancer cells in cocultures with O-ASCs had significantly higher NO levels compared to their non-coculture counterparts. We previously showed that NO synthesis upregulates Warburg effect in ovarian cancers [5]. Interestingly, coculture of cancer cells with O-ASCs reduced oxygen consumption rates (OCR) in cancer cells. We confirmed this shift in ovarian cancers’ metabolism from oxidative phosphorylation (OXPHOS) to glycolysis by comparing the contributions of both pathways towards cellular ATP generation and our data elucidated that O-ASCs increased NO synthesis in cancer cells, resulting in suppression of mitochondrial respiration. Previously, we showed that O-ASCs induced chemo-resistance in cancer cells [2]. Interestingly, in recent studies we found that O-ASCs-mediated chemoresistance can be deregulated by disrupting NO homeostasis [3]. We added L-arginase in direct-contact cocultures of O-ASCs and cancer cells. Both L-arginase (which depletes any secreted arginine by O-ASCs and thereby blocks NO synthesis in cancer cells) and L-NAME (a NO synthase inhibitor) in direct-contact cocultures of O-ASCs and ovarian cancer cells, increased chemosensitivity of paclitaxel in cancer cells. Our results suggest that combined approach of depleting arginine using L-arginase, along with inhibiting NO synthesis in cancer cells using L-NAME, may be a viable therapeutic approach for targeting ovarian cancers, to disrupt the communication between cancer cell and O-ASCs. Arginine is not the only player in the metabolic coupling of O-ASCs and ovarian cancers. Surprisingly, O-ASCs-secreted arginine when is used for NO synthesis in cancer cells, generates citrulline as a byproduct, which is secreted by cancer cells and in turn is ingested by O-ASCs. We hypothesized that this high amount of secreted citrulline concentrations may be beneficial for NO metabolism in O-ASCs. As mentioned before, O-ASCs are multipotent population of mesenchymal stem cells that can differentiate into adipocytes. Surprisingly, we found that secreted citrulline by cancer cells significantly increased adipogenesis capacity of O-ASCs and stimulated the lipid droplet accumulation within O-ASCs. Studies have shown that NO inhibits lipolysis of lipid depots in subcutaneous adipose tissue [6]. This highlights the metabolic symbiosis between omentum-derived ASCs and cancer cells in maintaining NO homeostasis in tumors. Future studies are needed to investigate the mechanism behind the modulation of ovarian tumor's metabolism by NO. The post-translational modification of metabolic enzymes caused by binding of NO with their cysteine residue (s-nitrosylation) may play a crucial role. S-nitrosylation has been known to alter the functions of enzymes in fatty acid metabolism as well as other central energy pathways [7]. The impact of obesity in ovarian cancer initiation and progression has been studied and further studies are needed to explore the role of NO metabolism in this modulation. Furthermore, the link between omentum and other hormonal tumors such prostate cancers needs to be further studied. Even as new mechanisms implicating omentum for ovarian cancer metastasis keep emerging, many aspect of its biology still remains to be unveiled [8]. Figure 1 Crosstalk between ovarian cancers and O-ASCs in tumor microenvironment

A recently developed dimerization/macrocyclization was employed to synthesize a series of macroheterocycles which were biologically evaluated, leading to the discovery of a number of potent cytotoxic agents (e.g., 27: GI50 = 51 nM against leukemia CCRF-CEM cell line; 29: GI50 = 99 nM against melanoma MDA-MB-435 cell line). Further biological studies support an apoptosis mechanism of action for these compounds involving deregulation of the tricarboxylic acid cycle activity and suppression of mitochondrial function in cancer cells.

