Metabolites In Cancer Cells Research Articles

Metabolic Signaling in the Tumor Microenvironment.

Cancer cells must reprogram their metabolism to sustain rapid growth. This is accomplished in part by switching to aerobic glycolysis, uncoupling glucose from mitochondrial metabolism, and performing anaplerosis via alternative carbon sources to replenish intermediates of the tricarboxylic acid (TCA) cycle and sustain oxidative phosphorylation (OXPHOS). While this metabolic program produces adequate biosynthetic intermediates, reducing agents, ATP, and epigenetic remodeling cofactors necessary to sustain growth, it also produces large amounts of byproducts that can generate a hostile tumor microenvironment (TME) characterized by low pH, redox stress, and poor oxygenation. In recent years, the focus of cancer metabolic research has shifted from the regulation and utilization of cancer cell-intrinsic pathways to studying how the metabolic landscape of the tumor affects the anti-tumor immune response. Recent discoveries point to the role that secreted metabolites within the TME play in crosstalk between tumor cell types to promote tumorigenesis and hinder the anti-tumor immune response. In this review, we will explore how crosstalk between metabolites of cancer cells, immune cells, and stromal cells drives tumorigenesis and what effects the competition for resources and metabolic crosstalk has on immune cell function.

Technologies for Decoding Cancer Metabolism with Spatial Resolution.

It is increasingly appreciated that cancer cells adapt their metabolic pathways to support rapid growth and proliferation as well as survival, often even under the poor nutrient conditions that characterize some tumors. Cancer cells can also rewire their metabolism to circumvent chemotherapeutics that inhibit core metabolic pathways, such as nucleotide synthesis. A critical approach to the study of cancer metabolism is metabolite profiling (metabolomics), the set of technologies, usually based on mass spectrometry, that allow for the detection and quantification of metabolites in cancer cells and their environments. Metabolomics is a burgeoning field, driven by technological innovations in mass spectrometers, as well as novel approaches to isolate cells, subcellular compartments, and rare fluids, such as the interstitial fluid of tumors. Here, we discuss three emerging metabolomic technologies: spatial metabolomics, single-cell metabolomics, and organellar metabolomics. The use of these technologies along with more established profiling methods, like single-cell transcriptomics and proteomics, is likely to underlie new discoveries and questions in cancer research.

ROS-dependent catalytic mechanism of melatonin metabolism and its application in the measurement of reactive oxygen.

Melatonin (Mel) is an endogenous active molecule whose metabolism progress significantly influences its bioactivity. However, the detailed metabolic pathway of Mel in the pathological state has not yet been fully illustrated. In this study, 16 metabolites of Mel in cancer cells and human liver microsomes were identified, of which seven novel metabolites were newly discovered. Among them, 2-hydroxymelatonin (2-O-Mel), as the major metabolite in cancer cells, was revealed for the first time, which was different from the metabolite found in the human liver. Furthermore, CYP1A1/1A2- and reactive oxygen species (ROS)-mediated 2-hydroxylation reactions of Mel were verified to be the two metabolic pathways in the liver and cancer cells, respectively. ROS-dependent formation of 2-O-Mel was the major pathway in cancer cells. Furthermore, the underlying catalytic mechanism of Mel to 2-O-Mel in the presence of ROS was fully elucidated using computational chemistry analysis. Therefore, the generation of 2-O-Mel from Mel could serve as another index for the endogenous reactive oxygen level. Finally, based on the ROS-dependent production of 2-O-Mel, Mel was successfully used for detecting the oxygen-carrying capacity of hemoglobin in human blood. Our investigation further enriched the metabolic pathway of Mel, especially for the ROS-dependent formation of 2-O-Mel that serves as a diagnostic and therapeutic target for the rational use of Mel in clinics.

Mapping a comprehensive targetome of glycolytic metabolites in cancer cells.

Mapping a comprehensive targetome of glycolytic metabolites in cancer cells.

A novel single-particle multiple-signal sensor array combined with multidimensional data mining for the detection of tricarboxylic acid cycle metabolites and discrimination of cells.

