Central Carbon Research Articles

Retraction: The lncRNA XIST/miR-29b-3p/COL3A1 axis regulates central carbon metabolism in head and neck squamous cell carcinoma and is associated with poor tumor prognosis.

[This retracts the article DOI: 10.21037/atm-23-30.].

Construction of halogenated tetrasubstituted carbon centers through copper(I)-catalyzed asymmetric alkylation of 2-azaarylesters

Construction of halogenated tetrasubstituted carbon centers through copper(I)-catalyzed asymmetric alkylation of 2-azaarylesters

Chemoselectivity of the CuAAC/Ring Cleavage/Cyclization Reaction between Enaminones and α-Acylketenimine.

Ketenimines represent an important class of reactive species, useful synthetic intermediates, and synthons. However, in general, ketenimines preferentially undergoes nucleophilic addition reactions with hydroxyl and amino groups, and carbon functional groups remain a less studied subset of such systems. Herein, we develop a straightforward syntheses of pyridin-4(1H)-imines that is achieved by cyclization of a reacting enaminone unit with α-acylketenimine which is generated from the reactions of sulfonyl azides and terminal ynones in situ (CuAAC/Ring cleavage reaction). The cascade process preferentially starts with the nucleophilic α-C of the enaminone unit instead of an amino group, attacking the electron-deficient central carbon of ketenimine, and the chemoselectivity unconventional products pyridin-4(1H)-imines were formed by intramolecular cyclization.

Prediction of positive pulmonary nodules based on machine learning algorithm combined with central carbon metabolism data

BackgroundLung cancer causes a huge disease burden, and early detection of positive pulmonary nodules (PPNs) as an early sign of lung cancer is extremely important for effective intervention. It is necessary to develop PPNs risk recognizer based on machine learning algorithm combined with central carbon metabolomics.MethodsThe study included 2248 participants at high risk for lung cancer from the Ma'anshan Community Lung Cancer Screening cohort. The Least Absolute Shrinkage and Selection Operator (LASSO) was used to screen 18 central carbon-related metabolites in plasma, recursive feature elimination (RFE) was used to select all 42 features, followed by five machine learning algorithms for model development. The performance of the model was evaluated using area under the receiver operator characteristic curve (AUC), accuracy, precision, recall, and F1 scores. In addition, SHapley Additive exPlanations (SHAP) was performed to assess the interpretability of the final selected model and to gain insight into the impact of features on the predicted results.ResultsFinally, the two prediction models based on the random forest (RF) algorithm performed best, with AUC values of 0.87 and 0.83, respectively, better than other models. We found that homogentisic acid, fumaric acid, maleic acid, hippuric acid, gluconic acid, and succinic acid played a significant role in both PPNs prediction model and NPNs vs PPNs model, while 2-oxadipic acid only played a role in the former model and phosphopyruvate only played a role in the NPNs vs PPNs model. This model demonstrates the potential of central carbon metabolism for PPNs risk prediction and identification.ConclusionWe developed a series of predictive models for PPNs, which can help in the early detection of PPNs and thus reduce the risk of lung cancer.

Transcriptomic and metabolomic analysis reveals the influence of carbohydrates on lignin degradation mediated by Bacillus amyloliquefaciens