Warburg effect has emerged as a potential hallmark of many cancers. However, the molecular mechanisms that led to this metabolic state of aerobic glycolysis, particularly in ovarian cancer (OVCA) have not been completely elucidated. HSulf-1 predominantly functions by limiting the bioavailability of heparan binding growth factors and hence their downstream signaling. Here we report that HSulf-1, a known putative tumor suppressor, is a negative regulator of glycolysis. Silencing of HSulf-1 expression in OV202 cell line increased glucose uptake and lactate production by upregulating glycolytic genes such as Glut1, HKII, LDHA, as well as metabolites. Conversely, HSulf-1 overexpression in TOV21G cells resulted in the down regulation of glycolytic enzymes and reduced glycolytic phenotype, supporting the role of HSulf-1 loss in enhanced aerobic glycolysis. HSulf-1 deficiency mediated glycolytic enhancement also resulted in increased inhibitory phosphorylation of pyruvate dehydrogenase (PDH) thus blocking the entry of glucose flux into TCA cycle. Consistent with this, metabolomic and isotope tracer analysis showed reduced glucose flux into TCA cycle. Moreover, HSulf-1 loss is associated with lower oxygen consumption rate (OCR) and impaired mitochondrial function. Mechanistically, lack of HSulf-1 promotes c-Myc induction through HB-EGF-mediated p-ERK activation. Pharmacological inhibition of c-Myc reduced HB-EGF induced glycolytic enzymes implicating a major role of c-Myc in loss of HSulf-1 mediated altered glycolytic pathway in OVCA. Similarly, PG545 treatment, an agent that binds to heparan binding growth factors and sequesters growth factors away from their ligand also blocked HB-EGF signaling and reduced glucose uptake in vivo in HSulf-1 deficient cells.

The effect on pH on protein binding in HIC systems was investigated. Isocratic experiments were carried out to determine the capacity factors of various proteins as a function of temperature, pH and salt type. This paper presents a framework based on the Maxwell linkage function for estimating the number of released water molecules during the adsorption/desorption process due to a change of buffer pH. This approach also enables one to predict the effect of pH change on the water released values upon binding at any temperature condition. The results indicate that the total number of released water molecules ( �ν ) for a pH change increased more on aromatic surfaces (phenyl Sepharose) than on aliphatic resins (butyl Sepharose). The results also indicate that the total number of released water molecules ( �ν ) for a pH change increased with salt concentration and when changing from chaotropic to kosmotropic salts. The (�ν ) values also increased as the buffer pH approached the protein’s pI, and decreased away from its pI. This work helps to establish a framework for the investigation of pH effects on protein selectivity in HIC systems. © 2005 Published by Elsevier B.V.

A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12 h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1 h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4 h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8 h, gluconeogenesis had switched to glycolysis. By 12 h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF.

The increase in the scale of preparative chromatographic processes for biopharmaceutical applications now necessitates the development of effective optimization strategies for large-scale processes in a manufacturing setting. The current state of the art for optimization of preparative chromatography has been limited to single objective functions. Further, there is a lack of understanding of when to use a particular objective, and how to combine and/or prioritize mutually competing objectives to achieve a true optimal solution. In this paper, these limitations are addressed by using a physical programming– based multiobjective optimization (MO) strategy. A set of Pareto solutions are first generated for model protein separations for both bi-objective (production rate and yield) and tri-objective (production rate, yield, and product pool concentration) scenarios. These Pareto frontiers are used to visualize the Pareto optimal surface for different components with various purity constraints and provide a qualitative framework to evaluate the optimal solutions. A physical programming– based multiobjective framework is then used for the quantitative evaluation of the optimal solutions for tertiary protein mixtures. This enables the interpretation of results for different sets of hierarchy and priority values assigned to the objective functions and constraints for the chromatographic processes. This novel multiobjective optimization approach computes the tradeoffs between the conflicting design objectives and helps in choosing an operating condition from infinite feasible optimal solutions. The combined quantitative and visualization framework presented in this paper sets the stage for the development of true optimal solutions for complex nonlinear preparative separations. © 2005 American Institute of Chemical Engineers AIChE J, 51: 511–525, 2005

A novel generalized run-to-run control (GR2R) control strategy is presented for the optimization and control of nonlinear preparative chromatographic processes. The GR2R approach synergistically employs a hybrid (both physical and empirical) model to control chromatographic processes in the presence of sporadic and autocorrelated disturbances. First, parameters of the physical model through experiments are determined, and then the physical model is used to estimate initial parameters of the nonlinear empirical model (Hammerstein) using orthogonal forward regression. Parameters of the nonlinear empirical model are updated at the end of each run using a nonlinear recursive parameter estimation method. The updated empirical model is then used in the control algorithm (model predictive control) to estimate operating conditions for the next batch. Processes operating under fixed optimal conditions are compared with those operating with GR2R control for both gradient and displacement chromatography. The GR2R outperforms the fixed conditions in the presence of various disturbances (such as bed capacity, column efficiency, and feed load) and is an effective strategy for the optimization and control of complex chromatographic processes.

