Metabolic Genes Research Articles

Genes involved in carbon, nitrogen, and sulfur cycling in an important estuarine ecosystem show coherent shifts in response to changes in environmental conditions

AbstractWhile metagenomics can provide insight into microbial community metabolic potential, understanding factors that influence gene abundance is necessary to maximize the information gained from this analysis. Gene abundances are influenced by chemical or physical conditions along with other factors, such as copy number variation between taxa, methodological biases, or issues associated with identification and classification. Here, we identify major drivers of spatiotemporal shifts in microbial gene relative abundance from multiple months, sites, and depths within Chesapeake Bay in 2017 using shotgun metagenomics. We compared changes in relative abundance of key genes for bacterial photosynthesis, nitrogen, and sulfur metabolism with each other and measured environmental variables. Major drivers of differences in key metabolic gene abundances are associated with environmental variables that largely change with depth and season (e.g., temperature, oxygen, phosphate). For sulfur oxidation, bacterial photosynthesis, and denitrification, genes within each process are generally significantly correlated with each other and with several environmental variables. For other processes, such as nitrification, nitrogen fixation, and dissimilatory nitrate reduction to ammonium, genes that encode enzymes within the same pathway are not well correlated. The lack of correlation typically results from differences in identified taxa carrying these genes, suggesting modular pathway structure, methodological errors, or discrepancies in gene copy number between taxonomic groups. To be suitable indicators of biogeochemical processes for models, genes or pathways should be strongly correlated with environmental variables and specific to and inclusive of all taxa mediating the associated process.

ABA and Melatonin: Players on the Same Field?

In plants, abscisic acid (ABA) and melatonin (MT) are conventionally treated as molecules mitigating stress responses. To understand the mechanisms of ABA–MT interplay, we examined the effects of ABA and MT treatment in ABA and MT loss-of-function mutants of Arabidopsis thaliana exposed to high light (HL) stress. ABA constantly suppressed ASMT encoding N-acetylserotonin methyltransferase in the context of differential responses of other MT biosynthesis genes in both the wild type (WT) and mutants. However, this response was absent in the mutant with the disrupted ABI4. Given that the ASMT promoter region contains several potential ABI4-binding elements, these data suggest that ASMT can be a potential target gene for ABI4. A role for ABI4 in the interactions between ABA and MT is supported by the finding that ABI4 is constitutively derepressed in the MT signaling mutants cand2 and gpa1, which exhibited elevated steady state levels of ABI4 transcripts and were not regulated by either stress or melatonin. In addition, the abi4 mutant showed increased modulations in the expression of the MT catabolic genes M2H and M3H in response to ABA treatment, inferring that this transcription factor is a negative regulator of ABA-dependent changes in MT content. Furthermore, all tested mutants with impaired ABA synthesis or signaling displayed elevated steady state MT levels compared to WT, while MT treatment contributed to the downregulation of key ABA synthesis and signaling genes. Collectively, our results suggest that ABA and melatonin act antagonistically, modulating the expression of ABA and MT signaling and metabolism genes. To understand the mechanisms of ABA–MT interactions, we studied the effects of ABA and MT treatment in ABA and MT loss-of-function mutants of Arabidopsis thaliana exposed to severe light stress (SLS).

Deciphering the anthocyanin metabolism gene network in tea plant (Camellia sinensis) through structural equation modeling.

Tea is an important cash crop that significantly contributes to rural development, poverty reduction and food security in many developing countries. It provides livelihoods for millions of smallholder producers and aids their economic stability. Anthocyanins in tea leaves provides excellent commercial quality and germplasm exploration potential. These compounds give tea leaves vibrant colors and increase health benefits. The current understanding of the synergistic regulation mechanisms responsible for color changes in purple tea, attributed to anthocyanin degradation, remains unclear. In this study, we have identified 30 gene families within the genome that are associated to with anthocyanin metabolism from tea. These gene families play distinct roles in the biosynthesis of anthocyanin including the formation of the core, structure, modification of the molecular framework, facilitation of transport process, regulation of gene expression, breakdown pathways, sugar transportation and iron ion respectively. Subsequently, we investigated the synergistic mechanisms of anthocyanin metabolism related gene families within tea leaves using structural equation modeling. The results showed that sugar transport positively affects anthocyanin transportation, and promotes anthocyanin degradation during leaf pigmentation, whereas, it inhibits anthocyanin degradation during the fading of leaf color. Further, Iron ions facilitate the degradation of anthocyanins during their deposition and conversely, impede this degradation process during digestion. These finding suggests that tea plants may regulate the synthesis and degradation of anthocyanins through sugar transport and iron ions ensure healthy levels and vibrant colors. Our study contributes valuable information into the dynamic equilibrium anthocyanin mechanism and sheds light on complex regulatory mechanisms that govern the synthesis, transport and degradation of these pigments. These insights could be further used to develop strategies for enhancing anthocyanins content in unique tea germplasm to aid tea industry in producing new tea products with increased health benefits and aesthetic appeals.

