Enzymes In Metabolic Pathways Research Articles

The influence of high frequency electromagnetic field on the growth of lactic acid bacteria used in the food industry

The objects of the study were lactic acid bacteria Lactobacillus lactis and Lactobacillus casei used in the food industry. It is proposed to use an extremely high frequency electromagnetic field (EMF EHF) with a wavelength of 4.9 mm for 5 minutes to activate the metabolism of lactic acid bacteria. The stimulating effect of EMF EHF on the growth of biomass and excretion of lactic acid during the growth was found. Physiological constants characterizing the development of microorganisms have been determined. It was found that the maximum yield from the consumed substrate Ymax increased by 40% and the maximum specific growth rate rmax by 70% when exposed to radiation, the consumption rate for maintenance ms decreased by 10% and the substrate saturation constant Ks by 23%. A change in the activity of key enzymes of metabolic pathways of lactose utilization immediately after the action of EMF of the EHF was revealed. The preservation of the achieved effect during the entire fermentation was observed. The activity of β-galactosidase and hexokinase after exposure to EMF EHF increases from 134% to 290%, depending on the strain and fermentation conditions. The increase in the activity of fumarate hydratase is 6-9%.

Microwave assisted Biology-Oriented Drug Synthesis (BIODS) of new N,N′-disubstituted benzylamine analogous of 4-aminoantipyrine against leishmaniasis – In vitro assay and in silico-predicted molecular interactions with key metabolic targets

Biology-Oriented Drug Synthesis (BIODS) deals with the simple chemical transformations on the commercially available drugs in order to enhance their new and diversified pharmacological profile. It opens new avenues for the rapid development of drug candidates for neglected tropical diseases (NTDs). Leishmaniasis is one of the NTDs which spread by the bite of sandflies (plebotomine). It ranges from cutaneous self-healing leishmaniasis to life threatening visceral leishmaniasis, known as kala-azar. The current treatment options include the use of pentamidine, miltefosine, and amphotericin B drugs. Unfortunately, all currently available drugs are associated with adverse effects, such as severe nephron- and cardiotoxicity, pancreatitis, and hepatotoxicity. This warrants the development of new drugs against leishmaniasis. Moreover, emergence of resistance against the current medications further worsens the conditions. With this objective, new N, N′-disubstituted benzylamine derivatives of ampyrone (4-aminoantipyrine) were synthesized by using ultrasonication, and microwave assistance. All derivatives were found to be new, except 1, 4, and 11. All the compounds were evaluated for their anti-leishmanial activity, and cellular cytotoxicity. Among them, compounds 4, 5, 8, and 9 showed a significant anti-leishmanial activity in vitro, in comparison to standard drug, miltefosine (IC50 = 25.78 ± 0.2 µM). These compounds were also docked against various metabolic enzymes to predict their interactions and mechanism of action, and were found to act via targeting important enzymes of various metabolic pathways.

One-Pot Biocatalytic In Vivo Methylation-Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L-DOPA.

Electron‐rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high‐value chemicals, such as α‐amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O‐methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co‐substrate S‐adenosyl‐l‐methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron‐rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one‐pot, two whole cell enzyme cascade to produce the l‐DOPA precursor l‐veratrylglycine from lignin‐derived ferulic acid.

One-Pot Biocatalytic In Vivo Methylation-Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L-DOPA.

Electron-rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high-value chemicals, such as α-amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O-methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co-substrate S-adenosyl-l-methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron-rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one-pot, two whole cell enzyme cascade to produce the l-DOPA precursor l-veratrylglycine from lignin-derived ferulic acid.

Immune, metabolism and therapeutic targets in RA (Rheumatoid Arthritis)

Rheumatoid arthritis is a classic autoimmune disease, the pathogenesis of which is closely linked to the auto-reactivity of immune cells and joint inflammation. Three cell types, namely T cells, macrophages and fibroblast-like synoviocytes (FLS), play an important role in the pathogenesis of RA. Numerous studies have pointed to a metabolic reprogramming of T cells, macrophages and FLS in the pathogenesis of RA arthritis, with alterations in different metabolic pathways of cells, mainly producing a shift from oxidative phosphorylation (OXPHOS) to glycolysis, in addition to lipid metabolism and amino acid metabolism which are also altered in the cellular activation state. Metabolic changes are regulated by metabolism-related signalling pathways, and RA is associated with two representative signalling pathways, namely the mTOR signalling pathway and the AMPK signalling pathway. In RA, both signalling pathways are activated or inhibited, and through a series of cascade reactions, different gene expressions are ultimately induced, altering intracellular metabolic pathways and promoting pro-inflammatory functions (e.g. pro-inflammatory cytokine release and FLS phenotypes), or inhibiting the expression of genes related to immune tolerance. Targeting key components of metabolic signalling pathways and key enzymes in cellular metabolic pathways in RA has emerged as a new way of finding drugs for RA, and many modulators targeting these targets have been extensively studied for their therapeutic effects in RA. In this article, we focus on cellular metabolic alterations in RA, related signalling pathways and possible drugs targeting RA metabolic pathways.