Cascade control is commonly used in the operation of chemical processes to reject disturbances that have a rapid effect on a secondary measured state, before the primary measured variable is affected. In this paper, we develop a state estimation-based model predictive control approach that has the same general philosophy of cascade control (taking advantage of secondary measurements to aid disturbance rejection), with the additional advantage of the constraint handling capability of model predictive control (MPC). State estimation is achieved by using a Kalman filter and appending modeled disturbances as augmented states to the original system model. The example application is an open-loop unstable jacketed exothermic chemical reactor, where the jacket temperature is used as a secondary measurement in order to infer disturbances in jacket feed temperature and/or reactor feed flow rate. The MPC-based cascade strategy yields significantly better performance than classical cascade control when operating close to constraints on the jacket flow rate.

The tumor microenvironment has a pleiotropic role in supporting cancer cell growth, metastasis and drug resistance. Exosomes from mesenchymal stromal cells (MSC) were found to regulate metabolism of acute myeloid leukemia (AML) cells that led to a chemoresistant phenotype. Exosomes carry a host of proteins, nucleotides and metabolites that can induce metabolic reprogramming recipient cells via direct supply of metabolite cargo or through signaling. We utilized 13C tracer techniques and two metabolic flux analysis techniques to reveal the mechanism of metabolic reprogramming induced by MSC-derived exosomes. First, 13C metabolic flux analysis was used to quantify intracellular fluxes of central carbon, amino acids and fatty acid metabolism in AML cells cultured with and without exosomes in media with 13C substrates. Second, we employed a novel technique, exosome-mediated metabolic flux analysis (Exo-MFA), to estimate direct support provided by metabolite supply from exosome cargo by introducing 13C-labeled exosomes. The combined analysis dissected the metabolite supply from exosome cargo from the overall reprogramming of metabolic pathways due to exosomes. Our results revealed key metabolic pathways that could be targeted to inhibit exosome-induced reprogramming to reverse the chemoresistant phenotype. Citation Format: Abhinav Achreja, Hongyun Zhao, Ziwen Zhu, Jonathan Gerszberg, Marina Y. Konopleva, Michael Andreeff, Deepak Nagrath. Metabolic flux analysis reveals targets to sensitize chemoresistance in acute myeloid leukemia induced by mesenchymal stromal cell-derived exosomes [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 439. doi:10.1158/1538-7445.AM2017-439