IntroductionLigninolytic bacteria can secrete extracellular enzymes to depolymerize lignin into small-molecular aromatics that are subsequently metabolized and funneled into the TCA cycle. Carbohydrates, which are the preferred carbon sources of bacteria, influence the metabolism of lignin-derived aromatics through bacteria.MethodsIn this study, untargeted metabolomics and transcriptomics analyses were performed to investigate the effect of carbohydrates on lignin degradation mediated by Bacillus amyloliquefaciens MN-13, a strain with lignin-degrading activity that was isolated in our previous work.ResultsThe results demonstrated that the cell growth of the MN-13 strain and lignin removal were promoted when carbohydrates such as glucose and sodium carboxymethyl cellulose were added to an alkaline lignin-minimal salt medium (AL-MSM) culture. Metabolomics analysis showed that lignin depolymerization took place outside the cells, and the addition of glucose regulated the uptake and metabolism of lignin-derived monomers and activated the downstream metabolism process in cells. In the transcriptomics analysis, 299 DEGs were screened after 24 h of inoculation in AL-MSM with free glucose and 2 g/L glucose, respectively, accounting for 8.3% of the total amount of annotated genes. These DEGs were primarily assigned to 30 subcategories, including flagellar assembly, the PTS system, RNA degradation, glycolysis/gluconeogenesis, the TCA cycle, pyruvate metabolism, and tryptophan metabolism. These subcategories were closely associated with the cell structure, generation of cellular energy, and precursors for biosynthetic pathways, based on a − log 10 (P adjust) value in the KEGG pathway analysis.ConclusionIn summary, the addition of glucose increased lignin degradation mediated by the MN-13 strain through regulating glycolysis, TCA cycle, and central carbon metabolism.

Bioplastic degradation and assimilation processes by a novel bacterium isolated from the marine plastisphere

Biosourced and biodegradable plastics offer a promising solution to reduce environmental impacts of plastics for specific applications. Here, we report a novel bacterium named Alteromonas plasticoclasticus MED1 isolated from the marine plastisphere that forms biofilms on foils of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Experiments of degradation halo, plastic matrix weight loss, bacterial oxygen consumption and heterotrophic biosynthetic activity showed that the bacterial isolate MED1 is able to degrade PHBV and to use it as carbon and energy source. The likely entire metabolic pathway specifically expressed by this bacterium grown on PHBV matrices was shown by further genomic and transcriptomic analysis. In addition to a gene coding for a probable secreted depolymerase, a gene cluster was located that encodes characteristic enzymes involved in the complete depolymerization of PHBV, the transport of oligomers, and in the conversion of the monomers into intermediates of central carbon metabolism. The transcriptomic experiments showed the activation of the glyoxylate shunt during PHBV degradation, setting the isocitrate dehydrogenase activity as regulated branching point of the carbon flow entering the tricarboxylic acid cycle. Our study also shows the potential of exploring the natural plastisphere to discover new bacteria with promising metabolic capabilities.

Silver-Catalyzed Dearomative Skeletal Editing of Indazoles by Donor Carbene Insertion.

Given the prevalence of heterocyclic scaffolds in drug-related molecules, converting these highly modular heterocyclic scaffolds into structural diversified and dearomatized analogs is an ideal strategy for improving their physicochemical and pharmacokinetic properties. Here, we described an efficient method for silver carbene-mediated dearomative N-N bond cleavage leading to skeletal hopping between indazole and 1,2-dihydroquinazoline via a highly selective single-carbon insertion procedure. Using this methodology, a series of dihydroquinazoline analogues with diarylmethylene-substituted quaternary carbon centers were constructed with excellent yields and good functional group compatibility, which was further illustrated by the late-stage diversification of important pharmaceutically active ingredients. DFT calculations indicated that the silver catalyst not only induces the formation of the silver carbene, but also activates the diazahexatriene intermediate, which plays a crucial role in the formation of the C-N bond.

Intramolecular carbon isotopic rollover in propane from natural gas reservoirs of the Santanghu Basin: Insights into chemical structure of kerogen

An intramolecular isotopic study was conducted on natural gases from the Santanghu Basin in China. Propane samples spanned a wide range of ΔC-T (δ13Ccentral-δ13Cterminal) values from –8.4 to 8.5 ‰, δ13Ccentral and δ13Cterminal values varied from –37.3 to –25.8 ‰ and –35.7 to –28.9 ‰, respectively. During thermal maturation, the position-specific carbon isotopic composition of propane shifted from δ13Ccentral < δ13Cterminal to δ13Ccentral > δ13Cterminal, and the δ13Cterminal decreased by 6.8 ‰ within a Ro range of 0.46–0.50 %. The intramolecular carbon isotopic rollover and a decrease in δ13Cterminal with a slight increase in maturation could be explained by kinetic effects in thermal cracking of kerogen with a low proportion of branched chains. As isomeric groups were rapidly exhausted, propane generation pathway rapidly changed and primary kinetic isotope effect completely shifted from central carbon position to terminal carbon positions in propane. These changes increased carbon isotope fractionation at terminal carbon positions, thereby reducing δ13Cterminal values and shifting δ13Ccentral < δ13Cterminal to δ13Ccentral > δ13Cterminal within a small maturity range. This work demonstrated that position-specific isotope distribution of propane can be applied to elucidate the chemical structure of kerogen.