Discovery and characterization of complete genomes of 38 head-tailed proviruses in four predominant phyla of archaea.

Archaea play a significant role in natural ecosystems and the human body. Archaeal viruses exert a considerable influence on the structure and composition of archaeal communities and their associated ecological environments. The present study revealed the complete genomes of 38 archaeal head-tailed proviruses through comprehensive data mining. The hosts of these proviruses were identified as belonging to the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. In addition to the 14 proviruses of halophilic archaea related to the Graaviviridae family, the remaining proviruses exhibited limited genetic similarities to known (pro)viruses, suggesting the existence of 14 potential novel families. Of the 38 archaeal proviruses, 30 have the potential to lyse host cells. Eleven proviruses contain genes linked to antiviral defense mechanisms, including those involved in restriction modification (RM), clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) nucleases, defense island system associated with restriction-modification (DISARM), and DNA degradation (Dnd). Moreover, auxiliary metabolic genes were identified in the proviruses of Bathyarchaeia and Halobacteriota archaea, including those involved in carbohydrate and amino acid metabolism. Our findings indicate the diversity of archaeal viruses, their interactions with archaeal hosts, and their roles in the adaptation of the host.IMPORTANCEThe field of archaeal virology has seen a rapid expansion through the use of metagenomics, yet the diversity of these viruses remains largely uncharted. In this study, the complete genomes of 38 novel archaeal proviruses were identified for the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. Two families and six genera of Archaea were the first to be identified as hosts for viruses. The proviruses were found to contain diverse genes that were involved in distinct adaptation strategies of viruses to hosts. Our findings contribute to the expansion of the lineages of archaeal viruses and highlight their intricate interactions and essential roles in enabling host survival and adaptation to diverse environmental conditions.

Metabolomic and transcriptomic analyses reveals candidate genes and pathways involved in secondary metabolism in Bergenia purpurascens.

Bergenia purpurascens is an important medicinal, edible and ornamental plant. The lack of omics information hinders the study of its metabolic pathways and related genes. In order to investigate candidate genes and pathways involved in secondary metabolism in B. purpurascens, roots, stems and leaves of B. purpurascens were subjected to metabolomic and transcriptomic analyses in this study. A total of 351 differentially accumulated secondary metabolites were identified. We identified 120 candidate genes involved in phenylpropanoid and flavonoid biosynthesis pathway, from which 29 key candidate genes were obtained by WGCNA. Five UDP-Glycosyltransferases and four O-methyltransferases were suggested to be the candidate enzymes involved in synthetic pathway from gallic acid to bergenin by correlation analysis between transcriptional and metabolic levels and phylogenetic analysis. This study provides data resources and new insights for further studies on the biosynthesis of major active components in B. purpurascens.

Abstract A018: Alternative Polyadenylation Regulates Pancreatic Cancer Fibroblast Metabolism

Abstract Dense, fibrotic stroma is a defining feature of pancreatic ductal adenocarcinoma (PDAC), comprising up to 90% of tumor volume. Multiple studies have shown that this stroma strongly influences cancer progression and response to treatment. Cancer Associated Fibroblasts (CAFs) make up the majority of the Tumor Microenvironment (TME) and directly alter the tumor landscape and response to therapeutics. CAFs are a heterogenous population comprising of a mixture of inflammatory, myofibroblastic, and antigen-presenting CAFs, with both pro- and anti- tumorigenic functions. While we have been able to define CAFs phenotypically, we lack a deeper understanding of the mechanisms underlying their transcriptional heterogeneity and ultimately their contribution to driving PDAC progression. Our lab has previously shown 3’UTR-Alternative Polyadenylation (APA) to be an important driver of genetic dysregulation in PDAC. APA is an important process regulating mRNA stability, protein localization, and expression, ultimately determining cellular processes such as proliferation, metastasis, and migration. We have also shown that increased 3’ UTR shortening is associated with the pro- tumorigenic inflammatory CAF subtype. However, how APA regulates CAF function is unknown. We are now trying to understand the possible mechanisms of these changes in vitro using human-derived CAF cell lines and treating them with a known pharmacological modulator of APA. We performed PAC-Seq analyses on these samples and show that important drivers of CAF metabolism are hindered by APA, ultimately affecting the ability of CAFs to support cancer cell growth. We find that inhibition of APA drives CAF migration and senescence phenotype and alters gene expression of critical genes required for CAF function, and metabolism. We identified a novel mechanism by which a key metabolic gene SLC7A11 is regulated by APA. To our knowledge, our work is one of the first studies to study how APA is an important regulator of CAF function. Citation Format: Aditi H Chaubey, Michael E Feigin, Eric J Wagner. Alternative Polyadenylation Regulates Pancreatic Cancer Fibroblast Metabolism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A018.