Tricarboxylic acid (TCA) metabolites in cancer cells show amarked difference from those in normal cells. Herein, we report a single-particle multiple-signal lanthanide/europium-based metal-organic framework (Tb/Eu MOF) sensor array for the detection of TCA metabolites and discrimination of cancer cells. In the presence of TCA metabolite, 6 characteristic peaks of Tb/Eu MOF showed dramatic changes due to host-guest interactions, allowing sensor array-based qualitative and quantitative detection to be performed. In the qualitative detection ability test, 18 TCA metabolites at 4 concentrations (50μM, 100μM, 200μM, 300μM) were accurately discriminated by the sensor array via linear discriminant analysis (LDA). Significantly, these 4 concentrations include the clinical detection criteria for most TCA metabolites. In the quantitative detection ability test, a good linear relationship between Euclidean distances and the concentrations of L-valine (Val) could be obtained in the range of 50 to 500μM (R2 = 0.9755). On this basis, theprovided method was successfully applied for theclassification of 2 normal cells and 5 cancer cells via principal components analysis (PCA), LDA and aradial basis function neural network (RBFN). What's more, by verifying the weight coefficient of each point, detection and discrimination results are proved as a trustworthy balanced evaluation of multiple factors. Depending on precise data processing, theexperimental operation was simplified on the premise of ensuring accuracy, soour method is a meaningful exploration for array design.

A rapid intracellular enrichment of alkylating payload is essential for melphalan flufenamide potency and mechanism of action

Despite decades of development of treatments and the successful application of targeted therapies for multiple myeloma, clinical challenges remain for patients with relapsed/refractory disease. A drug designed for efficient delivery of an alkylating payload into tumor cells that yields a favorable therapeutic window can be an attractive choice.Herein we describe melphalan flufenamide (melflufen), a drug with a peptide carrier component conjugated to an alkylating payload, and its cellular metabolism. We further underline the fundamental role of enzymatic hydrolysis in the rapid and robust accumulation of alkylating metabolites in cancer cells and their importance for downstream effects. The formed alkylating metabolites were shown to cause DNA damage, both on purified DNA and on chromatin in cells, with both nuclear and mitochondrial DNA affected in the latter. Furthermore, the rapid intracellular enrichment of alkylating metabolites is shown to be essential for the rapid kinetics of the downstream intracellular effects such as DNA damage signaling and induction of apoptosis. To evaluate the importance of enzymatic hydrolysis for melflufen's efficacy, all four stereoisomers of the compound were studied in a systematic approach and shown to have a different pattern of metabolism. In comparison with melflufen, stereoisomers lacking intracellular accumulation of alkylating payloads showed cytotoxic activity only at significantly higher concentration, slower DNA damage kinetics, and different mechanisms of action to reach cellular apoptosis.

Development of simultaneous quantitative analysis of tricarboxylic acid cycle metabolites to identify specific metabolites in cancer cells by targeted metabolomic approach

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Phosphomannose Isomerase High Expression Associated with Better Prognosis in Pancreatic Ductal Adenocarcinoma.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States. The need for increased patient survival has not been met for PDAC. The addition of mannose to conventional chemotherapy leads to accumulation of mannose metabolite in cancer cells and increases subsequent cell death. This susceptibility to mannose depends on the levels of phosphomannose isomerase (PMI). The cancer cells with lower levels of PMI are more sensitive to mannose than cells with higher levels. In this study, we investigated the association of PMI expression with clinical and pathological features of PDAC cases.MethodsPMI antibody immunohistochemistry (AbCam) was performed on tissue microarrays from 235 PDAC by a standard protocol on Ventana automated immunostainer. The PMI intensity was graded (0–3) and the proportion of positivity was scored. Correlation of PMI expression with staging and survival was analyzed.ResultsOf the 235 cases, 51.5% (n=121) cases demonstrated grade 2 intensity with 90.1% of these (n=109) showing positivity in ≥70% of tumor cells. Ninety-eight (41.7%) cases exhibited grade 3 intensity with 94.9% (n=93) of these cases showing ≥70% reactivity. Sixteen cases (6.8%) were nonreactive (intensity grade 0–1). Intensity of PMI expression was associated with significantly better prognosis as assessed by median survival in months (M): grade 0–1 intensity group: 11.2 M; grade 2 intensity group: 25.2 M; and grade 3 intensity group: 33.2 M (p=0.03). A minority (6.8%) of PDACs show non-high PMI expression with poorer prognosis.DiscussionMannose may be a particularly useful adjunct with chemotherapy to treat this aggressive subgroup. PMI expression is also a potential biomarker to predict the prognosis of PDAC.