Optimized Mass Spectrometry Detection of Thyroid Hormones and Polar Metabolites in Rodent Cerebrospinal Fluid.

Thyroid hormones (TH) are required for brain development and function. Cerebrospinal fluid (CSF), which bathes the brain and spinal cord, contains TH as free hormones or as bound to transthyretin (TTR). Tight TH level regulation in the central nervous system is essential for developmental gene expression, which governs neurogenesis, myelination, and synaptogenesis. This integrated function of TH highlights the importance of developing precise and reliable methods for assessing TH levels in CSF. We report an optimized liquid chromatography-mass spectrometry (LC-MS)-based method to measure TH in rodent CSF and serum, applicable to both fresh and frozen samples. Using this new method, we find distinct differences in CSF TH in pregnant dams vs. non-pregnant adults and in embryonic vs. adult CSF. Further, targeted LC-MS metabolic profiling uncovers distinct central carbon metabolism in the CSF of these populations. TH detection and metabolite profiling of related metabolic pathways open new avenues of rigorous research into CSF TH and will inform future studies on metabolic alterations in CSF during normal development.

Application of (bio) chemical engineering concepts and tools to model genetic regulatory circuits, and some essential central carbon metabolism pathways in living cells. Part 4. Applications in the design of some Genetically Modified Micro-Organisms (GMOs)

In the first part of this work, the general Chemical and Biochemical Engineering (CBE) concepts and rules are briefly reviewed, together with the rules of the control theory of Nonlinear Systems (NSCT), all in the context of (i) deriving deterministic Modular Structured Kinetic Models (MSDKM) to describe the dynamics of metabolic processes in living cells, and (ii) of Hybrid Structured Modular Dynamic Models (HSMDM) (with continuous variables, linking the cell-nano-scale MSDKM state variables to the macro-scale state variables of the bioreactor dynamic model). Thus, in the HSMDM model, both prediction quality and its validity range are improved. By contrast, the current (classical/default) approach in bioengineering practice for solving design, optimization, and control problems based on the math models of industrial biological reactors is to use unstructured Monod (for cell culture reactor) or simple Michaelis-Menten (if only enzymatic reactions are retained) global kinetic models by ignoring detailed representations of metabolic cellular processes. By contrast, as reviewed, and exemplified in the second part of this work, an accurate and realistic math modelling of the dynamic individual GERMs (gene expression regulatory module), or genetic regulatory circuits (GRC), and cell-scale CCM (central carbon metabolism) key-modules can be done by only using the novel holistic ’Whole-Cell Of Variable-Volume’ (WCVV) modelling framework, under isotonic/homeostatic conditions/constraints introduced and promoted by the author. An example was given in the same Part 2 for the case study of a dynamic model for the oscillating glycolysis coupled with the Tryptophan (TRP) oscillating synthesis in the E. coli cells.

Identification of key ferroptosis genes and mechanisms associated with breast cancer using bioinformatics, machine learning, and experimental validation.