Analysis of immune status and prognostic model incorporating lactic acid metabolism-associated genes.

Cancer development is intricately linked with metabolic dysregulation, including lactic acid metabolism, which plays a pivotal role in tumor progression and immune evasion. However, its specific implications in gastric adenocarcinoma (STAD) remain unclear. This study introduces a novel methodology to evaluate lactic acid metabolism comprehensively in STAD, aiming to elucidate its prognostic significance and impact on immunotherapy efficacy. Targeted therapies directed at key lactic acid metabolism genes (LMGs) identified within the tumor microenvironment (TME) hold promise for personalized treatment strategies. Lactic acid metabolism patterns were assessed in 415 STAD patients using a panel of 21 LMGs. Cox regression and Lasso regression analyses were employed to develop a predictive risk model based on differentially expressed genes (DEGs). Validation of the model was conducted using independent cohorts from the GEO and TCGA databases, as well as additional datasets focused on immunotherapy responses. Further investigations into TME dynamics of lactic acid metabolism included functional assays targeting SLC16A3, a pivotal gene identified through our analyses. Patients were stratified into distinct risk groups based on their lactic acid metabolism profiles. Low-risk patients exhibited attenuated lactic acid metabolism, correlating with favorable clinical outcomes characterized by prolonged survival and enhanced responsiveness to immunotherapy. Notably, tumor cells within the TME demonstrated heightened levels of active lactic acid metabolism, particularly impacting tumor-infiltrating lymphocytes such as CD8 + T cells and regulatory T cells. Mechanistically, SLC16A3 emerged as a critical regulator promoting STAD cell proliferation, invasion, and migration while modulating the metabolic landscape. This study underscores the prognostic value of a lactic acid metabolism-based model in STAD, providing insights into its potential as a predictive biomarker for patient stratification and therapeutic targeting. The findings highlight SLC16A3 as a promising candidate for therapeutic intervention aimed at modulating lactic acid metabolism in the TME, thereby advancing personalized treatment strategies in gastric cancer management.

Why Symptoms Linger in Quiescent Crohn's Disease: Investigating the Impact of Sulfidogenic Microbes and Sulfur Metabolic Pathways.

Even in the absence of inflammation, persistent symptoms in patients with Crohn's disease (CD) are prevalent and worsen quality of life. We previously demonstrated enrichment in sulfidogenic microbes in quiescent Crohn's disease patients with (qCD + S) vs without persistent GI symptoms (qCD-S). Thus, we hypothesized that sulfur metabolic pathways would be enriched in stool while differentially abundant microbes would be associated with important sulfur metabolic pathways in qCD + S. We performed a multicenter observational study nested within SPARC IBD. Quiescent inflammation was defined by fecal calprotectin level < 150 mcg/g. Persistent symptoms were defined by CD-PRO2. Active CD (aCD) and non-IBD diarrhea-predominant irritable bowel syndrome (IBS-D) were included as controls. Thirty-nine patients with qCD + S, 274 qCD-S, 21 aCD, and 40 IBS-D underwent paired shotgun metagenomic sequencing and untargeted metabolomic profiling. The fecal metabolome in qCD + S was significantly different relative to qCD-S and IBS-D but not aCD. Patients with qCD + S were enriched in sulfur-containing amino acid pathways, including cysteine and methionine, as well as serine, glycine, and threonine. Glutathione and nicotinate/nicotinamide pathways were also enriched in qCD + S relative to qCD-S, suggestive of mitochondrial dysfunction, a downstream target of H2S signaling. Multi-omic integration demonstrated that enriched microbes in qCD + S were associated with important sulfur metabolic pathways. Bacterial sulfur metabolic genes, including CTH, isfD, sarD, and asrC, were dysregulated in qCD + S. Finally, sulfur metabolites with and without sulfidogenic microbes showed good accuracy in predicting the presence of qCD + S. Microbial-derived sulfur pathways and downstream mitochondrial function are perturbed in qCD + S, which implicate H2S signaling in the pathogenesis of this condition. Future studies will determine whether targeting H2S pathways results in improved quality of life in qCD + S.