Abstract 931: From bench to bedside: Using ProTide chemistry to transform 3'-deoxyadenosine into the novel anti-cancer agent Nuc-7738

Abstract Background: 3'-deoxyadenosine (3'-dA; also known as cordycepin) is a nucleoside analog that has shown potent anti-cancer activity in non-clinical studies but has not been clinically developed because of its vulnerability to rapid degradation by the circulating enzyme adenosine deaminase (ADA) and its poor uptake into cancer cells. The ProTide NUC-7738 is a pre-activated and protected nucleotide analog (3'-dA 5'monophosphate; 3'-dAMP) specifically designed to overcome the limitations of 3'-dA. NUC-7738's phosphoramidate moiety renders it resistant to ADA degradation. Here we compared NUC-7738 to 3'-dA in several model systems prior to conducting a first-in-class dose-escalation/expansion study of NUC-7738 in patients with advanced cancers. Materials and Methods: To determine the potency of NUC-7738, IC50 values were measured in multiple cancer cell lines and compared to the parent compound, 3'-dA. Chemical inhibitors of ADA and other 3'-dA processing enzymes were applied to assess the relative ability of NUC-7738 to bypass these pathways. Using genome-wide gene-trap screens and RNA sequencing we compared mechanisms of action (MOA) for NUC-7738 and 3'-dA. Results: NUC-7738 demonstrated up to 185x greater anti-cancer potency than 3'-dA across a variety of cancer cells lines. Gene trap experiments showed that the intracellular activating enzyme adenosine kinase (ADK) and the hENT1 transporter were amongst the highest enriched genes for 3'-dA, whilst no enrichments for these genes were observed in NUC-7738 treated cells. In support of this, in vitro inhibition assays showed that unlike 3'-dA, NUC-7738 is resistant to ADA breakdown, is not reliant on hENT1 transport for its cellular uptake, and is independent of ADK for its activity. As expected, RNA sequencing analysis demonstrated overlap between the MOA of NUC-7738 and 3'-dA; both cause cancer cell death via the intrinsic apoptosis pathway and suppression of pro-survival signaling. Further investigation of gene candidates was employed in ex-vivo cancer kidney cancer samples. Conclusion: Phosphoramidate chemistry was used to transform the nucleoside analog 3'-dA into NUC-7738, rendering it resistant to degradation by ADA and enabling it to enter cancer cells independent of nucleoside transporters, both of which contribute to NUC-7738's substantially greater in vitro potency compared to 3'-dA. The gene trap approach allowed a sophisticated comparison of the MOA of NUC-7738 with 3'-dA. By overcoming the resistance mechanisms associated with 3'-dA, NUC-7738 generates higher levels of the active anti-cancer metabolite in cancer cells. These data supported the initiation of NuTide:701, a first-in-human Phase I study assessing the safety, tolerability, pharmacokinetics and pharmacodynamics of NUC-7738 in patients with advanced solid tumors that are resistant to conventional treatment. Citation Format: Hagen Schwenzer, Michaela Serpi, Valentina Ferrari, James Chettle, Josephine Morris, Ruud van Stiphout, Erica de Zan, Sebastian Nijman, Mustafa Elshani, Mary Kudsy, David Harrison, Gareth Bond, Sarah P. Blagden. From bench to bedside: Using ProTide chemistry to transform 3'-deoxyadenosine into the novel anti-cancer agent Nuc-7738 [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 931.

