Synthesis, evaluation and mechanistic insights of novel IMPDH inhibitors targeting ESKAPEE bacteria.

Antimicrobial resistance poses a significant threat to global health, necessitating the development of novel therapeutic agents with unique mechanisms of action. Inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme in guanine nucleotide biosynthesis, is a promising target for the discovery of new antimicrobial agents. High-throughput screening studies have previously identified several urea-based leads as potential inhibitors, although many of these are characterised by reduced chemical stability. In this work, we describe the design and synthesis of a series of heteroaryl-susbtituted analogues and the evaluation of their inhibitory potency against IMPDHs. Our screening targets ESKAPEE pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Several analogues with submicromolar inhibitory potency are identified and show no inhibitory potency on human IMPDH nor cytotoxic effects on human cells. Kinetic studies revealed that these molecules act as noncompetitive inhibitors with respect to the substrates and ligand virtual docking simulations provided insights into the binding interactions at the interface of the NAD+ and IMP binding sites on IMPDH.

Harnessing nucleotide metabolism and immunity in cancer: a tumour microenvironment perspective.

The tumour microenvironment (TME) is a dynamic nexus where cancer cell metabolism and the immune system intricately converge, with nucleotide metabolism (NM) playing a pivotal role. This review explores the critical function of NM in cancer cell proliferation and its profound influence on the TME and immune landscape. NM is essential for DNA and RNA synthesis and is markedly upregulated in cancer cells to meet the demands of rapid growth. This metabolic rewiring fuels cancer progression, but also shapes the TME, impacting the function and viability of immune cells. The altered nucleotide milieu in the TME can suppress immune response, aiding cancer cell evasion from immune surveillance. Drug discoveries in the field of NM have revealed different therapeutic strategies, including inhibitors of nucleotide synthesis and drugs targeting salvage pathways, which are discussed thoroughly in this review. Furthermore, the emerging strategy of combining NM-targeted therapies with immunotherapies is emphasised, particularly their effect on sensitising tumours to immune checkpoint inhibitors and enhancing overall treatment efficacy. The Human Genome Project paved the way for personalised medicine, countering the established 'one size fits all' approach to cancer treatment. Advances in understanding the TME and NM have spurred interest in personalised therapeutic strategies. This review highlights the potential of leveraging individual tumour metabolic profiles to guide treatment selection, aiming to optimise efficacy and minimise adverse effects. The strategic importance of targeting NM in cancer therapy and its synergistic potential with immunotherapies offers a path towards more effective and personalised cancer treatments.

Nuclear translocation of nucleotide enzyme Phosphoglucomutase 2 governs DNA damage response and anti-tumor immunity.

Targeting nucleotide enzymes emerges as a promising avenue for impeding tumor proliferation and fortifying anti-tumor immunogenicity. The non-canonical role of nucleotide enzymes remains poorly understood. In this study, we have identified that Phosphoglucomutase 2 (PGM2) rapidly accumulates at the DNA damage site to govern the DNA damage response mediated by the phosphorylation at Serine 165 and by forming a complex with Rho-associated coiled-coil-containing protein kinase 2 (ROCK2). Silencing PGM2 in Glioblastoma Multiforme (GBM) cells heightens DNA damage in vitro and enhances the sensitivity of temozolomide (TMZ) treatment by activating anti-tumor immunity in vivo. Furthermore, we demonstrate that pharmacological inhibition of ROCK2 synergistically complements TMZ treatment and pembrolizumab (PD-L1) checkpoint immunotherapy, augmenting anti-tumor immunity. This study reveals the non-canonical role of the nucleotide enzyme PGM2 in the regulation of DNA damage response and anti-tumor immunity, with implications for the development of therapeutic approaches in cancer treatment.

The Association between Gut Microbiota and Its Metabolites in Gestational Diabetes Mellitus.

Gut microbial metabolites have been demonstrated to play a role in diabetes mellitus and gestational diabetes mellitus (GDM). This study aimed to investigate gut microbiome, fecal metabolomics, and their association in pregnant women with and without GDM. The metabolome indicated that the top 2 differential metabolic pathways between control (Con) and GDM groups were phenylalanine metabolism and nucleotide metabolism. The increased Phenylalanylglycine, m-coumaric acid, and Phenylacetic acid were among the top differential metabolites between Con and GDM groups and involved in phenylalanine metabolism. Uracil and hypoxanthine were top differential metabolites in Con vs. GDM and involved in nucleotide metabolism. The proficiently altered gut microbiota at the class level was c_unclassified_ Firmicutes. Association analysis between gut microbiota and fecal metabolites indicated that the increased gut symbiont Clostridium belonged to Firmicutes and was linked to the dysregulation of phenylalanine metabolism in GDM. This study may provide the mechanism underlying how Clostridium-phenylalanine metabolism association contributes to GDM pathogenesis and also be a novel therapeutic strategy to treat GDM.