Comparative transcriptomic and metabolomic analysis of FTO knockout and wild-type porcine iliac artery endothelial cells.

The fat mass and obesity associated (FTO) gene, previously identified as a pivotal genetic locus associated with adiposity, has recently been linked to various cancers. In this study, we established an FTO knockout (KO) cell line in porcine iliac artery endothelial cells (PIECs) utilizing CRISPR/Cas9 technology to systematically investigate the gene's function and effect through transcriptomic and metabolomic analysis. Our results revealed significant gene expression and metabolic profiles differences between the FTO KO and wild-type (WT) cells. Furthermore, enrichment analysis highlighted the involvement of differentially expressed genes in metabolic processes, cellular components, and molecular functions, as well as in complement and coagulation cascades, mineral absorption, glutathione metabolism, insulin signaling, fluid shear stress, and atherosclerosis pathways. The metabolomic profiling revealed clear distinctions between the FTO KO and WT cells, indicating profound modifications in cellular metabolism. Correlation analysis of transcriptomic and metabolomic data revealed a significant association between six metabolites and twenty genes, with melatonin showing specific correlations with the expression of several genes, indicating a complex regulatory network between gene expression and metabolic changes. This study provides a foundation for further research on the FTO gene's role in cellular processes and molecular mechanisms underlying physiological and pathological conditions.

Lipid metabolism-related gene signature predicts prognosis and unveils novel anti-tumor drugs in specific type of diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma which possess highly aggressive and heterogeneous. Despite advances in understanding heterogeneity and development of novel targeted agents, the prognosis of DLBCL patients remains unsatisfied. Lipids are crucial components of biological membranes and signal transduction while accumulating evidence has supported the vital roles of abnormal lipid metabolism in tumorigenesis. Furthermore, some related pathways could serve as prognostic biomarkers and potential therapeutic targets. However, the clinical significance of abnormal lipid metabolism reprogramming in DLBCL has not been investigated. In the current study, we developed a prognostic risk model for DLBCL based on the abnormal expressed lipid metabolism genes and moreover based on our risk model we classified patients with DLBCL into novel subtypes and identified potential drugs for DLBCL patients with certain lipid metabolism profiles. We utilized univariate Cox regression analysis to identify the prognosis-related lipid metabolism genes, and then performed LASSO Cox regression to identify prognostic related lipid metabolism related genes. Multivariate cox regression was used to establish the prognostic model. Patients were divided in to high and low risk groups based on the median risk score. Immune cell infiltration and GSEA were used to identify the pathways between high and low risk groups. Oncopredict algorithm was utilized to identify potential drug for high-risk patients. In vitro cell apoptosis and viability analysis were employed to verify the specific tumor inhibition effects of AZD5153. Nineteen survival related lipid metabolism genes TMEM176B, LAYN, RAB6B, MMP9, ATAD3B, SLC2A11, CD3E, SLIT2, SLC2A13, SLC43A3, CD6, SIRPG, NEK6, LCP2, CTTN, CXCL2, SNX22, BCL6 and FABP4 were identified and subjected to build the prognostic model which was further verified in four external microarray cohorts and one RNA seq cohorts. Tumor immune microenvironment analysis and GSEA results showed that the activation of MYC targets genes rather than immunosuppression contribute to the poor survival outcome of patients in the high-risk group. AZD5153, a novel bivalent BET bromodomain inhibitor which could inhibit the transcription of MYC and E2F exhibited specific antitumor function for cells with high-risk score. Our results provide the first lipid metabolism-based gene signature for predicting the survival of patients with DLBCL. Furthermore, by determining novel subtypes with our lipid metabolism prognostic model we illustrated that drugs that compromising MYC target genes rather than immune checkpoint inhibitors may be beneficial to DLBCL patients with certain lipid metabolism profiles.