Advanced stage high grade serous ovarian cancer (HGSC) metastasizes preferentially to the omentum, which is a well-vascularized fold of peritoneal tissue and is a major site of adipose tissue accumulation. The mechanisms by which omental adipose tissue interact with ovarian cancer cells and promotes tumor growth and disease progression are not entirely clear. We previously showed that a novel adipokine called omentin (Intestinal Lactoferrin Receptor ITLN1) produced by the omental adipose tissue was significantly down-regulated in patients with HGSC compared with those in BMI matched healthy individuals, and omentin suppressed ovarian cancer cell growth only in the presence of adipocytes in vitro. Since we and others demonstrated that omentin induced insulin-dependent glucose uptake exclusively in adipocytes, we therefore hypothesized that omentin may suppress ovarian cancer growth via driving metabolic shift in ovarian cancer cells in the omental microenvironment. Using a cancer cell/adipocyte-co-culture model, we demonstrated that omentin reduced both glucose uptake and lactate secretion in ovarian cancer cells when they were co-cultured with adipocytes but not with other stromal cell types, suggesting that omentin-induced glucose uptake in adipocytes may deplete the surrounding glucose that fuels the glucose-addicted ovarian cancer cells in the omental microenvironment and thus drive metabolic shift in ovarian cancer cells. To delineate the underlying mechanism by which omentin induced glucose uptake in adipocytes, expression levels of the adipocyte-specific glucose transporter GLUT4 induced by omentin in adipocytes and the effect of GLUT4 silencing in adipocytes using GLUT4 specific siRNAs on ovarian cancer growth were examined. The results showed that omentin up-regulated GLUT4 in adipocytes and GLUT4 silencing in adipocytes abrogated the effects of omentin on glucose uptake in adipocytes and ovarian cancer cell growth in the co-culture model. Taken together, this study shows that omentin plays an important role in driving metabolic shift in ovarian cancer cells in the omental microenvironment. Therapeutic strategies based on up-regulating omentin in ovarian cancer patients may inhibit ovarian cancer progression and improve patient survival rates. Note: This abstract was not presented at the meeting. Citation Format: Chi Lam Au Yeung, Abhinav Achreja, Hongyun Zhao, Tsz-Lun Yeung, Rosemarie Schmandt, Daniel K. Yip, Karen H. Lu, Deepak Nagrath, Samuel C. Mok. Omentin drives metabolic shift in ovarian cancer cells in the omental tumor microenvironment [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 5911. doi:10.1158/1538-7445.AM2017-5911

Mesenchymal stromal cells play an important role in acute myeloid leukemia (AML) development. Altered cellular metabolism supports AML cells' survival in multiple aspects, such as drug resistance. Here, we demonstrate the role of MSC-derived exosomes in metabolic regulation of AML cells, and put forward a combinatorial strategy to sensitize AML cells to chemodrugs. Exosomes secreted by MSCs can reprogram the metabolic machinery following their internalization by AML cells. Through 13C tracing experiments and flux analysis, we elucidate that MSC-derived exosomes enhance oxidative phosphorylation and glutamine's entry into TCA cycle, which replenish the pool of carbon sources in mitochondria. Further, our work shows that inhibiting the interactions between MSCs and AML cells by targeting the metabolic regulation exerted by MSC-derived exosomes sensitizes AML cells to chemodrugs. Taken together, our work reveals a novel role of the TME in regulating the metabolic adaptation in AML cells and uncovers the improved strategy for AML therapy. Citation Format: Hongyun Zhao, Abhinav Achreja, Ziwen Zhu, Ahmed N. Rawi, Marina Konopleva, Michael Andreeff, Deepak Nagrath. Metabolic reprogramming in acute myeloid leukemia cells by mesenchymal stromal cell-derived exosomes induces chemoresistance [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 4323. doi:10.1158/1538-7445.AM2017-4323

