Our laboratory has established that the free radical signaling molecule nitric oxide (NO) is an endogenous regulator of epigenetic methylation through direct inhibition of the Fe(II)/2-oxoglutarate-dependent dioxygenase (2-ODD) family, including histone demethylases (KDMs)[1, 2], mRNA demethylases (FTO, ALKBH5) [3], and DNA demethylases (TETs, ALKBH2) [4]. We previously showed that NO alters 38 histone post-translational modifications (PTMs) and genome-wide transcription in breast cancer cells [3]. Here, we comprehensively quantify 76 histone modifications, including 30 combinatorial marks on adjacent residues not resolved in the original analysis, and systematically profile methyl-modifying enzyme expression.We identify four coordinated histone PTM axes converging on a chromatin compaction signature: (i) H3K9 methylation accumulation with selective K9me1K14ac remodeling, (ii) H4K20me1→me2/me3 conversion, (iii) a novel H3K27-K36 combinatorial landscape shift, and (iv) preferential depletion of hyperacetylated H4 species. A composite Chromatin Compaction Score integrating all 76 PTMs with literature-based valence assignments confirms progressive compaction, validated independently by PCA. ChIP-seq reanalysis reveals that H3K9me2 accumulates predominantly at intergenic regions while K9ac undergoes activity-dependent redistribution governed by baseline chromatin state, with near-perfect reciprocal H3K9me2 gain at silent loci. Systematic enzyme profiling shows coordinated upregulation of 13/18 detected KDMs alongside methyltransferase downregulation, a compensatory response that fails to overcome direct enzymatic inhibition by NO.This compaction signature is associated with tumor-permissive gene silencing: DNA repair genes are downregulated, chromatin remodeling complexes suppressed, and IDH1 downregulation creates a feed-forward loop compounding 2-ODD inhibition. These findings establish NO-mediated KMD inhibition as a driver of coordinated chromatin compaction linked to pro-tumorigenic gene silencing.

Nitric oxide (NO) is a pleiotropic free radical that functions as a master regulator of gene expression, and its sustained production within the tumor microenvironment reshapes the epitranscriptomic state of cancer cells. We previously demonstrated that NO inhibits the m6A mRNA demethylases FTO and ALKBH5 through dinitrosyliron complex formation while leaving the methyltransferase METTL3 intact, a demethylase-specific perturbation that increases global m6A on mRNA. Here, integrating m6A-RIP-seq and RNA-seq from triple-negative breast cancer cells, we show that chronic NO does not produce the uniform hypermethylation anticipated from demethylase inhibition. Instead, it redistributes m6A on mRNA, enriching the 5'UTR and coding sequence while depleting the 3'UTR and departing from the canonical stop-codon and 3'UTR topology. We found that the position of m6A, rather than its intensity or mere presence, shapes the outcome, in part by determining which reader protein is predicted to recognize it. In parallel, NO drives a canonical NF-κB and inflammatory transcriptional program. The transcriptional program is independent of the m6A methylome in both which genes respond and how strongly they respond, ruling out a linear methylome-to-transcriptome cascade; even so, m6A position remains associated with the direction of change among responding transcripts. The 3'UTR is the primary site of m6A loss and shows a suggestive computational link to miRNA-mediated regulation. Sense-antisense coordination reinforces the transcriptional response without bridging the two programs. These findings demonstrate that NO not only increases m6A abundance, but it also rewrites the m6A positional code, establishing spatial reprogramming of the epitranscriptome as a previously unrecognized mode of gene regulation.