Favorable inhibitory effect of clodronate on hepatic steatosis in short bowel syndrome model rats.

This study investigated the anti-inflammatory effect of clodronate, a vesicular nucleotide transporter (VNUT) inhibitor, on intestinal-failure-associated liver disease (IFALD) in a rat model of short bowel syndrome (SBS). The rats underwent jugular vein catheterization for continuous total parenteral nutrition (TPN) and 90% small bowel resection. The animals were divided into the following groups: TPN/SBS (Control group), TPN/SBS/intravenous administration of low-dose clodronate (20mg/kg twice per week; Low group), or TPN/SBS/intravenous administration of high-dose clodronate (60mg/kg twice per week; High group). On day 7, the rats were euthanized. Hepatic steatosis and hepatocellular injury were also assessed. Hepatic steatosis and lobular inflammation in the liver were observed in all groups. The High group showed histologically reduced hepatic steatosis compared with the Control group. IL-6 and Nlrp3 expression in the High group was significantly suppressed compared to that in the Control group. The expression of other inflammatory cytokines tended to be lower in the High dose group than in the control group. The lipid metabolism gene expression in the liver specimens showed no significant differences among the groups. The high-dose administration of clodronate may, therefore, inhibit hepatic steatosis and inflammation associated with IFALD in patients with SBS.

Plasticity and environment-specific relationships between gene expression and fitness in Saccharomyces cerevisiae.

The environment influences how an organism's genotype determines its phenotype and how this phenotype affects its fitness. Here, to better understand this dual role of environment in the production and selection of phenotypic variation, we determined genotype-phenotype-fitness relationships for mutant strains of Saccharomyces cerevisiae in four environments. Specifically, we measured how promoter mutations of the metabolic gene TDH3 modified expression level and affected growth for four different carbon sources. In each environment, we observed a clear relationship between TDH3 expression level and fitness, but this relationship differed among environments. Mutations with similar effects on expression in different environments often had different effects on fitness and vice versa. Such environment-specific relationships between phenotype and fitness can shape the evolution of phenotypic plasticity. We also found that mutations disrupting binding sites for transcription factors had more variable effects on expression among environments than those disrupting the TATA box, which is part of the core promoter. However, mutations with the most environmentally variable effects on fitness were located in the TATA box, because of both the lack of plasticity of TATA box mutations and environment-specific fitness functions. This observation suggests that mutations affecting different molecular mechanisms contribute unequally to regulatory sequence evolution in changing environments.

Integration of genomic and transcriptomic layers in RNA-Seq data leads to protein interaction modules with improved Alzheimer's disease associations.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and it is currently untreatable. RNA sequencing (RNA-Seq) is commonly used in the literature to identify AD-associated molecular mechanisms by analysing changes in gene expression. RNA-Seq data can also be used to detect genomic variants, enabling the identification of the genes with a higher load of deleterious variants in patients compared with controls. Here, we analysed AD RNA-Seq datasets to obtain differentially expressed genes and genes with a higher load of pathogenic variants in AD, and we combined them in a single list. We mapped these genes on a human protein-protein interaction network to discover subnetworks perturbed by AD. Our results show that utilizing gene pathogenicity information from RNA-Seq data positively contributes to the disclosure of AD-related mechanisms. Moreover, dividing the discovered subnetworks into highly connected modules reveals a clearer picture of altered molecular pathways that, otherwise, would not be captured. Repeating the whole pipeline with human metabolic network genes led to results confirming the positive contribution of gene pathogenicity information and enabled a more detailed identification of altered metabolic pathways in AD.

DDDR-07. FUNCTIONAL GENOMICSIN VIVO USING CRISPR/CAS9 IDENTIFIES POTENTIAL METABOLIC VULNERABILITIES WITHIN MELANOMA BRAIN METASTASIS