Nutritional Epigenetics: How Metabolism Epigenetically Controls Cellular Physiology, Gene Expression and Disease.

The regulation of gene expression is a dynamic process that is influenced by both internal and external factors. Alteration in the epigenetic profile is a key mechanism in the regulation process. Epigenetic regulators, such as enzymes and proteins involved in posttranslational modification (PTM), use different cofactors and substrates derived from dietary sources. For example, glucose metabolism provides acetyl CoA, S-adenosylmethionine (SAM), α- ketoglutarate, uridine diphosphate (UDP)-glucose, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), and fatty acid desaturase (FAD), which are utilized by chromatin-modifying enzymes in many intermediary metabolic pathways. Any alteration in the metabolic status of the cell results in the alteration of these metabolites, which causes dysregulation in the activity of chromatin regulators, resulting in the alteration of the epigenetic profile. Such long-term or repeated alteration of epigenetic profile can lead to several diseases, like cancer, insulin resistance and diabetes, cognitive impairment, neurodegenerative disease, and metabolic syndromes. Here we discuss the functions of key nutrients that contribute to epigenetic regulation and their role in pathophysiological conditions.

Practical aspects of therapy for glutaric aciduria type 1

Treatment of many of the diseases in the panel of expanded newborn screening includes dietary therapy. Glutaric aciduria type 1 (GA1) is a hereditary disorder caused by mutations in the gene GCDH, encoding glutaryl‑CoA dehydrogenase, an enzyme in the amino acid metabolic pathways. The decreased activity of the enzyme leads to accumulation of neuro‑ toxic metabolites. The recommended treatment approaches for GA1 are the prescription of specialized nutrition products, levocarnitine, and symptomatic management. In 2021, clinical guidelines for the treatment of this rear disease were published in Russian Federation. To provide for the timely treatment, it is essential for a practitioner involved in the care patients with such a rare disorder as GA1 to have the knowledge of the principles of management, as well as practical algorithms for diet calculation.The article gives a detailed case‑based description of management during metabolic decompensation and the choice of dietary therapy for GA1 patients of different age groups.

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the “Kennedy pathway” is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

The biochemical mechanism of enhancing the conversion of chicken manure to biogenic methane using coal slime as additive

To improve the efficiency of methane production from chicken manure (CM) anaerobic digestion, the mechanism of coal slime (CS) as an additive on methane production characteristics were investigated. The results showed that adding an appropriate amount of CS quickened the start of the fermentation and effectively increased the methane yield. In addition, the pH changed in a stable manner in the liquid phase, and the concentrations of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) were reduced. Moreover, organic matter was decomposed and volatile fatty acids (VFAs) were consumed effectively. The abundance of Bacteroides in the bacterial community and Methanosarcina in the archaea was increased. In addition, the reduction of CO2 was the main methanogenic pathway, and adding CS raised the abundance of genes for key enzymes in metabolic pathways during methane metabolism. The results provide a novel method for the efficient methane production from CM.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Functional characterization of arginine metabolic pathway enzymes in the antibacterial immune response of penaeid shrimp

Arginine metabolism pathway enzymes and products are important modulators of several physiological processes in animals, including immune response. Although some components of the arginine metabolic pathway have been reported in penaeid shrimps, no systematic study has explored all the key pathway enzymes involved in shrimp antimicrobial response. Here, we explored the role of the three key arginine metabolism enzymes (nitric-oxide synthase (NOS), arginase (ARG), agmatinase (AGM)) in Penaeus vannamei antimicrobial immunity. First, P. vannamei homologs of ARG and AGM (PvARG and PvAGM) were cloned and found to be evolutionally conserved with invertebrate counterparts. Transcript levels of PvARG, PvAGM, and PvNOS were ubiquitously expressed in healthy shrimp tissues and induced in hemocytes and hepatopancreas upon challenge with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Streptoccocus iniae) bacteria, suggesting their involvement in shrimp antimicrobial immune response. Besides, RNA interference knockdown and enzyme activity assay revealed an antagonistic relationship between PvARG/PvAGM and PvNOS, while this relationship was broken upon pathogen stimulation. Interestingly, knockdown of PvNOS increased Vibrio abundance in shrimp hemolymph, whereas knockdown of PvAGR reduced Vibrio abundance. Taken together, our present data shows that homologs of the key arginine metabolism pathway enzymes in penaeid shrimp (PvARG, PvAGM, and PvNOS) work synergistically and/or antagonistically to modulate antibacterial immune response.