Cancer Cell Metabolites: Updates on Current Tracing Methods.

Cancer is the second leading cause of death-1 in 6 deaths globally is due to cancer. Cancer metabolism is a complex and one of the most actively researched area in cancer biology. Metabolic reprogramming in cancer cells entails activities that involve several enzymes and metabolites to convert nutrient into building blocks that alter energy metabolism to fuel rapid cell division. Metabolic dependencies in cancer generate signature metabolites that have key regulatory roles in tumorigenesis. In this minireview, we highlight recent advances in the popular methods ingrained in biochemistry research such as stable and flux isotope analysis, as well as radioisotope labeling, which are valuable in elucidating cancer metabolites. These methods together with analytical tools such as chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry have helped to bring about exploratory work in understanding the role of important as well as obscure metabolites in cancer cells. Information obtained from these analyses significantly contribute in the diagnosis and prognosis of tumors leading to potential therapeutic targets for cancer therapy.

Cholesterol as a functional metabolite cooperates with metadherin in cancer cells

Protein-metabolite interactions (PMIs) play important roles in various biological processes, especially in disease progression. However, due to the complexity of living cells, it is very difficult to identify specific PMIs. Herein, we chose one oncogenic factor, metadherin (MTDH), as a bait to identify its in vivo interacting metabolites in cancer cells. Cholesterol is an important metabolite and essential structural component of cell membranes. It could also drive several diseases including cancer. Interestingly, we found that cholesterol robustly interacted with MTDH and downregulated the expression of MTDH in cancer cells. Furthermore, MTDH disturbed metabolite alterations under cholesterol treatment in MTDH transduced cancer cells. Collectively, our results uncover an undescribed PMI where MTDH, as an oncogenic factor, might positively regulate cancer progression by interacting with cholesterol. This study interprets the theoretical basis of PMI-oriented cancer progression and targeting therapies in clinic.

Abstract 4377: Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer

Abstract Solid tumors evolve significant changes in metabolic pathways during development by virtue of their specific biosynthetic demands, their particular microenvironment and the potential occurrence of toxic metabolites such as reactive oxygen species. However, the development of cancer treatment approaches that are based on the inhibition of biosynthetic routes is impaired due to the high plasticity of metabolic networks, e.g. resulting in activation of compensatory pathways or in an increased exchange of metabolites between cancer cells and the tumor environment. Therefore, such therapies could not be translated into efficient clinical applications so far. Here we show that an enhanced lipogenesis, triggered by a pharmacological activation of the Liver X receptor (LXR), represents a new therapeutic strategy for the treatment of liver carcinoma (HCC). A combination of LXR mediated fatty acid synthesis and concomitant Raf suppression results in oxidative stress, induction of a critical ER stress response and subsequently in apoptosis of different murine and human liver cancer cells. Our mechanistic studies identified Raf as an important regulator of lipid metabolism in liver cancer. We found that Raf-1 directly interacts with Stearoyl-CoA desaturase-1 (Scd1), the central enzyme for the conversion of saturated into mono-unsaturated fatty acids and thereby maintains Scd1 protein stability in HCC cells. Thus, inhibition of Raf by Sorafenib diminished Scd1 protein abundance leading to toxic accumulation of saturated fatty acids and metabolic stress in cancer cells under sustained lipogenesis. Treatment studies in autochthonous liver cancer mouse models and xenograft models of human HCC revealed that a combinatorial therapy, consisting of the LXR agonist T0901317 and Sorafenib is highly potent to suppress liver cancer development and to extend the survival of tumor bearing animals. Such a therapy was efficient against hepatic neoplasia with different metabolic phenotypes and well tolerated by mice, even by animals that already suffer from a fatty liver disease. Taken together, we here propose a pharmacologically induced accumulation of toxic metabolites in cancer cells as a new strategy for efficient metabolic targeting of therapy refractory solid tumors. Citation Format: Ramona Rudalska, Jule Harbig, Marteinn Snaebjoernsson, Lyudmyla Taranets, Florian Heinzmann, Stefan Zwirner, Wei Ciu Hu, Thales Kronenberger, Tae-Won Kang, Antti Poso, Stefan Laufer, Mathias Rosenfeldt, Nisar P. Malek, Bernd Pichler, Nikita Popov, Almut Schulze, Lars Zender, Daniel Dauch. Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer [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 4377.

