BACKGROUND:The metabolic landscape of biliary tract cancer (BTC) remains poorly characterized. This study aimed to identify tumor-specific metabolic alterations in BTC using paired tumor and adjacent normal tissues. METHODS:Metabolomic profiling was performed on paired tumor and adjacent normal tissues from 71 patients with BTC using capillary electrophoresis time-of-flight mass spectrometry. Differential metabolites were identified using paired statistical analysis with false discovery rate correction. Pathway enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes database. RESULTS:Seventeen metabolites were significantly altered between tumor and normal tissues. Pathway analysis identified glycerophospholipid metabolism as the most enriched pathway, driven by water-soluble precursor and intermediate metabolites, including phosphorylcholine, CDP-choline, and ethanolamine phosphate. Hierarchical clustering demonstrated partially distinct metabolic patterns between tumor and normal tissues, with substantial inter-sample variability observed among tumor samples. Metabolites related to amino sugar and nucleotide sugar metabolism were also increased in tumor tissues. Additional pathways, including nicotinate and nicotinamide metabolism and arginine and proline metabolism, were also enriched. Principal component analysis showed partial separation between tumor and normal samples, indicating global metabolic differences between the two groups. These findings indicate metabolic alterations across multiple pathways in BTC. CONCLUSIONS:Paired tissue metabolomics revealed coordinated metabolic alterations in BTC involving choline phospholipid precursor metabolism, amino sugar and nucleotide sugar metabolism, and additional amino acid-related pathways. These results highlight the presence of broad metabolic reprogramming in BTC and underscore the importance of tissue-based metabolomic profiling for characterizing tumor metabolism.

Therapy resistance in acute myeloid leukemia (AML) remains a major clinical obstacle, particularly because of the persistence of leukemia stem cells (LSC) capable of metabolic adaptation. Although venetoclax (Ven) inhibits oxidative phosphorylation (OXPHOS), we found that Ven-resistant LSC undergo glycolytic reprogramming to bypass OXPHOS inhibition. This metabolic shift is supported by enhanced ribosome biogenesis, which is sustained by upregulated de novo guanine nucleotide biosynthesis. Abundant guanine nucleotides suppress the impaired ribosome biogenesis checkpoint (IRBC), leading to TP53 destabilization and persistent MYC expression. The inhibition of inosine mono-phosphate dehydrogenases (IMPDH1/2) depletes guanine nucleotides, activates IRBC, stabilizes TP53, represses MYC, and impairs the metabolic shift to glycolysis. This metabolic rewiring disrupts LSC stemness and suppresses the reconstitution of human AML cells in xenotransplantation experiments. Notably, the suppression of LSC stemness was observed regardless of Ven resistance or the TP53 mutational status of AML cells. These findings reveal that mutation-independent TP53 inactivation is involved in resistant AML and suggest that targeting guanine nucleotide biosynthesis may offer a clinically actionable strategy to eradicate therapy-resistant LSC.

Abstract Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohn’s disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

Abstract Cancer stem cells (CSCs) potentially possess multiple mechanisms of resistance to therapies and contribute to cancer initiation and progression. However, effective therapies that target CSCs have not yet been developed, as underlying mechanisms of stemness maintance, differentiation induction and drug resistantce remain unclear. In this study, we aimed to establish a research platform for CSCs and to identify the underlying molecular mechanisms. We conducted a genome-wide gain-of-function screening using a CRISPR activation (CRISPR-a) library to explore candidate regulators of CSCs. We used the PEO1-NS cell line, which lacks CD133 expression and stemness that PEO1-SL, the counterpart maintaining stemness. A CRISPR-a library covering 23,430 human genes was introduced into the cells, allowing the expression of one gene per cell. Subsequently, cells in which CD133 expression was induced were isolated using a cell sorter, and the sgRNA sequences present in these cells were analyzed by Illumina sequencing. As a result, 13 candidate genes potentially involved in CSC regulation were identified. These includ genes related to energy metabolism, such as mitochondrial function and glycolysis, and were presumed to act through MYC regulation. For the validation study, we evaluated the GI50 of cisplatin in cells overexpressing each candidate gene. Several genes significantly reduced drug sensitivity. The candidate genes identified in this study may include key regulators involved in CSC maintenance and function via CD133 expression. Since changes in energy metabolism and mitochondrial dynamics are closely linked to CSC traits, our findings provide insight into the molecular basis regulating these processes. This study may offer a useful research platform for CSC research to identify regulatory mechanisms and develop novel therapiutic aveneus. Citation Format: Yuki Nishida, Kazuharu Arakawa, Osamu Nureki, Tomoyoshi Soga, Shojiro Kitajima. Exploring cancer stem cell regulators and its mechanisms using CRISPR-activation based screening [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P70.