The aim of this paper is to mine ferroptosis genes associated with breast cancer based on bioinformatics and machine learning, and to perform in vitro functional validation. Transcriptional and clinical data of breast cancer patients were downloaded from TCGA database and ferroptosis-related genes were obtained from FerrDB database. Significant differentially expressed ferroptosis-related genes between breast cancer tissues and adjacent normal tissues were selected. Functional enrichment analysis was performed on these differentially expressed genes. Four machine learning algorithms were used to identify key ferroptosis-related genes associated with breast cancer. A multi-factor Cox regression analysis was used to construct a risk score model for the key ferroptosis-related genes. The accuracy of the risk score model was validated using Kaplan-Meier survival curve analysis and receiver operating characteristic (ROC) curve analysis. Finally, cell experiments were conducted to validate the biological functions of the key ferroptosis-related genes in breast cancer cells MCF-7, further confirming the accuracy of the analysis results. A total of 52 significantly differentially expressed ferroptosis-related genes were identified, which were mainly enriched in cancer pathways, central carbon metabolism in cancer, HIF-1 signaling pathway, and NOD-like receptor signaling pathway. Three key ferroptosis-related genes (TXNIP, SLC2A1, ATF3) closely related to the occurrence, development, and prognosis of breast cancer were identified using machine learning algorithms. The risk model constructed using these three key ferroptosis-related genes showed that the prognosis of the low-risk group was better than that of the high-risk group (P < 0.001). The ROC curve analysis showed that the prognosis model had good predictive ability. In vitro experiments validated the reliability of the bioinformatics and machine learning screening results. Downregulation of SLC2A1 expression promoted ferroptosis and suppressed tumor cell growth in breast cancer cells (P < 0.01), while overexpression of TXNIP or ATF3 had the same effect (P < 0.01). This study identified three key ferroptosis-related genes (TXNIP, SLC2A1, ATF3) associated with breast cancer, which are closely related to the occurrence, development, and prognosis of breast cancer.

Formation of Sterically Congested C−N Bonds by Electrochemical Reductive Coupling of Amines and α‐Bromocarboxamides

AbstractNucleophilic substitution of α‐bromocarboxamide with amines have been demonstrated to synthesize sterically congested C−N bonds. Electrochemical reductive coupling is an efficient route to access hindered amino acid derivatives possessing a quaternary carbon center with excellent yields under mild reaction conditions. Cyclic voltammetry analyses indicated the cathodic reduction of α‐bromocarboxamides in the presence of amines as nucleophiles. The control experiment using the N‐methyl derivative of α‐bromocarboxamide suggested that the aziridinone is the most probable intermediate in this method.

Hydroalkylation of 1,1‐Diarylalkenes Mediated by Magnesium Hydride in Ethereal Solvents

AbstractA method for the hydroalkylation of 1,1‐diarylalkenes has been developed using magnesium hydride (MgH2) generated in situ by the solvothermal treatment of magnesium iodide (MgI2) with sodium hydride (NaH) in ethereal solvents. The process is initiated by the hydromagnesiation of 1,1‐diarylalkenes with MgH2 to generate 1,1‐diarylethylmagnesium species, which are immediately alkylated with ethereal solvents to construct a diaryl quaternary carbon center in the hydroalkylation products.

Benchtop Nickel Catalysis Invigorated by Electron-Deficient Diene Ligands.

ConspectusDue to the rarity of precious metals like palladium, nickel catalysis is becoming an increasingly important player in organic synthesis, especially for the formation of bonds with sp3-hybridized carbon centers. Traditionally, catalytic processes involving active Ni(0) species have relied on Ni(COD)2 or in situ reduction of Ni(II) salts. However, Ni(COD)2 is an air- and temperature-sensitive material that requires use in an inert-atmosphere glovebox, and in situ reduction protocols of Ni(II) salts using metallic or organometallic reductants add additional complications to reaction development.This Account chronicles the development of air-stable Ni(0) precursors as replacements for Ni(COD)2 or in situ reduction. Based on Schrauzer's seminal discovery of Ni(COD)(DQ) as an air-stable zerovalent organonickel complex, our research laboratories at Scripps Research and Bristol Myers Squibb have developed a class of precatalysts based on the Ni(COD)(EDD) (EDD = electron-deficient diene) framework, relying on the steric and electronic properties of the supporting diene to render the metal center stable to air, moisture, and even silica gel but reactive to ligand substitution and redox changes.The stable Ni(0) complexes can be accessed through ligand exchange with Ni(COD)2, through reduction of Ni(acac)2 using DIBAL-H, or electrochemically via cathodic reduction of Ni(acac)2 to Ni(COD)2, followed by addition of an EDD ligand in one pot. As a toolkit, the complexes demonstrate reactivity that is equivalent or enhanced compared to Ni(COD)2, catalyzing C-C and C-N cross-couplings, Miyaura borylations, C-H activations, and other transformations. Since the initial report on Ni(COD)(DQ), its reactivity in C(sp2)-CN activation, metallophotoredox, and electric field-induced cross-coupling have also been demonstrated.By incorporating the precatalyst toolkit into reaction discovery campaigns, our laboratories have been able to perform C(sp3)-S(alkyl) couplings and metallonitrenoid carboamination, both of which represent challenging transformations that were inaccessible with traditional phosphine, nitrogen, or electron-deficient olefin ligands. Computational and experimental studies demonstrate how the quinone ligands are hemilabile, adopting η1(O)-bound geometries to relieve steric strain or stabilize transition states and intermediates; redox-active, able to transiently oxidize the metal center; and electron-withdrawing or -donating, depending on metal oxidation state and coordination geometry. These studies show how the ligands enable key steps in catalysis beyond imparting air-stability.Since our report documenting the catalytic activity of Ni(COD)(DQ), many other laboratories have also observed unique reactivity with this precatalyst. Ni(COD)(DQ) was found to offer superior reactivity to Ni(COD)2 in C-N cross coupling to form N,N-diaryl sulfonamides and in preparation of biaryls from aryl halides and benzene through a Ni-mediated, base-assisted homolytic aromatic substitution.