Targeting dysregulated intracellular immunometabolism within synovial microenvironment in rheumatoid arthritis with natural products.

Immunometabolism has been an emerging hotspot in the fields of tumors, obesity, and atherosclerosis in recent decades, yet few studies have investigated its connection with rheumatoid arthritis (RA). In principle, intracellular metabolic pathways upstream regulated by nutrients and growth factors control the effector functions of immune cells. Dynamic communication and hypermetabolic lesions of immune cells within the inflammatory synovial microenvironment contributes to the development and progression of RA. Hence, targeting metabolic pathways within immune subpopulations and pathological cells may represent novel therapeutic strategies for RA. Natural products constitute a great potential treasury for the research and development of novel drugs targeting RA. Here, we aimed to delineate an atlas of glycolysis, lipid metabolism, amino acid biosynthesis, and nucleotide metabolism in the synovial microenvironment of RA that affect the pathological processes of synovial cells. Meanwhile, therapeutic potentials and pharmacological mechanisms of natural products that are demonstrated to inhibit related key enzymes in the metabolic pathways or reverse the metabolic microenvironment and communication signals were discussed and highlighted.

Integrated bioinformatics analysis of nucleotide metabolism based molecular subtyping and biomarkers in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD), a predominant subtype of non-small cell lung cancers, continues to challenge treatment outcomes due to its heterogeneity and complex tumor microenvironment (TME). Dysregulation in nucleotide metabolism has been identified as a significant factor in tumorigenesis, suggesting its potential as a therapeutic target. Methods: This study analyzed LUAD samples from The Cancer Genome Atlas (TCGA) using Non-negative Matrix Factorization (NMF) clustering, Weighted Correlation Network Analysis (WGCNA), and various machine learning techniques. We investigated the role of nucleotide metabolism in relation to clinical features and immune microenvironment through large-scale data analysis and single-cell sequencing. Using in vivo and in vitro experiments such as RT-qPCR, Western Blot, immunohistochemistry, and subcutaneous tumor formation in mice, we further validated the functions of key nucleotide metabolism genes in cell lines and animals. Results: Nucleotide metabolism genes classified LUAD patients into two distinct subtypes with significant prognostic differences. The 'C1' subtype associated with active nucleotide metabolism pathways showed poorer prognosis and a more aggressive tumor phenotype. Furthermore, a nucleotide metabolism-related score (NMRS) calculated from the expression of 28 key genes effectively differentiated between patient outcomes and predicted associations with oncogenic pathways and immune responses. By integrating various immune infiltration algorithms, we delineated the associations between nucleotide metabolism signature genes and the tumor microenvironment, and characterized their distribution differences at the cellular level by analyzing single-cell sequencing dataset related to immunochemotherapy. Finally, we demonstrated the differential expression of the key nucleotide metabolism gene AUNIP acts as an oncogene to promote LUAD cell proliferation and is associated with tumor immune infiltration. Conclusion: The study underscores the pivotal role of nucleotide metabolism in LUAD progression and prognosis, highlighting the NMRS as a valuable biomarker for clinical outcomes and therapeutic responses. Specifically, AUNIP functions as a critical oncogene, offering a promising target for novel treatment strategies in LUAD.

Appendage regeneration requires IMPDH2 and creates a sensitized environment for enzyme filament formation.

Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpole Xenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration.

Transcriptomic profiling identifies a nucleotide metabolism-related signature with prognostic power in gliomas.

OBJECTIVE: Nucleotide metabolic reprogramming as a hallmark of cancer is closely related to the occurrence and progression of cancer. We aimed to comprehensively analyze the nucleotide metabolism-related gene set and clinical significance in gliomas. METHODS: The RNA sequencing data of 702 gliomas from the Cancer Genome Atlas (TCGA) dataset were included as the training set, and the RNA sequencing data from the other three datasets (CGGA, GSE16011, and Rembrandt) were used as independent validation sets. Survival curve, Cox regression analysis, time-dependent ROC curve and nomogram model were performed to evaluate prognostic power of signature. R language was the main tool for bioinformatic analysis and graphical work. RESULTS: Based on the expression profiles of nucleotide metabolism-related genes, consensus clustering identified two robust clusters with different prognosis. We then developed a nucleotide metabolism-related signature that was closely related to clinical, pathological, and genomic characteristics of gliomas. And ROC curve showed that our signature was a potential biomarker for mesenchymal subtype. Survival curve and Cox regression analysis revealed signature as an independent prognostic factor for gliomas. In addition, we constructed a nomogram model to predict individual survival. Finally, functional analysis showed that nucleotide metabolism not only affected cell division and cell cycle, but also was associated with immune response in gliomas. CONCLUSION: We developed a nucleotide metabolism-related signature to predict prognosis and provided new insights into the role of nucleotide metabolism in gliomas.

