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Biology subjects

Denson, L.

Publications and source records attributed to Denson, L..

4 recordsLinked to original sources

Sodium Aurothiomalate Induces Ferroptosis by Targeting GPX4 via Gold-Dependent Thiomalate Covalent Modification

Ferroptosis, an iron-dependent form of oxidative cell death, is predominantly regulated by glutathione peroxidase 4 (GPX4), making it a promising target for cancer therapy. However, the majority of GPX4 inhibitors, most of which contain a chloroacetamide moiety such as RSL3, are limited by poor pharmacokinetic properties and off-target effects, hindering their preclinical translation. Utilizing a range of interdisciplinary methodologies, we show that sodium aurothiomalate (ATM), a drug approved by many agencies, induces ferroptosis by covalently targeting GPX4 via formation of a selenenylsulfide bond. In preclinical models of neuroblastoma and acute myeloid leukemia (AML), ATM combined with ferric ammonium citrate (FAC) yields a synergistic effect, resulting in a significant reduction in tumor growth. Mechanistically, ATM disrupts GPX4 activity by covalently binding thiomalate to the active site selenocysteine, while modification of specific cysteine residues leads to destabilization of the protein and impaired binding to phospholipids. We propose that these covalent modifications are achieved through a unique reaction mechanism, in which the gold component of ATM acts as a thiol-masking carrier and is only transiently present, being subsequently displaced and allowing the reaction of the thiomalate moiety with the target selenocysteine or cysteine. Our data lay the foundation for development of novel, drug-like thiol-based GPX4 inhibitors.

biochemistry↗

Epithelial-intrinsic nitric oxide synthase 2 sustains host-microbiota dynamics that promote colitis

The microbiota influence disease pathogenesis and treatment, however we have limited ability to assess patient status in relation to the microbiota. Here we find that the nitric oxide generating enzyme, nitric oxide synthase 2 (Nos2), is transcriptionally primed in intestinal epithelial cells (IECs), as opposed to immune cells, in inflammatory bowel disease (IBD) patients. Generation of IEC-specific Nos2 knockout mice revealed that epithelial Nos2 activity promoted susceptibility to intestinal disease and sustained a colitogenic microbiota. Epithelial Nos2 increased levels of nitric oxide-derived nitrates and nitrate-metabolizing bacteria in the intestine. Unexpectedly, extra-intestinal nitrates also reflected IEC-intrinsic Nos2 expression, and systemic nitrate concentrations in patients paralleled intestinal Nos2 activation. In fact, temporally inhibiting epithelial Nos2 was sufficient to alter intestinal nitrate homeostasis and inflammation in mice, as well as restrict nitrate production by human intestinal organoids. These data reveal that epithelial nitric oxide metabolism directs host-microbiota dynamics that can alter disease and that monitoring and targeting this axis may benefit patients with IBD.

immunology↗

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohns Disease

Background and AimsWe previously identified small molecules predicted to reverse an ileal gene signature for future Crohns Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). MethodsInduced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. ResultsiPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ConclusionsETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

molecular biology↗

Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy

Clinical benefits to cytokine blockade in ileal Crohns disease (iCD) have been limited to a subset of patients. Whether cellular and molecular heterogeneity contributes to variability in treatment responses has been unclear. Using single cell technologies combining scRNAseq, CyTOF and multiplex tissue imaging, we mapped the cellular landscape of inflamed ileum lesions, adjacent non-inflamed ileum and matched circulating blood cells of iCD patients. In inflamed tissues, we identified a pathogenic module characterized by an inflammatory mononuclear phagocyte (Inf.MNP)-associated cellular response organized around inflammatory macrophages and mature dendritic cells in a subset of iCD patients. We confirmed the Inf.MNP-associated cellular response in 4 independent iCD cohorts (n=441) and showed that presence of this pathogenic module at diagnosis correlated with primary resistance to anti-TNF therapy. Single cell mapping of iCD tissues identifies a complex cellular signature of anti-TNF resistance thereby revealing novel biomarkers of treatment response and tailored therapeutic opportunities.

immunology↗