Abstract BACKGROUND Inhibition of the oxidative phosphorylation (OxPhos) pathway in mice suppressed the growth of melanoma brain metastasis but not extracranial or primary skin melanoma. This indicates that melanoma brain metastasis may have targetable metabolic vulnerabilities. Human clinical trials, however showed toxicities following OxPhos pathway inhibition. This suggests that more selective metabolic targets are needed to minimize off target effects. METHODS We designed a focused CRISPR guide RNA (gRNA) lentiviral knockout library targeting 106 metabolism genes significantly enriched in human melanoma brain metastasis (n=88) relative to extracranial (n=50) or primary skin melanoma (n=52). We infected mouse B16F10 melanoma cell lines with the CRISPR library and implanted 1X106 cells subcutaneously or intracerebrally into male (n=3) and female (n=3) C57/Bl6 mice. DNA from cell lines and tumor samples were sent for next generation sequencing. Candidate metabolism genes that may drive growth of melanoma lesion within the brain were identified based on loss of gRNA copies in brain relative to skin. RESULTS The CRISPR/Cas9 in vivo screen identified metabolic genes whose knockout resulted in loss of gRNA clones within established B16F10 melanoma brain lesions relative to skin lesions. These putative metabolism targets included specific components in the oxidative phosphorylation pathway, choline metabolism, and ubiquitin cascade activation. CONCLUSION Using a focused CRISPR/Cas9 in vivo screen, we uncovered potential metabolic targets within established mouse melanoma brain lesions. vWe are currently performing the in vivo screen in melanoma mouse models with spontaneous brain metastasis. Top 3 hits across all models will be validated individually prior to a compound screen for inhibitors.

Grape-Seed Proanthocyanidin Extract (GSPE) Modulates Diurnal Rhythms of Hepatic Metabolic Genes and Metabolites, and Reduces Lipid Deposition in Cafeteria-Fed Rats in a Time-of-Day-Dependent Manner.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health issue with increasing prevalence. Polyphenols, such as grape seed proanthocyanidin extract (GSPE), are bioactive compounds present in plants and represent an interesting therapeutical approach for MASLD. This study questioned whether the timing of GSPE administration impacts liver diurnal metabolism and steatosis in a rat obesity model. Results from hepatic lipid profiling and diurnal metabolic gene expression and metabolomics reveal that rats fed with a cafeteria (CAF) diet show impaired glucose homeostasis and enhanced lipogenesis in the liver, contributing to liver steatosis. Chronic consumption of GSPE in the inactive or active phase is associated with beneficial effects as the restoration of rhythms of transcripts and metabolites is observed. However, only when given in the active phase, GSPE treatment decreases hepatic triglyceride levels. Using an in vitro hepatocyte model, the study identifies that catechin, one of the main phenolic compounds found in the GSPE extract, is a potential mediator in ameliorating the effects of CAF-induced liver steatosis. Taken altogether, the findings show that the beneficial effects of GSPE on MASLD development depend on the treatment time.

IMMU-33. REPROGRAMMING OF MYELOID CELL METABOLISM VIA HEME OXYGENASE-1 INHIBITION POTENTIATES ANTI-TUMOR IMMUNITY IN A MURINE GLIOMA MODEL

Abstract Cancer cells exhibit a preference for aerobic glycolysis, a phenomenon known as the Warburg effect. Emerging evidence suggests that immune cells within the tumor microenvironment (TME) may employ a similar metabolic adaptation. Heme oxygenase-1 (HMOX-1), a metabolic regulatory gene, is elevated in myeloid cells of glioblastoma (GBM), yet its precise role in modulating immune suppression and energy utilization remains unclear. Our objective was to explore how targeting HMOX-1 activity in the GBM TME affects immune response and energy metabolism. We utilized syngeneic glioma models with C57BL/6J mice implanted with CT-2A cells, which were randomized into four treatment arms: intracranial (IC) injection of the HMOX-1 inhibitor zinc protoporphyrin (ZnPP), intraperitoneal (IP) injection of anti-PD-1, IC ZnPP + IP anti-PD-1, or IC saline (sham). For mechanistic studies, we developed a myeloid-specific knockdown model via adoptive cell transfer of myeloid-derived suppressor cells (MDSCs) transfected with Hmox-1 siRNA into CSF-1R myeloid-depleted mice. Immunophenotyping was conducted with flow cytometry. The Seahorse mitochondrial stress test was used for evaluation of energy dynamics. Downregulation of myeloid Hmox-1 expression and inhibition of HMOX-1 via ZnPP both led to heightened T cell activation, with increased IFN-γ expression in brain compared to untreated mice (P=0.039, 0.02, respectively). Notably, ZnPP treatment induced a compensatory elevation of PD-L1 in myeloid cells (P&amp;lt;0.0001). Both ZnPP monotherapy and anti-PD-1 + ZnPP combination therapy demonstrated enhanced median overall survival rates in comparison to sham and anti-PD-1 monotherapy groups, with the combined approach yielding the most significant survival advantage (P&amp;lt;0.0001). Metabolic analyses following Hmox-1 knockdown and ZnPP treatment revealed a shift away from cancer-like glycolytic shunting in MDSCs. Inhibiting the HMOX-1 pathway facilitated T cell activation and reversal of glycolysis-predominant metabolism in MDSCs, resulting in improved survival outcomes in a murine glioma model. Modulation of metabolic reprogramming in myeloid cells may potentiate anti-tumor immunity, especially in conjunction with checkpoint blockade.