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. We previously showed that nicotinic acid riboside (NAR) sustains NAD biosynthesis in vivo and compensates for NAMPT inhibition in SCLC models. Here, we evaluated combining NAMPT inhibition with dietary nicotinic acid (NA) restriction to suppress NAR-dependent NAD biosynthesis. This combination showed enhanced antitumor activity consistent with synthetic lethality in SCLC CDX models and showed robust efficacy in PDX models, including those derived from chemotherapy-refractory tumors. In most CDX models examined, combination therapy showed greater antitumor activity than standard chemotherapy with cisplatin and etoposide. Although we did observe transient leukopenia, the treatment was otherwise better tolerated than chemotherapy, with less impact on body weight and platelet counts. These findings support NAD metabolism-targeted therapy as a promising strategy to treat SCLC.

Abstract The impact of the metabolic microenvironment on epigenetically plastic cancer cells underpins phenotypic heterogeneity, a major cause of metastasis and therapy resistance. Nutrient limitation is a key microenvironmental stress, and can cause cells to transition from proliferative to invasive phenotypes, however, whether cancer cells have the capacity to delay phenotype switching remains unknown. Here, using melanoma as a model, we reveal that the ability to buffer glucose availability by accumulating and mobilizing glycogen can determine cancer cell phenotypic transitions. While proliferating cells contain high levels of glycogen, invasive cells are marked by depleted glycogen stores. Accordingly, the inability to store and metabolize glycogen leads to phenotype instability and a switch from proliferation to invasion. The amount of stored glycogen inversely correlates with tissue invasion depth in primary melanomas, and reduced expression of the glycogen phosphorylases PYGB/L and phosphoglucomutase 1 (PGM1) is associated with worse patient survival. Together, we identify metabolic glucose buffering as a determinant of invasive phenotype transitions in skin cancer, suggesting similar paradigms in other cancer types.

Pathway enrichment analysis is a crucial method for the biological interpretation of metabolomic data by identifying associations between altered metabolites and biological pathways. However, such traditional approaches often rely on a limited set of predefined metabolic pathways, resulting in a low likelihood of discovering pathways associated with a given metabolic profile. To overcome this limitation, we extended our previously developed iDMET methodology to incorporate a broader range of metabolite sets, including those derived from differential metabolomic profiles. This enhanced approach, termed iDMET+, significantly expands dataset diversity and size, increasing the likelihood of discovering associated metabolite sets for a given metabolic profile, thereby enables more biological insights to be obtained from the metabolic profile. We validated iDMET+ through case studies on three diseases: clear cell renal cell carcinoma, colorectal cancer, and small cell lung cancer. First, using a clear cell renal cell carcinoma study as input, iDMET+ correctly identified another study of the same disease that involved metabolomic analysis. This pair of studies was identified as relevant in our previous iDMET results, showing the consistency between iDMET+ and iDMET. Second, using the metabolomic profile of colorectal cancer as input, iDMET+ identified not only another metabolomic study of the same cancer but, surprisingly, also metabolomic studies on prostate cancer and a high-fat diet. These studies focused on MYC-driven metabolic reprogramming, which was also a major focus of the input study. In both case studies, related studies were enriched because the differential metabolomic profiles of directly associated studies were part of the metabolite set. In contrast, the small cell lung cancer study highlighted limitations in dataset coverage—the absence of directly relevant differential metabolomic profiles resulted in fewer enriched metabolite sets. Nevertheless, the analysis of commonly altered metabolites still yielded some meaningful results. Metabolite alterations associated with inhibition of the purine salvage pathway were observed, suggesting potential involvement in tumor metabolic reprogramming. These results demonstrate that iDMET+ offers broader biologically relevant information than the conventional pathway-based approaches and has the potential to uncover biologically significant findings by searching across diverse datasets. This work also identifies areas of improvements for iDMET+.