Nitric oxide synthase 2 (NOS2) and cyclooxygenase 2 (COX2) lie at a critical intersection between inflammation, metabolism, and oncogenic signaling, where they cooperatively promote and establish a Nitric Oxide (NO)-driven Warburg phenotype in advanced cancers. Early work in macrophages established NOS2-derived NO as both a signaling molecule and metabolic stressor that inhibits oxidative phosphorylation (OXPHOS) by targeting iron-sulfur enzymes and respiratory complexes, forcing neighboring cells to rewire metabolism. In human tumors, sustained NOS2 expression in cancer cells and tumor-associated macrophages (TAMs) enforces a Warburg-like state characterized by high glycolytic flux, glutamine dependence, and enhanced NADPH production, supporting proliferation, biosynthesis, and resistance to oxidative stress. At nitrosative-signaling concentrations (≈100-500 nM), NO breaks carbon entry into the TCA cycle at aconitase and pyruvate dehydrogenase, progressively disables dehydrogenase complexes containing dihydrolipoamide dehydrogenase (DLD) and electron-transport complexes (ETCs), and activates hypoxia-inducible factor 1-alpha (HIF-1), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), extracellular signal-regulated kinase (ERK)/pyruvate kinase M2 (PKM2)/c-Myc signaling axis, nuclear factor erythroid 2-related factor 2 (Nrf2), and transforming growth factor Beta (TGF-β)/SMAD pathways. These biochemical and signaling effects convert transient glycolytic adaptation into chemically enforced dependency, further stabilized by metabolite-driven inhibition of ten-eleven translocation (TET) and Jumonji demethylases, creating an "epigenetic lock" that maintains oncogenic transcriptional programs. NOS2 and COX2 form a reciprocal feed-forward circuit in which NO, prostaglandin E2 (PGE2), interleukin (IL)-6, and IL-8 reinforce one another, driving tumor-promoting inflammation, immunosuppression, angiogenesis, and metastasis while depleting nutrients and acidifying the tumor interstitial fluid. Spatially, NOS2/COX2 niches at the tumor-stroma interface and within immune deserts generate gradients of NO, PGE2, oxygen, and metabolites that partition tumors into microdomains with distinct metabolic states, immune composition, and therapeutic vulnerabilities. Integrating these insights with Hanahan's updated hallmarks of cancer, we propose that NOS2-derived NO functions as a node synchronizing deregulated energetics, inflammation, immune evasion, plasticity, and therapy resistance within the tumor microenvironment (TME). Targeting the NOS2-COX2 axis and its downstream NO-iron-epigenetic circuitry may therefore disrupt multiple hallmarks and reveal combinatorial strategies to exploit NO-induced metabolic liabilities in cancer.

Nitric oxide (NO) is a free radical signaling molecule with multiple biological functions. As part of the innate immune system, NO has antimicrobial properties playing an important role in host defense. Mechanisms of NO cytotoxicity result from its ability to bind metals and inhibit enzyme function or by increasing nitrosative and oxidative stress within cells. One of the primary biological targets of NO is the chelatable iron pool (CIP) which is quantitatively converted to dinitrosyliron complexes (DNIC) when it reacts with NO. Despite the numerous purported mechanisms attributed to NO's bactericidal properties, DNIC formation and its ability to restrict iron bioavailability from pathogenic bacteria has not been directly tested. Streptococcus pyogenes is a human pathogen that causes a range of diseases spanning from pharyngitis and impetigo to soft tissue necrosis and toxic shock. S. pyogenes employs the Rgg2/Rgg3 quorum sensing (QS) system to regulate aspects of its virulence potential, including biofilm formation, lysozyme resistance, and modulation of host innate immune response. Previous studies found that iron and manganese restriction induced Rgg2/Rgg3 QS, leading us to test whether NO-dependent iron restriction mediated by DNIC formation was sufficient to induce QS and related iron-starvation phenotypes. Here, we demonstrate that DNIC are formed in S. pyogenes exposed to physiologically relevant NO concentrations. The DNIC are formed from the CIP, and formation led to a significant reduction in the CIP, which correlated to a concomitant activation of QS and iron-regulated gene expression. These studies are the first to demonstrate that restriction of iron bioavailability mediated by DNIC formation is a functional mechanism by which NO can regulate QS, gene expression, and cell growth in bacteria.

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

Supplementary Materials and Methods. Figure S1. Nitric oxide induces significant changes in histone posttranslational modifications. Figure S2. NO-mediated gene expression changes are consistent with the induction of an oncogenic phenotype. Figure S3. Nitric oxide alters the distribution patterns of H3K9ac and H3K9me2 across the genome. Figure S4. NO-upregulated genes with a new promoter-associated H3K9ac peak are involved in critical tumorigenic pathways. Table S1. NO-mediated changes in global levels of histone acetylation. Table S2. List of NO-regulated miRNAs (1.5 Fold Change cut-off). Table S3. Control ChIP-PCR primer sequences. Table S4. List of transcription factors with binding motifs found exclusively around promoters of upregulated genes with a new NO-induced H3K9ac peak.

Nitric oxide (NO) and the enzyme that synthesizes it, nitric oxide synthase 2 (NOS2), have emerged as key players in inflammation and cancer. Expression of NOS2 in tumors has been correlated both with positive outcomes and with poor prognoses. The chemistry of NO is the major determinate to the biological outcome and the concentration of NO, which can range over five orders of magnitude, is critical in determining which pathways are activated. It is the activation of specific oncogenic and immunological mechanisms that shape the outcome. The kinetics of specific reactions determine the mechanisms of action. In this review, the relevant reactions of NO and related species are discussed with respect to these oncogenic and immunological signals.