Lung cancer continues to be fatal, in part due to the inability to prevent and treat metastases. Highly metastatic cancers exhibit enhanced glucose uptake to sustain proliferation and importantly, tumor invasion. Among the SLC2A family of facilitative glucose transporters, GLUT1 is largely attributed to be responsible for increased glucose uptake of cancer cells. GLUT1 is however responsible for glucose transport across the blood-brain barrier, expressed in many normal cell types and therefore a less desirable therapeutic target. We previously reported that multiple myeloma cells rely on overexpression and constitutive plasma membrane localization of insulin-responsive glucose transporter, GLUT4. In this study we investigated a role for GLUT4 in lung cancer. To interrogate contributions of GLUT1 and GLUT4 in proliferation, invasion and migration we generated H1299 and A549 GLUT 1 or GLUT4 knockdowns. Knockdown (KD) of GLUT4 did not inhibit proliferation but suppressed migration and invasion assessed through scratch and Boyden chamber assays, respectively. On the contrary, knockdown of GLUT1 reduced proliferation of these lines. Treatment of H1299 and A549 with our newly developed GLUT4-selective inhibitors also reduced invasion, phenocopying the effects detected with GLUT4 KD. GLUT4 inhibition also reduced H1299 invasion in a spheroid invasion model. We utilized H1299 cells to isolate highly invasive less proliferative “leader cells” and less invasive but highly proliferative “follower cells”. Interestingly, examination of these two cell types exhibited a differential expression pattern of GLUT1/GLUT4. Leader cells have elevated expression of GLUT4 and decreased GLUT1. On the contrary, follower cells have high GLUT1 and low GLUT4 expression. Leader cells are more sensitive to GLUT4 inhibitors indicating they are more dependent on GLUT4 than follower cells. In addition, leader cells are more sensitive to mitochondrial complex I inhibitors compared to follower cells, suggesting they rely more on oxidative phosphorylation. A differential reliance on glycolysis/OXPHOS was further supported by evaluation of glucose uptake/oxygen consumption. Isotope tracer and bioenergetics analyses further support altered nutrient dependencies of leader and follower cells. Lastly, we found that GLUT4 is expressed in patient lung adenocarcinoma specimens including more aggressive micropapillary lung adenocarcinoma. Examination of collective invasion packs in human adenocarcinoma demonstrated patchy GLUT1 expression suggestive of a subset of more proliferative “follower” cells. These results suggest that in a lung cancer population a subset of more invasive cells are reliant on GLUT4 with reduced GLUT1 expression while more proliferative cells rely on high GLUT1 expression, making GLUT4 a promising candidate for targeting metastasis in lung cancer. Citation Format: Changyong Wei, Abhinav Achreja, Jessica Konen, Gabriel Sica, Melissa Gilbert-Ross, Deepak Nagrath, Adam Marcus, Mala Shanmugam. GLUT4 exhibits a non-canonical role of regulating lung cancer metastasis [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 4904. doi:10.1158/1538-7445.AM2017-4904

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine9s entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

The behavior and phenotypic changes of cells are governed by a cellular circuitry that represents a set of biochemical reactions. Based on biological functions, this circuitry is divided into three types of networks, each encoding for a major biological process: signal transduction, transcription regulation, and metabolism. This division has generally enabled taming computational complexity dealing with the entire system, allowed for using modeling techniques that are specific to each of the components, and achieved separation of the different time scales at which reactions in each of the three networks occur. Nonetheless, with this division comes loss of information and power needed to elucidate certain cellular phenomena. Within the cell, these three types of networks work in tandem, and each produces signals and/or substances that are used by the others to process information and operate normally. Therefore, computational techniques for modeling integrated cellular machinery are needed. In this work, we propose an integrated hybrid model (IHM) that combines Petri nets and Boolean networks to model integrated cellular networks. Coupled with a stochastic simulation mechanism, the model simulates the dynamics of the integrated network, and can be perturbed to generate testable hypotheses. Our model is qualitative and is mostly built upon knowledge from the literature and requires fine-tuning of very few parameters. We validated our model on two systems: the transcriptional regulation of glucose metabolism in human cells, and cellular osmoregulation in S. cerevisiae. The model produced results that are in very good agreement with experimental data, and produces valid hypotheses. The abstract nature of our model and the ease of its construction makes it a very good candidate for modeling integrated networks from qualitative data. The results it produces can guide the practitioner to zoom into components and interconnections and investigate them using such more detailed mathematical models., Author Summary Within the cell of an organism, three networks—signaling, transcriptional, and metabolic—are always at work to determine the response of the cell to signals from its environment, and consequently, its fate. Evidence from experimental studies is painting a picture of complex crosstalk among these networks. Thus, while a wide array of computational techniques exist for analyzing each of these network types, there is clear need for new modeling techniques that allow for simultaneously analyzing integrated networks, which combine elements from all three networks. Here, we provide a step towards achieving this task by combining two population modeling techniques—Petri nets and Boolean networks—to produce an integrated hybrid model. We demonstrate the accuracy and utility of this model on two biological systems: transcriptional regulation of glucose metabolism in human cells, and cellular osmoregulation in yeast.