Comprehensive analysis the prognostic and immune characteristics of mitochondrial transport-related gene SFXN1 in lung adenocarcinoma

BackgroundMitochondria, which serve as the fundamental organelle for cellular energy and metabolism, are closely linked to the growth and survival of cancer cells. This study aims to identify and assess Sideroflexin1 (SFXN1), an unprecedented mitochondrial gene, as a potential prognostic biomarker for lung adenocarcinoma (LUAD).MethodsThe mRNA and protein levels of SFXN1 were investigated based on the Cancer Genome Atlas (TCGA) LUAD dataset, and then validated by real-time quantitative PCR, Western Blotting and immunohistochemistry from our clinical samples. The clinical correlation and prognostic value were evaluated by the TCGA cohort and verified via our clinical dataset (n = 90). The somatic mutation, drug sensitivity data, immune cell infiltration and single-cell RNA sequencing data of SFXN1 were analyzed through public databases.ResultsSFXN1 was markedly upregulated at both mRNA and protein levels in LUAD, and high expression of SFXN1 were correlated with larger tumor size, positive lymph node metastasis, and advanced clinical stage. Furthermore, SFXN1 upregulation was significantly associated with poor clinical prognosis. SFXN1 co-expressed genes were also analyzed, which were mainly involved in the cell cycle, central carbon metabolism, DNA repair, and the HIF-1α signaling pathway. Additionally, SFXN1 expression correlated with the expression of multiple immunomodulators, which act to regulate the tumor immune microenvironment. Results also demonstrated an association between SFXN1 expression and increased immune cell infiltration, such as activated CD8 + T cells, natural killer cells (NKs), activated dendritic cells (DCs), and macrophages. LUAD patients with high SFXN1 expression exhibited heightened sensitivity to multiple chemotherapies and targeted drugs and predicted a poor response to immunotherapy. SFXN1 represented an independent prognostic marker for LUAD patients with an improved prognostic value for overall survival when combined with clinical stage information.ConclusionsSFXN1 is frequently upregulated in LUAD and has a significant impact on the tumor immune environment. Our study uncovers the potential of SFXN1 as a prognostic biomarker and as a novel target for intervention in LUAD.