Drug discovery of anticancer drugs targeting methylenetetrahydrofolate dehydrogenase 2

Many anticancer drugs have serious adverse effects; therefore, it is necessary to target features specific to cancer cells to minimize the effects on healthy cells. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was reported to be specifically enhanced in cancer. We confirmed the validity of MTHFD2 as a drug discovery target using clinical data. In addition, we performed in silico screening to design an anticancer drug specifically targeting MTHFD2. Analysis of the clinical data indicated that MTHFD2 was enhanced in most cancers compared with normal tissues, and affected the prognosis in cancer patients. Candidate compounds for MTHFD2 inhibitors were identified using in silico drug discovery techniques, and the important interactions for MTHFD2 binding were determined. In addition, these candidate compounds decreased levels of MTHFD2 metabolites in cancer cells. The findings of the present study may help to develop anticancer drugs targeting MTHFD2, with a view to minimizing the adverse effects of anticancer drugs.

Identification of Intracellular and Extracellular Metabolites in Cancer Cells Using 13C Hyperpolarized Ultrafast Laplace NMR.

Ultrafast Laplace NMR (UF-LNMR), which is based on the spatial encoding of multidimensional data, enables one to carry out 2D relaxation and diffusion measurements in a single scan. Besides reducing the experiment time to a fraction, it significantly facilitates the use of nuclear spin hyperpolarization to boost experimental sensitivity, because the time-consuming polarization step does not need to be repeated. Here we demonstrate the usability of hyperpolarized UF-LNMR in the context of cell metabolism, by investigating the conversion of pyruvate to lactate in the cultures of mouse 4T1 cancer cells. We show that 13C ultrafast diffusion–T2 relaxation correlation measurements, with the sensitivity enhanced by several orders of magnitude by dissolution dynamic nuclear polarization (D-DNP), allows the determination of the extra- vs intracellular location of metabolites because of their significantly different values of diffusion coefficients and T2 relaxation times. Under the current conditions, pyruvate was located predominantly in the extracellular pool, while lactate remained primarily intracellular. Contrary to the small flip angle diffusion methods reported in the literature, the UF-LNMR method does not require several scans with varying gradient strength, and it provides a combined diffusion and T2 contrast. Furthermore, the ultrafast concept can be extended to various other multidimensional LNMR experiments, which will provide detailed information about the dynamics and exchange processes of cell metabolites.

Metabolomics analysis of the potential anticancer mechanism of annonaceous acetogenins on a multidrug resistant mammary adenocarcinoma cell

Although annonaceous acetogenins (ACGs) have been reported to have antitumor activity for over three decades, and many of the underlying mechanism of ACGs on cancer have been clarified, there are still outstanding issues. In particular, the changes of small metabolite in cancer cells, caused by ACGs intake, have been reported rarely. Recent research has showed that cellular metabolic profiling coupled with ultra-flow liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UFLC-Q-TOF-MS) and multivariable statistical analysis enables a good understanding of ACGs' effects on multidrug resistant human mammary adenocarcinoma (MCF-7/Adr) cells. As a result, 23 potential biomarkers (p < 0.05, VIP >1) were identified, and 5 pathways (impact-value > 0.10) identified. The differential metabolites suggested that ACGs affected metabolomics pathways, including arginine and proline metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism and D-Glutamine and D-glutamate metabolism.

Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated.