PATH-52. DIFFERENCES IN GENE EXPRESSION, GENOME-WIDE METHYLATION AND METABOLITES IN ADULT DIFFUSE GLIOMAS

Abstract Metabolic reprogramming is a hallmark of cancer, including diffuse gliomas. IDH1 and IDH2 are key enzymes linking cellular metabolism to epigenetic regulation and redox states. IDH1/IDH2 enzymes catalyze the conversion of isocitrate to α-ketoglutarate, while the mutant enzymes increase the production of D-2-hydroxyglutarate, an oncometabolite that induces epigenetic changes, leading to gene expression alterations. Understanding how epigenetic alterations affect metabolic gene expression and metabolite levels in diffuse gliomas could help identify diagnostic biomarkers and therapeutic targets. We studied the expression of metabolism-related genes, metabolites, and genome-wide methylation status, which will facilitate an integral understanding of the metabolic changes in IDH-mutant and IDH-wildtype gliomas. Study design included 47 fresh frozen tissue samples. RNA was extracted and used for gene expression analysis using the NanoString platform (nCounter® Metabolic Pathways Panel), while extracted DNA was used for targeted sequencing with a panel interrogating 600 genes and for genome-wide methylation analysis using the Illumina Epic v2. In addition, unbiased metabolomic analysis by mass spectrometry was performed in 25 tissue and CSF samples out of 47 samples. Methylation status, gene expression and metabolite levels were compared between IDH-mutant and IDH-wildtype gliomas. Results showed significant differences in DNA methylation, gene expression and metabolites between IDH-mutant and IDH-wildtype gliomas. UPP1, an enzyme involved in uracil metabolism, was studied in depth to understand the interplay between DNA methylation, gene expression, and metabolic reprogramming in diffuse glioma. Epigenetic analysis revealed that the CpG sites within the UPP1 gene displayed reduced methylation in IDH wild-type compared to IDH-mutant tumors. IDH wild-type tumors showed higher UPP1 expression compared to their IDH-mutant counterparts. Consequently, we observed higher levels of uracil in tissue and CSF samples from patients with IDH wild-type gliomas. These data provide a framework to study the relationship between mutations, methylation, gene expression and metabolism in diffuse gliomas.

EPCO-17. CARM1 METHYLATES GLIOBLASTOMA TUMOR SUPPRESSOR QUAKING TO REGULATE ITS TRANSCRIPTIONAL COACTIVATOR FUNCTION

Abstract Glioblastoma (GBM) represents the most prevalent and deadly primary brain tumor in adults, and it remains highly refractory to current therapeutic interventions. Significant intratumor heterogeneity drives rapid invasion, immune escape, and therapeutic resistance. Therapeutic paradigms based on deconvoluting key molecular mechanisms driving gliomagenesis are urgently needed. Quaking (Qki, encoded by Qk) is frequently altered in GBM patients; Qk mutations or deletions occur in 35% percent of cases, and the Qki protein is absent in 50-60% of cases. Our lab has characterized Qki as important tumor suppressor in GBM, where deletion of Qk enables pre-malignant neural stem cells to expand outside their niche and develop into tumors that recapitulate human GBM. We discovered that Qki functions as a transcriptional coactivator of lipid metabolism genes, whereby Qki loss disrupts lipid homeostasis and compromises the integrity of various membrane structures. Several downstream processes governed by Qki have been delineated; however, upstream regulators of Qki have yet to be identified. Recurrent cancer mutations to specific Qki arginine sites implicate the dysregulation of arginine methylation, a common post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Leveraging in vitro methylation assays, we demonstrate that Qki is selectively methylated by the PRMT coactivator-associated arginine methyltransferase 1 (CARM1), specifically in the Quaking regulatory domain where cancer mutations are reported. We observe that Qki interacts with known CARM1 substrates, functioning in the transcriptional complex assembled by CARM1. Through mutagenesis studies, we identified arginine 242 (R242) and arginine 256 (R256) as specific CARM1-mediated methylation sites on Qki. Importantly, mutations to these sites compromise Qki’s activity as a transcriptional coactivator. Moreover, brain-specific deletion of CARM1 in vivo results in neurological phenotypes reminiscent of Qki deletion. Altogether, we provide evidence that CARM1 represents a novel tumor suppressor in GBM that functions through the arginine methylation of Qki.