Gas chromatography/mass spectrometry (GC/MS) is widely employed in metabolomics owing to its high resolution, reproducibility, and compatibility with volatile and semi-volatile compounds. While helium has traditionally been used as the carrier gas, increasing costs and global shortages have prompted the exploration of hydrogen gas as an alternative. Hydrogen offers faster analysis times and reduced ion source contamination but presents challenges, including ion source reactions, which can change compound fragmentation patterns. To address this, a hydrogen-deactivated electron ionization (EI) source has been developed to minimize undesired chemical interactions and improve spectral integrity. In this study, we systematically evaluated the effect of extraction lens sizes (3, 6, and 9 mm) under hydrogen carrier gas conditions using n-alkane and human metabolomic standard solutions. The hydrogen-deactivated ion source equipped with a 3 mm extraction lens provided superior peak symmetry and the highest signal intensities, with acceptable relative standard deviation (RSD) values (i.e., below 30%). In addition, the reproducible fragmentation patterns as those observed with helium carrier gas were obtained for phosphorylated metabolites. Using this optimized configuration, we applied hydrogen gas-based GC/MS to metabolomic profiling of the human colon cancer cell line HCT116 with mutant isocitrate dehydrogenase 1 (IDH1). Key metabolic alterations, including increased levels of the known biomarker 2-hydroxyglutaric acid, were detected in IDH1-mutant cells compared with wild-type cells. These findings establish hydrogen gas-based GC/MS with a hydrogen-deactivated ion source as a robust and reliable platform for metabolomics, offering an effective alternative to helium-based systems.

Phosphoinositides coordinate membrane trafficking and bioenergetic homeostasis, and many tumors rely on elevated phosphoinositide flux to sustain growth. Therapeutic development has largely pursued single-phosphoinositide kinase inhibition, whereas polypharmacologic strategies that perturb the broader network remain underexplored. FTY720 (fingolimod), a clinically approved sphingosine-1-phosphate receptor modulator, shows anti-tumor activity at micromolar concentrations, but its non-canonical mechanisms remain incompletely defined. Building on our work with the structurally related compound KRP203, we show that high-dose FTY720 produces isozyme-divergent modulation across phosphoinositide kinases and biases PIKFYVE activity toward phosphatidylinositol, a pattern we term ASURA (Asymmetric Simultaneous Uncoupling of Related Activities). FTY720 induces vacuolization and endomembrane remodeling in cancer cells, and suppresses macropinocytic ruffling as demonstrated by tracer uptake and scanning ion conductance microscopy analyses. Quantitative metabolomics revealed depletion of intracellular amino acids and ribonucleoside triphosphates, coupled with reduced glycolysis. Concurrently, FTY720 induced extensive rewiring of the hexosamine pathway, nitrogen metabolism, and tricarboxylic acid (TCA)-cycle anaplerosis, along with redox signatures indicating oxidative stress despite a nutrient-replete medium. The metabolites depleted by FTY720 showed extensive, directionally concordant overlap with those depleted by PTEN induction. Patient-derived glioblastoma (GBM) neurospheres were sensitive to FTY720, and co-treatment with a PI3Kα-selective inhibitor augmented growth suppression in U87MG cells. Together, these data support a model in which ASURA-dose FTY720 disrupts phosphoinositide-regulated trafficking and nutrient access, imposing intracellular nutrient stress that culminates in tumor-cell death.