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N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m6A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m6A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6A-mRNA levels and transcriptional changes of m6A-associated genes. These results build a direct link between NO and m6A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome.

GO terms associated with upregulated genes bearing new ∙NO-induced H3K9ac peaks around promoter regions

GO terms associated with ∙NO-regulated genes

Nitric oxide (NO) and hydrogen sulfide (H2S) were previously only known for their toxic properties. Now they are regarded as potent gaseous messenger molecules (gasotransmitters) that rapidly transverse cell membranes and transduce cellular signals through their chemical reactions and modifications to protein targets. Both are known to regulate numerous physiological functions including angiogenesis, vascular tone, and immune response, to name a few. NO and H2S often work synergistically and in competition to regulate each other's synthesis, target protein activity via posttranslational modifications (PTMs), and chemical interactions. In addition to their canonical modes of action, increasing evidence has demonstrated that NO and H2S share another signaling mechanism: epigenetic regulation. This review will compare and contrast biosynthesis and metabolism of NO and H2S, their individual and shared interactions, and the growing body of evidence for their roles as endogenous epigenetic regulatory molecules.

Triple-negative breast cancers (TNBC) that produce nitric oxide (NO) are more aggressive, and the expression of the inducible form of nitric oxide synthase (NOS2) is a negative prognostic indicator. In these studies, we set out to investigate potential therapeutic strategies to counter the tumor-permissive properties of NO. We found that exposure to NO increased proliferation of TNBC cells and that treatment with the histone deacetylase inhibitor Vorinostat (SAHA) prevented this proliferation. When histone acetylation was measured in response to NO and/or SAHA, NO significantly decreased acetylation on histone 3 lysine 9 (H3K9ac) and SAHA increased H3K9ac. If NO and SAHA were sequentially administered to cells (in either order), an increase in acetylation was observed in all cases. Mechanistic studies suggest that the “deacetylase” activity of NO does not involve S -nitrosothiols or soluble guanylyl cyclase activation. The observed decrease in histone acetylation by NO required the interaction of NO with cellular iron pools and may be an overriding effect of NO-mediated increases in histone methylation at the same lysine residues. Our data revealed a novel pathway interaction of Vorinostat and provides new insight in therapeutic strategy for aggressive TNBCs.

Nitric oxide (NO), the endogenously produced free radical signaling molecule, is generally thought to function via its interactions with heme-containing proteins, such as soluble guanylyl cyclase (sGC), or by the formation of protein adducts containing nitrogen oxide functional groups (such as S-nitrosothiols, 3-nitrotyrosine, and dinitrosyliron complexes). These two types of interactions result in a multitude of down-stream effects that regulate numerous functions in physiology and disease. Of the numerous purported NO signaling mechanisms, epigenetic regulation has gained considerable interest in recent years. There is now abundant experimental evidence to establish NO as an endogenous epigenetic regulator of gene expression and cell phenotype. Nitric oxide has been shown to influence key aspects of epigenetic regulation that include histone posttranslational modifications, DNA methylation, and microRNA levels. Studies across disease states have observed NO-mediated regulation of epigenetic protein expression and enzymatic activity resulting in remodeling of the epigenetic landscape to ultimately influence gene expression. In addition to the well-established pathways of NO signaling, epigenetic mechanisms may provide much-needed explanations for poorly understood context-specific effects of NO. These findings provide more insight into the molecular mechanisms of NO signaling and increase our ability to dissect its functional role(s) in specific micro-environments in health and disease. This review will summarize the current state of NO signaling via epigenetic mechanisms (the "third pillar" of NO signaling).

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

Nitric oxide (NO) is involved in numerous physiologic functions ranging from regulation of cardiovascular functions to participating in memory (1–4). In the immune system, this diatomic radical is involved in host defense and has tumoricidal functions (5,6) However, despite these properties, which are critical in maintaining homeostasis, NO has been implicated as a participant or causative agent in a variety of pathophysiologic conditions (7,8). Defining the exact role of NO under pathophysiologic conditions is further complicated by the fact that it has been shown to be both protective as well as deleterious even in the context of the same biologic setting. Therefore, the search for mechanistic explanations to account for these differing effects is ongoing. Unlike other biologic mediators, the main determinants of the biologic effects of NO are its chemical and physical properties. In addition to the numerous potential reactions of NO in biologic systems, many of its effects can be attributed to additional reactive nitrogen oxide species (RNOS) that are formed and that have their own selective reactivity. Although a large variety of chemical reactions relate to NO in biologic systems, it is difficult to determine which ones are pertinent. In an attempt to sort out the diverse chemical reactions and their importance, we have developed the concept of the “chemical biology of nitric oxide” (9–11). The chemical biology of NO sepa2