TMET-17. LIPID METABOLIC HETEROGENEITY OF GLIOMA CELLULAR STATES REVEALS ENVIRONMENTAL DEPENDENCIES

Abstract Malignant growth and survival of cancer is supported through reprogrammed lipid metabolism. In gliomas, genetic alterations can drive lipid metabolic dysregulation revealing therapeutic opportunities to exploit lipid metabolic vulnerabilities within genetically defined subsets of glioma tumors. However, it remains unclear whether inter- and intra-tumoral transcriptomic heterogeneity in gliomas relates to distinct lipid programs and potential dependencies. Here we set out to characterize pan-glioma lipid metabolic heterogeneity through genomic, transcriptomic, and lipidomic profiling of a large and diverse cohort of patient tumor samples. We define key axes of lipid metabolic variability across gliomas and identify relationships between lipid metabolic gene expression programs and glioma lipidomic profiles. Intriguingly, intersection of lipid gene expression signatures with single cell RNA sequencing revealed patterns of lipid metabolic heterogeneity that distinguish neurodevelopmental cellular states of gliomas, mirroring patterns of lipid metabolic diversity across cell types within the normal brain. Consequently, tumors with opposing cellular state enrichment have distinct lipid metabolism and environmental dependencies. Tumors enriched for Radial Glia-like states have high capacity for de novo fatty acid synthesis while tumors enriched for oligodendrocyte progenitor-like states are dependent on exogenous sources of lipids for survival and growth. These findings connect inter- and intra-tumoral heterogeneity of cellular states to variability in lipid metabolic reprogramming to reveal future avenues for precision medicine.

Modification of Glucose Metabolic Pathway to Enhance Polyhydroxyalkanoate Synthesis in Pseudomonas putida

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are semi-crystalline elastomers with a low melting point and high elongation at break, allowing for a wide range of applications in domestic, agricultural, industrial, and mainly medical fields. Utilizing low-cost cellulose hydrolyzed sugar as a carbon source and metabolic engineering to enhance synthesis in Pseudomonas putida is a promising strategy for commercializing mcl-PHAs, but little has been attempted to improve the utilization of glucose for synthesizing mcl-PHAs. In this study, a multi-pathway modification was performed to improve the utilization of substrate glucose and the synthesis capacity of PHAs. To enhance glucose metabolism to flow to acetyl-CoA, which is an important precursor of mcl-PHA, multiple genes in glucose metabolism were inactive (branch pathway and negative regulatory) and overexpressed (positive regulatory) in this study. The two genes, gcd (encoding glucose dehydrogenase) and gltA (encoding citrate synthase), involved in glucose peripheral pathways and TCA cycles were separately and jointly knocked out in Pseudomonas putida QSRZ6 (ΔphaZΔhsdR), and the mcl-PHA synthesis was improved in the mutants; particularly, the mcl-PHA titer of QSRZ603 (ΔgcdΔgltA) was increased by 33.7%. Based on the glucose branch pathway truncation, mcl-PHA synthesis was further improved with hexR-inactivation (encoding a negative regulator in glucose metabolism). Compared with QSRZ603 and QSRZ6, the mcl-PHA titer of QSRZ607 (ΔgcdΔgltAΔhexR) was increased by 62.8% and 117.5%, respectively. The mutant QSRZ609 was constructed by replacing the endogenous promoter of gltB encoding a transcriptional activator of the two-component regulatory system GltR/GltS with the ribosome subunit promoter P33. The final mcl-PHA content and titers of QSRZ609 reached 57.3 wt% and 2.5 g/L, an increase of and 20.9% and 27.3% over that of the parent strain QSRZ605 and an increase of 110.4% and 159.9% higher as compared to QSRZ6, respectively. The fermentation was optimized with a feeding medium in shaker flacks; then, the mcl-PHA contents and titer of QSRZ609 were 59.1 wt% and 6.8 g/L, respectively. The results suggest that the regulation from glucose to acetyl-CoA by polygenic modification is an effective strategy for enhancing mcl-PHA synthesis, and the mutants obtained in this study can be used as chassis to further increase mcl-PHA production.