Spatial transcriptome and single-cell reveal the role of nucleotide metabolism in colorectal cancer progression and tumor microenvironment.

BACKGROUND: The intricacies of nucleotide metabolism within tumor cells specific to colorectal cancer (CRC) remain insufficiently characterized. A nuanced examination of particular tumor clusters and their dynamic interplay with the tumor microenvironment (TME) may yield profound insights into these therapeutically auspicious communicative networks. METHODS: By integrating ten types of single-cell enrichment scoring methods, we carried out enrichment analysis on CRC cell types, which was validated through four additional single-cell cohorts. Groups of tumor cells were determined using the average values of the scores. Using cellphonedb, monocle, inferCNV, SCENIC, and Cytotrace, functional analyses were performed. Utilizing the RCTD approach, single-cell groupings were mapped onto spatial transcriptomics, analyzing cell dependency and pathway activity to distinguish between tumor cell subtypes. Differential expression analysis identified core genes in nucleotide metabolism, with single-cell and spatial transcriptomics analyses elucidating the function of these genes in tumor cells and the immune microenvironment. Prognostic models were developed from bulk transcriptome cohorts to forecast responses to immune therapy. Laboratory experiments were conducted to verify the biological function of the core gene. RESULTS: Nucleotide metabolism is significantly elevated in tumor cells, dividing them into two groups: NUhighepi and NUlowepi. The phenotype NUhighepi was discerned to exhibit pronounced malignant attributes. Utilizing the analytical tool stlearn for cell-to-cell communication assessment, it was ascertained that NUhighepi engages in intimate interactions with fibroblasts. Corroborating this observation, spatial transcriptome cell interaction assessment through MISTy unveiled a particular reliance of NUhighepi on fibroblasts. Subsequently, we pinpointed NME1, a key gene in nucleotide metabolism, affirming its role in thwarting metastasis via in vitro examination. Utilizing multiple machine learning algorithms, a stable prognostic model (NRS) has been developed, capable of predicting survival and responses to immune therapy. In addition, targeted drugs have been identified for both high and low scoring groups. Laboratory experiments have revealed that NME1 can inhibit the proliferation and invasion of CRC tumor cells. CONCLUSION: Our study elucidates the potential pro-tumor mechanism of NUhighepi and the role of NME1 in inhibiting metastasis, further deepening the understanding of the role of nucleotide metabolism in colorectal cancer, and providing valuable targets for disrupting its properties.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

Live-cell analysis of IMPDH protein levels during yeast colony growth provides insights into the regulation of GTP synthesis.

The purine nucleotides ATP and GTP are made from the common precursor inosine monophosphate (IMP). Maintaining the correct balance of these nucleotides for optimal cell growth is controlled in part by the enzyme IMP dehydrogenase (IMPDH), which catalyzes the first dedicated step of GTP biosynthesis. The regulation of IMPDH mRNA and protein levels in the yeast S. cerevisiae grown in liquid culture has been studied in some detail, but regulation of IMPDH protein under conditions of cellular crowding on a solid substrate has not been examined. Here, we report real-time, live-cell analysis of the accumulation of the Imd2 isoform of IMPDH in yeast cells forming a monolayer colony in a microfluidic device over a 50-hour time course. We observe two distinct phases of increased Imd2 accumulation: a guanine-insensitive phase early in outgrowth and a guanine-sensitive phase later, when cells become crowded. We show that the IMPDH inhibitor mycophenolic acid enhances both phases of increase. Deletion of a transcription attenuator upstream of the mRNA start site that decreases Imd2 mRNA synthesis in the presence of high GTP increases the baseline level of Imd2 protein 10-fold and abolishes guanine-sensitive but not guanine-insensitive induction. Our results suggest that at least two mechanisms of yeast Imd2 regulation exist, the known GTP-dependent attenuation of RNA polymerase II elongation and a GTP concentration-independent pathway that may be controlled by cell growth state. Live-cell analysis of IMPDH protein levels in a growing yeast colony confirms a known mechanism of regulation and provides evidence for an additional mode of regulation. IMPORTANCE: This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability.