Abstract 2830: The major human metabolites of abemaciclib are inhibitors of CDK4 and CDK6

Abstract Abemaciclib (LY2835219) is an ATP-competitive inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6) which is currently undergoing clinical evaluation for the treatment of breast and lung cancers. A radiolabeled disposition study following a single 150-mg oral dose of [14C]LY2835219 in healthy subjects indicated that in plasma, in addition to parent drug, the presence of 5 metabolites denoted as M1, M2, M18, M20 and M22. Abemaciclib (34%), M20 (26%), M2 (13%), and M18 (5%) constituted the majority of the plasma exposure. This study investigated the in vitro biological activity of these human circulating metabolites, with the exception of the trace metabolite, M1, and compared their potencies with the parent drug abemaciclib. Specifically non-small cell lung cancer (NSCLC) cells, colorectal cancer (CRC) cells and breast cancer cell lines were evaluated for growth inhibition, cell cycle inhibition and biomarker expression following treatment with abemaciclib and the metabolites. The metabolites were also profiled and compared to abemaciclib for inhibition of CDK4, CDK6, CDK1, and CDK9 in cell-free biochemical kinase assays. The IC50 values for the inhibition of CDK4 and CDK6, for metabolites M2, M18, and M20, but not M22, were between 1 and 3 nM and were nearly equivalent in potency to abemaciclib. Likewise, metabolites M2, M20, and M18 inhibited cell growth and cell cycle progression in a concentration-dependent manner that was consistent with the inhibition of CDK4 and CDK6 since these outcomes correlated with the concentration-dependent inhibition of various biomarkers such a phospho-serine 780-Rb (pRb), topoisomerase II-alpha (Topo IIα), and phospho-serine 10-histone H3 (pHH3). In this regard, metabolites M2 and M20 showed potencies nearly identical with abemaciclib in the cancer cell lines evaluated, whereas depending on the endpoint measured, the potency of M18 was approximately 3-20-fold lower than abemaciclib. M22 showed the least potency for growth inhibition and little or no inhibition of biomarker expression or cell cycle progression at concentrations below 2 μM. Although the cell-free kinase assays showed that like abemaciclib, M2, M18, and M20 had potential to inhibit CDK9, no measurable inhibition of CDK9 by any of these compounds was observed in cancer cells, indicating that the primary targets driving cell cycle inhibition for these metabolites in cancer cells were CDK4 and CDK6 and not CDK9. Studies with abemaciclib, M2 or M20 in breast cancer cells showed that all 3 compounds induced senescence in addition to growth inhibition following 6-8 days of treatment at 200 and 500 nM. In total the results indicated that the major human metabolites of abemaciclib, M2 and M20, are effective inhibitors of CDK4 and CDK6 that are remarkably similar to abemaciclib in regards to their effects in cancer cells on growth, senescence, and other phenotypic responses. Citation Format: Teresa Burke, Raquel Torres, Ann McNulty, Jack Dempsey, Stanley Kolis, Palaniappan Kulanthaivel, Richard Beckmann. The major human metabolites of abemaciclib are inhibitors of CDK4 and CDK6. [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 2830.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Glutaminases in slowly proliferating gastroenteropancreatic neuroendocrine neoplasms/tumors (GEP-NETs): Selective overexpression of mRNA coding for the KGA isoform

Glutamine (Gln) is a crucial metabolite in cancer cells of different origin, and the expression and activity of different isoforms of the Gln-degrading enzyme, glutaminase (GA), have variable implications for tumor growth and metabolism. Human glutaminases are encoded by two genes: the GLS gene encodes the kidney-type glutaminases, KGA and GAC, while the GLS2 gene encodes the liver-type glutaminases, GAB and LGA. Recent studies suggest that the GAC isoform and thus high GAC/KGA ratio, are characteristic of highly proliferating tumors, while GLS2 proteins have an inhibitory effect on tumor growth. Here we analyzed the expression levels of distinct GA transcripts in 7 gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with low proliferation index and 7 non-neoplastic tissues. GEP-NETs overexpressed KGA, while GAC, which was the most abundant isoform, was not different from control. The expression of the GLS2 gene showed tendency towards elevation in GEP-NETs compared to control. Collectively, the expression pattern of GA isoforms conforms to the low proliferative capacity of GEP-NETs encompassed in this study.

Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.