The clinical and biological effects of metabolic alterations in cancer are not fully understood.In high-grade serous ovarian cancer (HGSOC) samples (n = 101), over 170 metabolites were profiled and compared with normal ovarian tissues (n = 15). To determine NAT8L gene expression across different cancer types, we analyzed the RNA expression of cancer types using RNASeqV2 data available from the open access The Cancer Genome Atlas (TCGA) website (http://www.cbioportal.org/public-portal/). Using NAT8L siRNA, molecular techniques and histological analysis, we determined cancer cell viability, proliferation, apoptosis, and tumor growth in in vitro and in vivo (n = 6-10 mice/group) settings. Data were analyzed with the Student's t test and Kaplan-Meier analysis. Statistical tests were two-sided.Patients with high levels of tumoral NAA and its biosynthetic enzyme, aspartate N-acetyltransferase (NAT8L), had worse overall survival than patients with low levels of NAA and NAT8L. The overall survival duration of patients with higher-than-median NAA levels (3.6 years) was lower than that of patients with lower-than-median NAA levels (5.1 years, P = .03). High NAT8L gene expression in other cancers (melanoma, renal cell, breast, colon, and uterine cancers) was associated with worse overall survival. NAT8L silencing reduced cancer cell viability (HEYA8: control siRNA 90.61% ± 2.53, NAT8L siRNA 39.43% ± 3.00, P.001; A2780: control siRNA 90.59% ± 2.53, NAT8L siRNA 7.44% ± 1.71, P.001) and proliferation (HEYA8: control siRNA 74.83% ± 0.92, NAT8L siRNA 55.70% ± 1.54, P.001; A2780: control siRNA 50.17% ± 4.13, NAT8L siRNA 26.52% ± 3.70, P.001), which was rescued by addition of NAA. In orthotopic mouse models (ovarian cancer and melanoma), NAT8L silencing reduced tumor growth statistically significantly (A2780: control siRNA 0.52 g ± 0.15, NAT8L siRNA 0.08 g ± 0.17, P.001; HEYA8: control siRNA 0.79 g ± 0.42, NAT8L siRNA 0.24 g ± 0.18, P = .008, A375-SM: control siRNA 0.55 g ± 0.22, NAT8L siRNA 0.21 g ± 0.17 g, P = .001). NAT8L silencing downregulated the anti-apoptotic pathway, which was mediated through FOXM1.These findings indicate that the NAA pathway has a prominent role in promoting tumor growth and represents a valuable target for anticancer therapy.Altered energy metabolism is a hallmark of cancer (1). Proliferating cancer cells have much greater metabolic requirements than nonproliferating differentiated cells (2,3). Moreover, altered cancer metabolism elevates unique metabolic intermediates, which can promote cancer survival and progression (4,5). Furthermore, emerging evidence suggests that proliferating cancer cells exploit alternative metabolic pathways to meet their high demand for energy and to accumulate biomass (6-8).

Anoikis resistance, or the ability for cells to live detached from the extracellular matrix, is a property of epithelial cancers. The “Warburg effect,” or the preference of cancer cells for glycolysis for their energy production even in the presence of oxygen, has been shown to be evident in various tumors. Since a cancer cell's metastatic ability depends on microenvironmental conditions (nutrients, stromal cells, and vascularization) and is highly variable for different organs, their cellular metabolic fluxes and nutrient demand may show considerable differences. Moreover, a cancer cell's metastatic ability, which is dependent on the stage of cancer, may further create metabolic alterations depending on its microenvironment. Although recent studies have aimed to elucidate cancer cell metabolism under detached conditions, the nutrient demand and metabolic activity of cancer cells under nonadherent conditions remain poorly understood. Additionally, less is known about metabolic alterations in ovarian cancer cells with varying invasive capability under anoikis conditions. We hypothesized that the metabolism of highly invasive ovarian cancer cells in detachment would differ from less invasive ovarian cancer cells and that ovarian cancer cells will have altered metabolism in detached vs. attached conditions. To assess these metabolic differences, we integrated a secretomics-based metabolic footprinting (MFP) approach with mitochondrial bioenergetics. Interestingly, MFP revealed higher pyruvate uptake and oxygen consumption in more invasive ovarian cancer cells than their less invasive counterparts. Furthermore, ATP production was higher in more invasive vs. less invasive ovarian cancer cells in detachment. We found that pyruvate has an effect on highly invasive ovarian cancer cells' migration ability. Our results are the first to demonstrate that higher mitochondrial activity is related to higher ovarian cancer invasiveness under detached conditions. Importantly, our results bring insights regarding the metabolism of cancer cells under nonadherent conditions and could lead to the development of therapies for modulating cancer cell invasiveness.