Abstract A059: Targeting pancreatic cancer metabolic dependencies through glutamine antagonism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. A factor that contributes to the poor prognosis of the disease is the complex tumor microenvironment (TME). The PDAC TME is composed of excessive fibrosis and desmoplasia, which creates a harsh environment resulting in hypoxia and altered nutrient availability. To promote survival and proliferation in this environment, PDAC cells can reprogram glutamine (Gln) metabolism. Previous studies have demonstrated that PDAC cells use Gln to support proliferation and redox balance. However, earlier attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. We hypothesized that a Gln analogue, such as 6-Diazo-5-oxo-L-norleucine (DON), could broadly target Gln metabolism in PDAC and prevent rapid adaptation. Indeed, DON treatment led to a significant decrease in PDAC proliferation and a profound reduction of various metabolites involved in central carbon and nucleotide metabolism, suggesting that DON creates a metabolic crisis. In addition, we observed a significant decrease in tumor growth in various in vivo models (syngeneic, immunodeficient and PDXs) using DRP-104 (sirpiglenastat), a novel pro-drug version of DON that was designed to circumvent DON associated GI toxicity and allow the therapeutic exploration of broad Gln antagonism. Mechanistically, we found that ERK signaling is increased as a compensatory mechanism through the increased activity of receptor tyrosine kinases (RTKs). Combinatorial treatment of DRP-104 and Trametinib (MEK1/2) inhibitor led to a significant increase in survival in a syngeneic model PDAC. Taken together, these pre-clinical results suggest that broadly targeting Gln metabolism could provide a new therapeutic avenue for PDAC and that the combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome. Citation Format: Joel Encarnacion-Rosado, Alec C. Kimmelman, Robert Wild. Targeting pancreatic cancer metabolic dependencies through glutamine antagonism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A059.

Abstract PR10: Uridine-to-ribose axis supports glucose-restricted pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a 5-year survival rate of 12% in the USA. PDA highly rely on glucose but is surrounded by a nutrient-limited microenvironment. How PDA survive such condition, and the metabolites enabling that process, is unclear. To determine how PDA adapt to starvation, we profiled the utility of &amp;gt;175 metabolites by 21 pancreatic cell lines cultured in glucose limited condition. This nutrient profiling assay revealed that PDA use uridine when glucose is depleted. Uridine utilization strongly correlated with uridine phosphorylase 1 (UPP1) – the enzyme that breaks down uridine to uracil and ribose-1-phosphate. Metabolomics, including stable isotope tracing, revealed that when glucose is limited, PDA cells use the ribose from uridine to sustain central carbon metabolism and that this process is blocked upon the knockout of UPP1. We found that UPP1 is regulated by KRAS, is high in PDA, and correlates with poor survival in patients. Moreover, deletion of UPP1 blunted tumor growth in mice. Our data identify the uridine-ribose axis as a crucial compensatory pathway in nutrient-deprived PDA, suggesting a potential therapeutic target. Citation Format: Zeribe C. Nwosu, Matthew H. Ward, Peter Sajjakulnukit, Pawan Poudel, Chanthirika Ragulan, Steven Kasperek, Megan Radyk, Damien Sutton, Rosa E. Menjivar, Anthony Andren, Juan J. Apiz-Saab, Zachary Tolstyka, Kristee Brown, Ho-Joon Lee, Lindsey Dzierozynski, Xi He, Hari Ps, Julia Ugras, Gift Nyamundanda, Li Zhang, Christopher J. Halbrook, Eileen Carpenter, Jiaqi Shi, Leah P. Shriver, Gary J. Patti, Alexander Muir, Marina Pasca di Magliano, Anguraj Sadanandam, Costas A. Lyssiotis. Uridine-to-ribose axis supports glucose-restricted pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR10.

Abstract A006: Differential gene expression to delineate racial disparities in the molecular landscape of pancreatic ductal adenocarcinoma