Protective effects of all-trans retinoic acid against gastric premalignant lesions by repressing exosomal LncHOXA10-pyruvate carboxylase axis.

Long noncoding RNAs (LncRNAs) play a pivotal role in gastric tumorigenesis, while exosomes facilitate the LncRNAs transferring to recipient cells. However, the roles of exosomal LncRNAs in gastric premalignant lesions (GPL) remain unclear. We analyzed the expression of LncHOXA10 and its role in GPL progression. The protective effect of all-trans retinoic acid (ATRA) on GPL was explored in vitro and in vivo. Here, we found that LncHOXA10 expression was obviously increased in serum exosomes and gastric tissues from individuals with GPL, and exosomal LncHOXA10 from patients with GPL markedly promoted the malignant progression of human gastric epithelial cell line GES-1. Furthermore, RNA-pulldown assay revealed that LncHOXA10 mainly interacted with pyruvate carboxylase (PC), an essential enzyme in various cellular metabolic pathways. In gastric tissues from patients with GPL and gastric cancer (GC), PC was also upregulated and positively correlated with LncHOXA10 expression, which predicted a poor prognosis as well. Moreover, PC silencing attenuated the malignant effects of exosomal LncHOXA10 on GES-1 cells. ATRA also ameliorated the deterioration of GPL and prevented the malignant progression of GPL by reducing exosomal LncHOXA10 and PC expression. Collectively, the LncHOXA10-PC axis participated in the early stage of GC tumorigenesis, and ATRA might be useful to prevent GPL from developing into GC because it targets this axis.

Storage of carbon reserves in spruce trees is prioritized over growth in the face of carbon limitation

Climate change is expected to pose a global threat to forest health by intensifying extreme events like drought and insect attacks. Carbon allocation is a fundamental process that determines the adaptive responses of long-lived late-maturing organisms like trees to such stresses. However, our mechanistic understanding of how trees coordinate and set allocation priorities among different sinks (e.g., growth and storage) under severe source limitation remains limited. Using flux measurements, isotopic tracing, targeted metabolomics, and transcriptomics, we investigated how limitation of source supply influences sink activity, particularly growth and carbon storage, and their relative regulation in Norway spruce (Picea abies) clones. During photosynthetic deprivation, absolute rates of respiration, growth, and allocation to storage all decline. When trees approach neutral carbon balance, i.e., daytime net carbon gain equals nighttime carbon loss, genes encoding major enzymes of metabolic pathways remain relatively unaffected. However, under negative carbon balance, photosynthesis and growth are down-regulated while sucrose and starch biosynthesis pathways are up-regulated, indicating that trees prioritize carbon allocation to storage over growth. Moreover, trees under negative carbon balance actively increase the turnover rate of starch, lipids, and amino acids, most likely to support respiration and mitigate stress. Our study provides molecular evidence that trees faced with severe photosynthetic limitation strategically regulate storage allocation and consumption at the expense of growth. Understanding such allocation strategies is crucial for predicting how trees may respond to extreme events involving steep declines in photosynthesis, like severe drought, or defoliation by heat waves, late frost, or insect attack.

Tryptophan and Its Metabolites in Lung Cancer: Basic Functions and Clinical Significance.

Lung cancer is the most lethal malignancy worldwide. Recently, it has been recognized that metabolic reprogramming is a complex and multifaceted factor, contributing to the process of lung cancer. Tryptophan (Try) is an essential amino acid, and Try and its metabolites can regulate the progression of lung cancer. Here, we review the pleiotropic functions of the Try metabolic pathway, its metabolites, and key enzymes in the pathogenic process of lung cancer, including modulating the tumor environment, promoting immune suppression, and drug resistance. We summarize the recent advance in therapeutic drugs targeting the Try metabolism and kynurenine pathway and their clinical trials.

A high extra-virgin olive oil diet induces changes in metabolic pathways of experimental mammary tumors

Breast cancer is the most common malignancy in women worldwide, and environmental factors, especially diet, have a role in the etiology of this disease. This work aimed to investigate the influence of high fat diets (rich in corn oil or extra virgin olive oil -EVOO-) and the timing of dietary intervention (from weaning or after induction) on tumor metabolism in a seven,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer model in rat. The effects of lipids (oils and fatty acids) have also been investigated in MCF-7 cells. The results have confirmed different effects on tumor progression depending on the type of lipid. Molecular analysis at mRNA, protein and activity level of enzymes of the main metabolic pathways have also shown differences among groups. Thus, the animals fed with the EVOO-enriched diet developed tumors with less degree of clinical and morphological malignancy and showed modified glucose and mitochondrial metabolism when compared to the animals fed with the corn oil-enriched diet. Paradoxically, no clear influence on lipid metabolism by the high fat diets was observed. Considering previous studies on proliferation and apoptosis in the same samples, the results suggest that metabolic changes have a role in the molecular context that results in the modulation of different signaling pathways. Moreover, metabolic characteristics, without the context of other pathways, may not reflect tumor malignancy. The time of dietary intervention plays also a role, suggesting the importance of metabolic plasticity and the relation with mammary gland status when the tumor is induced.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

ABSTRACTThe ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2.IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.