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.

This study implicates the glucose deprivation response in breast cancer cell resistance to lapatinib and high relapse rates in Her2-positive patients. Identification of these compensatory networks suggests novel strategies to target cancer signaling and metabolism., Increased expression of the glucose deprivation response network, including glucagon signaling, glucose uptake, gluconeogenesis and unfolded protein response genes is found in breast cancer cells with acquired resistance to lapatinib. The glucose deprivation response gene network correlated significantly with high clinical relapse rates in ErbB2-positive breast cancer patients. Chemical genomics bioinformatics data mining identified drugs that target the glucose deprivation response networks to reduced survival of resistant cells., Dynamic interactions between intracellular networks regulate cellular homeostasis and responses to perturbations. Targeted therapy is aimed at perturbing oncogene addiction pathways in cancer, however, development of acquired resistance to these drugs is a significant clinical problem. A network-based computational analysis of global gene expression data from matched sensitive and acquired drug-resistant cells to lapatinib, an EGFR/ErbB2 inhibitor, revealed an increased expression of the glucose deprivation response network, including glucagon signaling, glucose uptake, gluconeogenesis and unfolded protein response in the resistant cells. Importantly, the glucose deprivation response markers correlated significantly with high clinical relapse rates in ErbB2-positive breast cancer patients. Further, forcing drug-sensitive cells into glucose deprivation rendered them more resistant to lapatinib. Using a chemical genomics bioinformatics mining of the CMAP database, we identified drugs that specifically target the glucose deprivation response networks to overcome the resistant phenotype and reduced survival of resistant cells. This study implicates the chronic activation of cellular compensatory networks in response to targeted therapy and suggests novel combinations targeting signaling and metabolic networks in tumors with acquired resistance.

Transcriptional motifs are small regulatory interaction patterns that regulate biological functions in highly-interacting cellular networks. Recently, attempts have been made to explain the significance of transcriptional motifs through dynamic function. However, fundamental questions remain unanswered. Why are certain transcriptional motifs with similar dynamic function abundant while others occur rarely? What are the criteria for topological generalization of these motifs into complex networks? Here, we present a novel paradigm that combines non-equilibrium thermodynamics with multiobjective-optimality for network analysis. We found that energetic cost, defined herein as specific dissipation energy, is minimal at the optimal environmental conditions and it correlates inversely with the abundance of the network motifs obtained experimentally for E. coli and S. cerevisiae. This yields evidence that dissipative energetics is the underlying criteria used during evolution for motif selection and that biological systems during transcription tend towards evolutionary selection of subgraphs which produces minimum specific heat dissipation under optimal conditions, thereby explaining the abundance/rare occurrence of some motifs. We show that although certain motifs had similar dynamical functionality, they had significantly different energetic cost, thus explaining the abundance/rare occurrence of these motifs. The presented insights may establish global thermodynamic analysis as a backbone in designing and understanding complex networks systems, such as metabolic and protein interaction networks.

The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0-4], pre-stable stage [day 5-7] and the stable stage [day 8-10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.

The development of liver support systems has been in intensive investigation for over 40 years. The main driving force is the shortage of donor organs for orthotopic liver transplantation. Liver cell transplantation and extracorporeal bioartificial livers (BAL) may bridge patients with end-stage liver diseases to successful orthotopic liver transplantation, support patients with acute liver failure to recover, and provide a curing method to patients with certain liver metabolic diseases. Another frontier of current liver tissue engineering is to construct many functional liver units in vitro for drug toxicity and metabolism screening. Much progress has been made, with several artificial liver dialysis devices on the market, a few BAL systems in clinical trials, and other in vitro micro-liver models in development. On the other hand, many lessons have been learned as well. In this chapter, we will focus on the review of advancement, challenges and the critical issues that have to be solved in the development of BAL systems and hepatic cell transplantation as well as in vitro micro-liver models from a tissue engineering perspective.