Abstract Introduction: In the US, there are racial disparities in PDAC incidence and mortality, higher among blacks than whites. In addition to socioeconomic status, lifestyles, and age, genetics also contributes to these disparities. Thus, we conducted transcriptomic analyses (RNA-seq) of PDAC samples collected from African American (AA) and Caucasian (CA) patients to identify race/ethnicity-specific gene expression profiles and their related pathways to find determinants that contribute to the aggressive phenotypes of PDACs. This study is relevant to the UAB catchment area, since about 30% of patients with PDAC are AAs. Methodology: Histologically confirmed PDACs (n=40) from AA (9 PDAC and 3 matching normal tissues) and CA (31 PDAC and 5 matching normal tissues) were included in this study. FFPE sections of PDACs and their corresponding normal tissues were macro-dissected for RNA isolation. Whole transcriptomic sequencing was performed with a NextSeq 500/550 platform. Trimmed reads were mapped to a human reference genome (hg38) using HISAT, and gene level read count data were obtained using HTSeq. Differential expression analysis was performed using the DESeq2 bioconductor package. ClusterProfiler/DOSE R packages were used for gene ontology and KEGG pathway enrichment analyses. Genes with log 2-fold change of ≥1 and adjusted P-value &amp;lt;0.05 were considered as differentially expressed. Results: Among the top upregulated genes altered in only AA PDACs, compared to their normal tissues, were RNF144B, TESPA1, KLHL17, H2AX, MDGA1, CDK20, PHLDA3, SLC6A16, PARVG, GATD3, NUDT16, RENBP, RTL8C, C1QTNF1, HLA−DRB5, PARP15, PPP1R16B, RASGRP2, and CHST15. Of note, targeting CHST15, by an RNA oligonucleotide, STNM01 in Phase I/IIa trial on unresectable PDAC patients showed improved overall survival. Additionally, inhibition of KLHL17, an upstream activator of Ras/MAPK, could be a candidate target in PDAC. The KEGG pathways altered in AA PDACs, were glycerophospholipid metabolism; bile secretion; retinol metabolism; regulation of lipolysis in adipocytes; and pantothenate and CoA biosynthesis. In CAs, the top upregulated genes in PDACs, compared to their normal tissues, were PPY, UGT1A10, GPR20, SDR16C5, KLK7, MYBPC1, ITLN1, PADI1, CLCA1, UGT1A9, and SERPINB3.The KEGG pathways altered in CA PDACs, were cellular senescence; AGE−RAGE signaling pathway in diabetic complication; PD−L1 and PD−1 checkpoint pathway; and central carbon metabolism. There were 13 genes differentially modulated in AA PDACs as compared to CA PDACs. The 6 down-regulated were USP17L1, C2CD4D, MMP13, DCUN1D5, and 2 genes without annotations (ENSG00000276345 and ENSG00000280966); the 7 up-regulated genes were SEMA4A, LETM2, HMOX1, OTUB2, MEDAG, VEGFD, and HBA1. Immunohistochemical validation of these markers is in progress. Conclusions: Findings of this study showed distinct gene expression profiles and differentially modulated pathways in AA and CA PDAC patients. These results will aid in identifying aggressive phenotypes and new targets for developing race/ethnicity-based therapeutic interventions. Citation Format: Prachi Bajpai, Ravi Paluri, Sameer Al Diffalha, Darshan S. Chandrashekar, Farrukh Afaq, Ryan Bash, C. Ryan Miller, Sooryanarayana Varambally, Moh’d Khushman, Upender Manne. Differential gene expression to delineate racial disparities in the molecular landscape of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A006.

Abstract B088: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M.O. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B088.

Lighting up Pyruvate Metabolism in Saccharomyces cerevisiae by a Genetically Encoded Fluorescent Biosensor.

Monitoring intracellular pyruvate is useful for the exploration of fundamental metabolism and for guiding the construction of yeast cell factories for chemical production. Here, we employed a genetically encoded fluorescent Pyronic biosensor to light up the pyruvate metabolic state in the cytoplasm, nucleus, and mitochondria of Saccharomyces cerevisiae BY4741. A strong correlation was observed between the pyruvate fluctuation in mitochondria and cytoplasm when exposed to different metabolites. Further metabolic analysis of pyruvate uptake and glycolytic dynamics showed that glucose and fructose dose-dependently activated cytoplasmic pyruvate levels more effectively than direct exposure to pyruvate. Meanwhile, the Pyronic biosensor could visually distinguish phenotypes of the wild-type S. cerevisiae BY4741 and the pyruvate-hyperproducing S. cerevisiae TAM at a single-cell resolution, having the potential for high-throughput screening. Overall, Pyronic biosensors targeting different suborganelles contribute to mapping and studying the central carbon metabolism in-depth and guide the design and construction of yeast cell factories.