Conserved Metabolic and Evolutionary Themes in Microbial Degradation of Carbamate Pesticides.

Carbamate pesticides are widely used as insecticides, nematicides, acaricides, herbicides and fungicides in the agriculture, food and public health sector. However, only a minor fraction of the applied quantity reaches the target organisms. The majority of it persists in the environment, impacting the non-target biota, leading to ecological disturbance. The toxicity of these compounds to biota is mediated through cholinergic and non-cholinergic routes, thereby making their clean-up cardinal. Microbes, specifically bacteria, have adapted to the presence of these compounds by evolving degradation pathways and thus play a major role in their removal from the biosphere. Over the past few decades, various genetic, metabolic and biochemical analyses exploring carbamate degradation in bacteria have revealed certain conserved themes in metabolic pathways like the enzymatic hydrolysis of the carbamate ester or amide linkage, funnelling of aryl carbamates into respective dihydroxy aromatic intermediates, C1 metabolism and nitrogen assimilation. Further, genomic and functional analyses have provided insights on mechanisms like horizontal gene transfer and enzyme promiscuity, which drive the evolution of degradation phenotype. Compartmentalisation of metabolic pathway enzymes serves as an additional strategy that further aids in optimising the degradation efficiency. This review highlights and discusses the conclusions drawn from various analyses over the past few decades; and provides a comprehensive view of the environmental fate, toxicity, metabolic routes, related genes and enzymes as well as evolutionary mechanisms associated with the degradation of widely employed carbamate pesticides. Additionally, various strategies like application of consortia for efficient degradation, metabolic engineering and adaptive laboratory evolution, which aid in improvising remediation efficiency and overcoming the challenges associated with in situ bioremediation are discussed.

Abstract 2495: SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming

Abstract Bladder cancer (BC) is the sixth most common cancer in the United States. Approximately, 75% of BC patients present with non-muscle invasive bladder cancer (NMIBC), which is responsive to local treatment and surveillance. However, recurrence is common with progression to muscle-invasive bladder cancer (MIBC). Forty percent of MIBC patients develop distant metastases, with few treatment options and poor survival. Limited advances have been made in BC therapy with only a few FDA-approved new treatments for advanced disease since 2016 with response rates hovering around 15%. Thus, there is an unmet need for better understanding of the pathobiology of the disease to develop more effective therapeutics. Metabolic re-programming of cancer is increasingly recognized as a hallmark of cancer as cancer cells take advantage of metabolic products to meet the increasing demands of proliferating cells. Our lab has identified the Secreted Protein Acidic Rich in Cysteine (SPARC) as a multi-faceted tumor suppressor within the BC tumor microenvironment. Herein, we sought to elucidate the role of SPARC in regulating metabolic programming in BC and identify pathways that may serve as diagnostic and therapeutic targets. We used three BC cell lines UMUC3, T24 (SPARChigh) and its isogenic SPARClow T24T. We treated BC cells with recombinant SPARC protein and genetically manipulated SPARC expression in these cells. We performed real-time monitoring of the effect of SPARC on bioenergetics using Seahorse assays. In tandem, we profiled transcriptomes of T24, T24T, and T24shSP-cell lines to identify aggressive phenotype signatures using GSEA. We then compared signatures with survival data from BC cohorts from the GEO and TCGA. We found that SPARC inhibited ATP production, basal and maximal respiration, and spare respiratory capacity in BC cell lines. This effect is more pronounced in more aggressive SPARClow T24T cells compared to its isogenic SPARChigh T24 cells. Knockdown of SPARC in BC cell lines not only induced an aggressive phenotype, but also increased ATP production and mitochondrial respiration. Integrated transcriptomic profiling indicated that SPARC-loss is associated with enriched glycolytic, lipidomic, and mitochondrial metabolic pathways. Importantly, in patient samples, SPARC expression negatively correlated with rate limiting enzymes in metabolic pathways associated with lower survival. SPARC loss also associated with oncogenic signatures of EMT, PI3K-AKT-mTOR signaling, and G1/S cell cycle arrest. Our data reveals a novel function of SPARC inhibiting mitochondrial bioenergetics and ATP production that fuel growth and invasion in BC cells. The SPARC-regulated metabolic profile highlights potential vulnerabilities that can be exploited as therapeutic targets. Citation Format: Sameh Waleed Almousa, Alia Ghoneum, Hesham Afify, Neveen Said. SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming [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 2495.

Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.MethodsDNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated (>1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.