Integration of mult-omics and nucleotide metabolism reprogramming signature analysis reveals gastric cancer immunological and prognostic features.

BACKGROUND: Gastric cancer is a frequent and lethal solid tumor that has a poor prognosis and treatment result. Reprogramming of nucleotide metabolism is a characteristic of cancer development and progression. METHODS: We used a variety of machine learning techniques to create a novel nucleotide metabolism-related index (NMRI) using gastric cancer sample data obtained from the TCGA and GEO databases. This index is based on genes associated to nucleotide metabolism. Gastric cancer patients were categorized into high and low NMRI groups based on NMRI results. The clinical features, tumor immune microenvironment, response to chemotherapy, and response to immunotherapy were then thoroughly examined. In vitro experiments were then used to confirm the biological role of SERPINE1 in gastric cancer. RESULTS: The four nucleotide metabolism-related genes that make up NMRI (GAMT, ORC1, CNGB3, and SERPINE1) were verified in an external dataset and are a valid predictor of prognosis for patients with gastric cancer. The high NMRI group was more responsive to immunotherapy and had greater levels of immune cell infiltration than the low NMRI group. The proliferation and migration of stomach cancer was shown to be decreased by SERPINE1 knockdown in vitro. CONCLUSIONS: This study's NMRI can reliably predict a patient's prognosis for stomach cancer and pinpoint the patient group that will benefit from immunotherapy, offering important new information on the clinical treatment of stomach cancer.

Mass Spectrometric Analysis of Purine Intermediary Metabolism Indicates Cyanide Induces Purine Catabolism in Rabbits.

Purines are the building blocks of DNA/RNA, energy substrates, and cofactors. Purine metabolites, including ATP, GTP, NADH, and coenzyme A, are essential molecules in diverse biological processes such as energy metabolism, signal transduction, and enzyme activity. When purine levels increase, excess purines are either recycled to synthesize purine metabolites or catabolized to the end product uric acid. Purine catabolism increases during states of low oxygen tension (hypoxia and ischemia), but this metabolic pathway is incompletely understood in the context of histotoxic hypoxia (i.e., inhibition of oxygen utilization despite normal oxygen tension). In rabbits exposed to cyanide-a classical histotoxic hypoxia agent-we demonstrated significant increases in several concordant metabolites in the purine catabolic pathway (including plasma levels of uric acid, xanthosine, xanthine, hypoxanthine, and inosine) via mass spectrometry-based metabolite profiling. Pharmacological inhibition of the purine catabolic pathway with oxypurinol mitigated the deleterious effects of cyanide on skeletal muscle cytochrome c oxidase redox state, measured by non-invasive diffuse optical spectroscopy. Finally, plasma uric acid levels correlated strongly with those of lactic acid, an established clinical biomarker of cyanide exposure, in addition to a tissue biomarker of cyanide exposure (skeletal muscle cytochrome c oxidase redox state). Cumulatively, these findings not only shed light on the in vivo role(s) of cyanide but also have implications in the field of medical countermeasure (MCM) development.

Unveiling the metabolic landscape of pulmonary hypertension: insights from metabolomics.

Pulmonary hypertension (PH) is regarded as cardiovascular disease with an extremely poor prognosis, primarily due to irreversible vascular remodeling. Despite decades of research progress, the absence of definitive curative therapies remains a critical challenge, leading to high mortality rates. Recent studies have shown that serious metabolic disorders generally exist in PH animal models and patients of PH, which may be the cause or results of the disease. It is imperative for future research to identify critical biomarkers of metabolic dysfunction in PH pathophysiology and to uncover metabolic targets that could enhance diagnostic and therapeutic strategies. Metabolomics offers a powerful tool for the comprehensive qualitative and quantitative analysis of metabolites within specific organisms or cells. On the basis of the findings of the metabolomics research on PH, this review summarizes the latest research progress on metabolic pathways involved in processes such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in the context of PH.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS.

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

Risk assessment model based on nucleotide metabolism-related genes highlights SLC27A2 as a potential therapeutic target in breast cancer.

