Background and Purpose Pro-inflammatory macrophages drive obesity-associated metabolic inflammation. Identifying key regulators of their activation is crucial for new therapies. This study aimed to identify novel regulators of pro-inflammatory adipose tissue macrophage (ATM) activation and assess their therapeutic potential.Experimental Approach We combined genome-wide CRISPR-Cas9 screening with scRNA-seq data from human adipose tissue and identified long-chain acyl-CoA synthetase 4 (ACSL4) as critical for pro-inflammatory ATM activation. Validation used myeloid-specific Acsl4 knockout mice (Acsl4 fl/fl Lyz2 Cre) and shRNA-mediated knockdown in macrophage cell lines (RAW264.7, THP-1, and iBMDM). Mechanisms were explored via mutagenesis, immunostaining, co-immunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFC), and transcriptional activity assays. A high-throughput screen of 8,685 DrugBank compounds was performed to identify candidates targeting ACSL4.Key Results ACSL4 deficiency markedly reduced pro-inflammatory factor expression (Tnf alpha, Il1 beta, and Il6). Myeloid ACSL4 deletion protected against high-fat diet (HFD)-induced obesity, correlated with reduced pro-inflammatory ATMs and enhanced adipocyte lipolysis and thermogenesis, without affecting food intake, water intake, or locomotor activity in mice. However, glucose tolerance and insulin sensitivity were not significantly affected. Mechanistically, ACSL4 contributed to inflammation independent of its enzymatic/ferroptotic functions (Thr328 mutation, troglitazone insensitivity). Instead, ACSL4 translocated to the nucleus in a karyopherin beta 1 (KPNB1)-dependent manner. DrugBank screening identified abemaciclib as a potential inhibitor of the ACSL4-KPNB1 interaction. Notably, abemaciclib treatment reduced macrophage inflammation and attenuated HFD-induced obesity in mice.Conclusions and Implications ACSL4 promotes pro-inflammatory macrophage activation through its KPNB1-dependent nuclear translocation and cooperation with IRF5. Targeting the ACSL4-KPNB1 interaction may represent a potential therapeutic strategy for metabolic inflammation.
Background and Purpose Atherosclerosis, a chronic inflammatory disease characterised by lipid accumulation and endothelial dysfunction, remains a leading cause of cardiovascular morbidity and mortality. While cannabinoid 2 (CB2) receptor agonists show therapeutic promise in preclinical studies, their systemic application is hindered by poor bioavailability and off-target effects. Consequently, targeted delivery systems are essential to enhance therapeutic efficacy and mitigate adverse effects.Experimental Approach This study presents a targeted nanotherapeutic strategy for localising the CB2 receptor agonist directly at atherosclerotic lesions. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), encapsulating the selective agonist JWH-133 and functionalised with a VCAM-1 binding peptide (VBP) for selective binding to vascular cell adhesion molecule-1 (VCAM-1) (VBP-JWH-133-NPs), were developed. In vivo experiments were conducted in both male and female apolipoprotein E-deficient (ApoE-/-) mice to assess the anti-inflammatory and lipid-modulating effects of the nanotherapy. Serum lipid profiles, aortic plaque burden and key inflammatory markers were analysed.Key Results VBP-JWH-133-NPs significantly reduced foam cell-rich depositions and modulate systemic inflammatory markers in both sexes, demonstrating superior anti-atherogenic efficacy compared with free JWH-133. The therapeutic benefits are notably more pronounced in male mice, which exhibit greater initial plaque area and lipid deposition.Conclusion and Implications These findings underscore the potential of VBP-JWH-133-NPs as an efficient and superior delivery system for cannabinoid therapies in atherosclerosis. By addressing sex as a biological variable, this approach offers a nuanced strategy for developing more effective and precise cardiovascular interventions.
Background and Purpose Tocopherols (vitamin E) are well characterized as essential lipophilic antioxidant agents. However, recent evidence suggests that tocopherols may also regulate cellular signalling through direct interactions with specific protein targets. This study aimed to investigate the molecular mechanism by which α‐tocopherol regulates programmed cell death and to identify its direct molecular target. Experimental Approach A high‐throughput screen identified α‐tocopherol as an inhibitor of TNF‐α/SMAC‐mimetic‐induced cell death. The mechanism(s) underlying this action of α‐tocopherol was investigated using western blotting, immunoprecipitation, ubiquitination assays, drug affinity responsive target stability, cellular thermal shift assay, isothermal titration calorimetry and molecular docking. Its efficacy, in vivo, was evaluated in a murine model of ulcerative colitis, induced by dextran sulphate sodium (DSS). Key Results α‐Tocopherol selectively inhibited TNF‐α/SMAC‐mimetic‐induced cell death by directly and competitively binding to the BIR3 domain of the inhibitor of apoptosis protein‐1 (cIAP1). This interaction stabilized cIAP1, prevented the formation of RIPK1‐dependent death complexes and suppressed apoptosis and necroptosis. In a murine model of DSS‐induced colitis, α‐tocopherol significantly alleviated disease severity while preserving cIAP1 protein expression. Conclusion and Implications Our findings identified cIAP1 as a previously unrecognized molecular target of α‐tocopherol and demonstrated that cIAP1 stabilization contributed to the suppression of RIPK1‐dependent cell death. These findings expand the current understanding of the biological functions of tocopherols and provide a mechanistic basis for their potential therapeutic application in inflammatory diseases associated with aberrant cIAP1 degradation.
BACKGROUND AND PURPOSE Cancer cells exhibit the Warburg effect, but oxidative phosphorylation (OXPHOS) is also important for cancer cell proliferation. Metabolic reprogramming from glycolysis to OXPHOS is one mechanism of resistance to anti-tumour drugs. Here, we have synthesized and characterized OPB-111077, a novel, first-in-class orally available small-molecule compound, and investigated its anti-tumour activity and therapeutic potential in combination with alkylating agents. EXPERIMENTAL APPROACH Inhibition of mitochondrial function by OPB-111077 were assessed by measuring the oxygen consumption rate, ATP production and Complex I enzymatic activity. anti-tumour effects were evaluated in human haematological and solid tumour cell lines and in cell line-derived xenograft and lymphoma patient-derived xenograft models. Mechanistic studies included CRISPR screening, metabolomic analyses, and measurements of the oxygen consumption and extracellular acidification rates. Effects of OPB-111077 combined with alkylating agents were also assessed in mice. KEY RESULTS OPB-111077 inhibited mitochondrial respiratory chain Complex I, suppressed OXPHOS, and exerted anti-tumour effects in several models. OPB-111077 demonstrated synergism with the alkylating agent bendamustine, with simultaneous inhibition of both OXPHOS and glycolysis. Bendamustine reprogrammed cancer cells into an OXPHOS-dominant state, thereby enhancing the efficacy of OPB-111077. CONCLUSION AND IMPLICATIONS Based on these findings, we propose a novel anticancer therapeutic strategy of combining OXPHOS inhibitors with alkylating agents. OPB-111077 has demonstrated potential in combination therapy and was recently evaluated with bendamustine and rituximab in a Phase 1 clinical trial for patients with relapsed/refractory diffuse large B-cell lymphoma (NCT04049825).