Depression represents a major global disease burden. While the Center for Epidemiologic Studies Depression Scale (CES-D) is widely used, it relies on subjective self-reporting. Saliva enables non-invasive metabolite analysis, but existing metabolomics studies have focused on clinical depression using blood or urine, with limited investigation of salivary metabolites in community populations. This study investigated associations between CES-D scores and salivary metabolite concentrations in Tokyo residents. This cross-sectional study collected CES-D responses and morning saliva samples from Tokyo residents. Salivary metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA) for overall metabolite-CES-D associations, pathway enrichment analysis using fast gene set enrichment analysis (FGSEA), and principal component analysis, adjusted for age and sex. Analysis included 107 participants (40 males, 67 females; mean age 55.3 ± 14.2 years). PERMANOVA revealed significant association between 112 salivary metabolites and CES-D scores (F = 2.870, p = 0.003), with metabolites explaining 7.7

Abstract One-carbon (1C) metabolism is frequently upregulated in cancer to support anabolic growth and nucleotide biosynthesis. However, the contribution of mitochondrial 1C metabolism to cancer stemness and metastatic progression remains incompletely understood. Here, we identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), a mitochondrial 1C metabolic enzyme, as a critical regulator of breast cancer stemness, tumor initiation, and lung metastasis. Genetic depletion of MTHFD1L in triple-negative breast cancer (TNBC) cell lines and patient-derived breast cancer models markedly impaired proliferation, sphere formation, tumorigenicity, and lung colonization. Metabolomic profiling revealed extensive metabolic rewiring following MTHFD1L loss, characterized by accumulation of the purine biosynthetic intermediates SAICAR and AICAR together with perturbations in glycolytic and pentose phosphate pathways. Importantly, suppression of lung metastasis was accompanied by reduced expression of the stemness-associated transcription factor SOX2 and decreased proliferative activity in metastatic lesions. Collectively, our findings establish MTHFD1L as a key metabolic dependency linking mitochondrial 1C metabolism to stem-like properties and metastatic progression in breast cancer, and highlight MTHFD1L as a promising therapeutic target in metastatic TNBC.

Environmental exposures can influence offspring health through epigenetic alterations in the male germline. Folate deficiency, a dietary perturbation that disrupts one-carbon metabolism and S-adenosylmethionine (SAM) production, has been linked to altered histone methylation and developmental abnormalities in offspring. However, when and how folate availability shapes the germline epigenome during spermatogenesis remains unclear. In this study, unbiased metabolomic profiling of spermatogenic cells uncovers stage-specific metabolic remodeling, including downregulation of serine-glycine-one-carbon (SGOC) metabolism in meiotic spermatocytes. Using a post-weaning folate-deficient mouse model, we investigate how folate availability influences germline epigenome establishment during spermatogenesis. Consistent with this metabolic transition, genome-wide chromatin accessibility profiling demonstrates extensive, stage-dependent remodeling under folate-deficient conditions, particularly in meiotic spermatocytes and post-meiotic spermatids. These accessibility changes display cell-type-specific genomic distributions and preferential localization to repressive chromatin compartments in post-meiotic cells. Histone modification analyses further reveal bidirectional redistribution of the active histone mark H3K4me3 in round spermatids. Although genome-wide distribution of the repressive mark H3K27me3 remains largely stable, folate deficiency alters its nuclear organization. Notably, a subset of H3K4me3 alterations established in post-meiotic cells is retained in mature sperm, providing a mechanistic link between paternal metabolic perturbation and the germline epigenome. Together, these findings demonstrate that folate availability shapes germline epigenome establishment through stage-specific metabolic and chromatin remodeling during spermatogenesis, revealing a metabolic basis for paternal environmental effects on the germline epigenome.