PURPOSE: Breast cancer (BC) is the most prevalent malignant tumor worldwide among women, with the highest incidence rate. The mechanisms underlying nucleotide metabolism on biological functions in BC remain incompletely elucidated. MATERIALS AND METHODS: We harnessed differentially expressed nucleotide metabolism-related genes from The Cancer Genome Atlas-BRCA, constructing a prognostic risk model through univariate Cox regression and LASSO regression analyses. A validation set and the GSE7390 dataset were used to validate the risk model. Clinical relevance, survival and prognosis, immune infiltration, functional enrichment, and drug sensitivity analyses were conducted. RESULTS: Our findings identified four signature genes (DCTPP1, IFNG, SLC27A2, and MYH3) as nucleotide metabolism-related prognostic genes. Subsequently, patients were stratified into high- and low-risk groups, revealing the risk model's independence as a prognostic factor. Nomogram calibration underscored superior prediction accuracy. Gene Set Variation Analysis (GSVA) uncovered activated pathways in low-risk cohorts and mobilized pathways in high-risk cohorts. Distinctions in immune cells were noted between risk cohorts. Subsequent experiments validated that reducing SLC27A2 expression in BC cell lines or using the SLC27A2 inhibitor, Lipofermata, effectively inhibited tumor growth. CONCLUSIONS: We pinpointed four nucleotide metabolism-related prognostic genes, demonstrating promising accuracy as a risk prediction tool for patients with BC. SLC27A2 appears to be a potential therapeutic target for BC among these genes.

Targeting nucleotide metabolic pathways in colorectal cancer by integrating scRNA-seq, spatial transcriptome, and bulk RNA-seq data.

BACKGROUND: Colorectal cancer is a malignant tumor of the digestive system originating from abnormal cell proliferation in the colon or rectum, often leading to gastrointestinal symptoms and severe health issues. Nucleotide metabolism, which encompasses the synthesis of DNA and RNA, is a pivotal cellular biochemical process that significantly impacts both the progression and therapeutic strategies of colorectal cancer METHODS: For single-cell RNA sequencing (scRNA-seq), five functions were employed to calculate scores related to nucleotide metabolism. Cell developmental trajectory analysis and intercellular interaction analysis were utilized to explore the metabolic characteristics and communication patterns of different epithelial cells. These findings were further validated using spatial transcriptome RNA sequencing (stRNA-seq). A risk model was constructed using expression profile data from TCGA and GEO cohorts to optimize clinical decision-making. Key nucleotide metabolism-related genes (NMRGs) were functionally validated by further in vitro experiments. RESULTS: In both scRNA-seq and stRNA-seq, colorectal cancer (CRC) exhibited unique cellular heterogeneity, with myeloid cells and epithelial cells in tumor samples displaying higher nucleotide metabolism scores. Analysis of intercellular communication revealed enhanced signaling pathways and ligand-receptor interactions between epithelial cells with high nucleotide metabolism and fibroblasts. Spatial transcriptome sequencing confirmed elevated nucleotide metabolism states in the core region of tumor tissue. After identifying differentially expressed NMRGs in epithelial cells, a risk prognostic model based on four genes effectively predicted overall survival and immunotherapy outcomes in patients. High-risk group patients exhibited an immunosuppressive microenvironment and relatively poorer prognosis and responses to chemotherapy and immunotherapy. Finally, based on data analysis and a series of cellular functional experiments, ACOX1 and CPT2 were identified as novel therapeutic targets for CRC. CONCLUSION: In this study, a comprehensive analysis of NMRGs in CRC was conducted using a combination of single-cell sequencing, spatial transcriptome sequencing, and high-throughput data. The prognostic model constructed with NMRGs shows potential as a standalone prognostic marker for colorectal cancer patients and may significantly influence the development of personalized treatment approaches for CRC.

SIRT4 loss reprograms intestinal nucleotide metabolism to support proliferation following perturbation of homeostasis.

The intestine is a highly metabolic tissue, but the metabolic programs that influence intestinal crypt proliferation, differentiation, and regeneration are still emerging. Here, we investigate how mitochondrial sirtuin 4 (SIRT4) affects intestinal homeostasis. Intestinal SIRT4 loss promotes cell proliferation in the intestine following ionizing radiation (IR). SIRT4 functions as a tumor suppressor in a mouse model of intestinal cancer, and SIRT4 loss drives dysregulated glutamine and nucleotide metabolism in intestinal adenomas. Intestinal organoids lacking SIRT4 display increased proliferation after IR stress, along with increased glutamine uptake and a shift toward de novo nucleotide biosynthesis over salvage pathways. Inhibition of de novo nucleotide biosynthesis diminishes the growth advantage of SIRT4-deficient organoids after IR stress. This work establishes SIRT4 as a modulator of intestinal metabolism and homeostasis in the setting of DNA-damaging stress.