Natural and synthetic biomaterials utilized in tissue engineering applications require a dynamic interplay of complex macromolecular compositions of hydrated extracellular matrices (ECMs) and soluble growth factors. The challenges in utilizing synthetic ECMs is the effective control of temporal and spatial complexity of multiple signal presentation, as compared to natural ECMs that possess the inherent properties of biological recognition, including presentation of receptor-binding ligands, susceptibility to cell-triggered proteolytic degradation, and remodeling. We have developed a murine preadipocyte differentiation system for generating a natural basement membrane extract (Adipogel) comprising ECM proteins (collagen IV, laminin, hyaluronan, and fibronectin) and including relevant growth factors (hepatocyte growth factor, vascular endothelial growth factor, and leukemia inhibitory factor). We have shown the effective utilization of the growth factor-enriched extracellular matrix for enhanced albumin synthesis rate of primary hepatocyte cultures for a period of 10 d as compared to collagen sandwich cultures and comparable or higher function as compared to Matrigel cultures. We have also demonstrated comparable cytochrome P450 1A1 activity for the collagen-Adipogel condition to the collagen double-gel and Matrigel culture conditions. A metabolic analysis revealed that utilization of Adipogel in primary hepatocyte cultures increased serine, glycine, threonine, alanine, tyrosine, valine, methionine, lysine, isoleucine, leucine, phenylalanine, taurine, cysteine, and glucose uptake rates to enhance hepatocyte protein synthesis as compared to collagen double-gel cultures. The demonstrated synthesis, isolation, characterization, and application of Adipogel provide immense potential for tissue engineering and regenerative medicine applications.—Sharma, N. S., Nagrath, D., Yarmush, M. L. Adipocyte-derived basement membrane extract with biological activity: applications in hepatocyte functional augmentation in vitro.

Fatty liver is a significant risk factor for liver transplantation, and accounts for nearly half of the livers rejected from the donor pool. We hypothesized that metabolic preconditioning via ex vivo perfusion of the liver graft can reduce fat content and increase post-transplant survival to an acceptable range. We describe a perfusate medium containing agents that promote the defatting of hepatocytes and explanted livers. Defatting agents were screened on cultured hepatocytes made fatty by pre-incubation with fatty acids. The most effective agents were then used on fatty livers. Fatty livers were isolated from obese Zucker rats and normothermically perfused with medium containing a combination of defatting agents. This combination decreased the intracellular lipid content of cultured hepatocytes by 35% over 24 hours, and of perfused livers by 50% over 3 hours. Metabolite analysis suggests that the defatting cocktail upregulated both lipid oxidation and export. Furthermore, gene expression analysis for several enzymes and transcription factors involved in fatty acid oxidation and triglyceride clearance were elevated. We conclude that a cocktail of defatting agents can be used to rapidly clear excess lipid storage in fatty livers, thus providing a new means to recondition donor livers deemed unacceptable or marginally acceptable for transplantation.

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine-addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Recently, we uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low-invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high-invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the cellular NAD+/NADH homeostasis, which mediates cancer metastasis and progression. On the other hand, the overexpression of NAD+ biosynthesis pathway enhances glutamine's entry into TCA cycle for cancer metastasis, as well as chemo-drug resistance. Our findings suggest that a combined approach of targeting high-invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting NAD+ biosynthesis pathway may lead to potential therapeutic approaches for treating OVCAs. Our insights will present a unique opportunity for overcoming the drug resistance limitation in clinical trials in ovarian cancers. Citation Format: Lifeng Yang, Abhinav Achreja, Tyler Moss, Joelle Baddour, Katherine Stilles, Lisa Chiba, Sun Hye Kim, Josh Morse, Juan Marini, Anil K. Sood, Prahlad T. Ram, Deepak Nagrath. Glutamine modulates cellular NAD+/NADH homeostasis thereby regulating cancer metastasis, drug sensitivity in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1208. doi:10.1158/1538-7445.AM2015-1208