CONTEXT:Previous metabolomics studies suggest potential associations between menopausal changes in lipids and an increased risk of metabolic syndrome (MetS). However, longitudinal data on other key metabolites, such as branched-chain amino acids (BCAAs) and homocysteine, remain limited, and most studies lack long-term follow-up across the menopause transition. OBJECTIVE:This study aimed to investigate longitudinal changes in circulating metabolites during menopause over a mean follow-up of 5 years and assess their associations with subsequent MetS development. METHODS:Premenopausal women from the Tsuruoka Metabolomics Cohort Study who participated in at least one follow-up survey were included. Menopausal status, data on MetS, and plasma metabolites profiled using capillary electrophoresis mass spectrometry were assessed at each visit. Thirty-one metabolites were examined for associations with menopausal status using mixed-effects models. The association of these menopause-related metabolites with MetS development was examined via logistic regression analysis adjusted for follow-up duration. RESULTS:Among 953 women (aged 43.8 ± 5.4 years), 316 (33.2%) reached menopause during follow-up (5.0 ± 1.1 years). Eighteen metabolites changed significantly with menopause, particularly those related to BCAA metabolism, urea cycle, and homocysteine metabolism. Of 695 women without MetS at baseline, 65 (9.4%) developed MetS. Glutamate (odds ratio [95% CI]: 1.95 [1.49-2.57]) was associated with higher MetS risk. Higher levels of glutamate, valine, leucine, and cystine were significantly associated with the development of hyperglycemia. CONCLUSION:Longitudinal changes in charged metabolites occur across the menopausal transition, with specific metabolites such as glutamate possibly contributing to the metabolic alterations underlying increased MetS risk.

To evaluate hexosamine biosynthetic pathway (HBP) involvement in high-fluorodeoxyglucose (FDG)-uptake lung adenocarcinoma. We conducted metabolomic analysis to evaluate the HBP in patients with lung adenocarcinoma, who underwent preoperative 18-FDG positron emission tomography. Capillary electrophoresis-time-of-flight mass spectrometry was done to obtain 511 small-molecule metabolite spectra, and a principal component analysis was performed. We examined 80 tissue samples: 40 tumor-adjacent non-tumor tissue samples and 40 resected lung adenocarcinomas. The principal component analysis confirmed good clustering between the tumor and non-tumor tissues. The non-tumor tissues comprised uniform materials, whereas the tumor tissues comprised a mixture of materials. Heatmaps for 50 metabolites revealed lower glucose and citrate levels and higher levels of lactate, glycolysis metabolites, succinate, fumarate, adenosine di- and -monophosphate, and all essential amino acids in the tumor tissues than the non-tumor tissues. HBP intermediate and uridine diphosphate N-acetylglucosamine levels were also higher in the tumor tissues. Both lactate and HBP intermediate levels were higher in hypermetabolic tumor tissues (standardized uptake value ≥ 3) than in non-hypermetabolic tumor tissues (standardized uptake value < 3). Low-FDG-uptake cells showed strong expression for glucose transporter SLC2A1 and weak expression for O-linked N-acetylglucosamine, whereas high-FDG-uptake cells showed strong expression for both markers. Hypermetabolic adenocarcinoma may be associated with intensified glycolysis and HBP activation.

The heat shock protein beta-1 (HSPB1/HSP27) is highly expressed and phosphorylated in cancer tissues. However, the precise role of HSPB1 in cancer remains unclear. In this study, we report the unexpected findings elucidating the essential role of HSPB1 in adapting amino acid deficiency by upregulating amino acid transporter SLC7A5 function. HSPB1 regulates estrogen receptor-positive (ER+) breast cancer cell proliferation in a SLC7A5-dependent manner. In response to cellular stress, which is specified as amino acid-deficient conditions, HSPB1 was phosphorylated at Ser 78 residue by stress MAPK p38. SLC7A5 is associated with phosphorylated HSPB1 for its functional activation, leading to upregulated amino acid incorporation. In addition, HSPB1 and SLC7A5 overexpression increased acetylated α-tubulin levels. SLC7A5 overexpression did not change acetyl-CoA level, but SLC7A5 knockdown decreased ATAT1 and induced HDAC6 upregulation. Furthermore, HSPB1 and SLC7A5 induced paclitaxel and tamoxifen resistance. Therefore, the HSPB1-SLC7A5 axis contributes to the acquisition of tolerance to both tamoxifen and paclitaxel in breast cancer cells, uncovering a novel therapeutic target against drug resistance in breast cancer.