Mechanical ventilation with hyperoxia is the major supportive measure to treat patients with acute lung injury and acute respiratory distress syndrome (ARDS). However, prolonged exposure to hyperoxia can induce oxidative inflammatory lung injury. Previously, we have shown that high levels of airway high-mobility group box 1 protein (HMGB1) mediate hyperoxia-induced acute lung injury (HALI). Using both ascorbic acid (AA, also known as vitamin C) and sulforaphane (SFN), an inducer of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), we tested the hypothesis that dietary antioxidants can mitigate HALI by ameliorating HMGB1-compromised macrophage function in phagocytosis by attenuating hyperoxia-induced extracellular HMGB1 accumulation. Our results indicated that SFN, which has been shown to attenute HALI in mice exposed to hyperoxia, dose-dependently restored hyperoxia-compromised macrophage function in phagocytosis (75.9 ± 3.5% in 0.33 µM SFN versus 50.7 ± 1.8% in dimethyl sulfoxide (DMSO) control, p < 0.05) by reducing oxidative stress and HMGB1 release from cultured macrophages (47.7 ± 14.7% in 0.33 µM SFN versus 93.1 ± 14.6% in DMSO control, p < 0.05). Previously, we have shown that AA enhances hyperoxic macrophage functions by reducing hyperoxia-induced HMGB1 release. Using a mouse model of HALI, we determined the effects of AA on hyperoxia-induced inflammatory lung injury. The i.p. administration of 50 mg/kg of AA to mice exposed to 72 h of ≥98% O2 significantly decreased hyperoxia-induced oxidative and nitrosative stress in mouse lungs. There was a significant decrease in the levels of airway HMGB1 (43.3 ± 12.2% in 50 mg/kg AA versus 96.7 ± 9.39% in hyperoxic control, p < 0.05), leukocyte infiltration (60.39 ± 4.137% leukocytes numbers in 50 mg/kg AA versus 100 ± 5.82% in hyperoxic control, p < 0.05) and improved lung integrity in mice treated with AA. Our study is the first to report that the dietary antioxidants, ascorbic acid and sulforaphane, ameliorate HALI and attenuate hyperoxia-induced macrophage dysfunction through an HMGB1-mediated pathway. Thus, dietary antioxidants could be used as potential treatments for oxidative-stress-induced acute inflammatory lung injury in patients receiving mechanical ventilation.

Mechanical ventilation (MV) with supraphysiological levels of oxygen (hyperoxia) is a life-saving therapy for the management of patients with respiratory distress. However, a significant number of patients on MV develop ventilator-associated pneumonia (VAP). Previously, we have reported that prolonged exposure to hyperoxia impairs the capacity of macrophages to phagocytize Pseudomonas aeruginosa (PA), which can contribute to the compromised innate immunity in VAP. In this study, we show that the high mortality rate in mice subjected to hyperoxia and PA infection was accompanied by a significant decrease in the airway levels of nitric oxide (NO). Decreased NO levels were found to be, in part, due to a significant reduction in NO release by macrophages upon exposure to PA lipopolysaccharide (LPS). Based on these findings, we postulated that NO supplementation should restore hyperoxia-compromised innate immunity and decrease mortality by increasing the clearance of PA under hyperoxic conditions. To test this hypothesis, cultured macrophages were exposed to hyperoxia (95% O-2) in the presence or absence of the NO donor, (Z)-1-[N (2 aminoethyl)-N-(2-ammonioethyeaminoldiazen-1-ium-1,2-diolate (DETA-NONOate/D-NO). Interestingly, D-NO (up to 37.5 mu M) significantly attenuated hyperoxia-compromised macrophage migratory, phagocytic, and bactericidal function. To determine whether the administration of exogenous NO enhances the host defense in bacteria clearance, C57BL/6 mice were exposed to hyperoxia (99% O-2) and intranasally inoculated with PA in the presence or absence of D-NO. D-NO (300 mu M-800 mu M) significantly increased the survival of mice inoculated with PA under hyperoxic conditions, and significantly decreased bacterial loads in the lung and attenuated lung injury. These results suggest the NO donor, D-NO, can improve the clinical outcomes in VAP by augmenting the innate immunity in bacterial clearance. Thus, provided these results can be extrapolated to humans, NO supplementation may represent a potential therapeutic strategy for preventing and treating patients with VAP.