IAP x MLKL

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with a complex pathogenesis and limited treatment options. Recently, necroptosis has been found to play a significant role in UC. This study aimed to investigate necroptosis-related mechanisms and hub targets in UC, and to screen natural potential inhibitors. Firstly, transcriptomic and single-cell analyses were used to explore the molecular and cellular mechanisms of necroptosis in UC and identify hub targets. Subsequently, virtual screening and molecular dynamics were performed. The results indicated that twenty-three necroptosis-related differentially expressed genes (DEGs) were predicted as diagnostic biomarkers in the best machine-learning model (GBM). Furthermore, four hub targets (IL1B, MLKL, STAT1, and BIRC3) were computationally prioritized and their overexpression might promote pro-inflammatory activity (neutrophils/M1 macrophages) while suppressing anti-inflammatory responses (Tregs/M2 macrophages), aggravating UC progression. Single-cell analysis revealed reduced epithelial cells and increased fibroblasts, endothelial cells, and immune cells in UC tissues, suggesting disruption of the intestinal epithelial barrier, exacerbation of fibrosis, and activation of the immune system. The high abundance of endothelial cells and monocytes expressing necroptosis-related DEGs suggested the important role of necroptosis in UC. Moreover, eight natural products were screened with strong binding affinity to MLKL, whose motion trajectories and energy trajectories reached equilibrium within 10 ns. Among them, the potential of trifolirhizin and curcumin as natural inhibitors was particularly prominent. Conclusively, this study computationally predicts four hub DEGs and eight potential natural necroptosis inhibitors, may provide a basis for future therapeutic exploration.

Cholangiocarcinoma (CCA), a heterogeneous tumor arising in the bile ducts, is associated with unfavorable prognosis and high mortality rates due to limited effective treatment options. Though promising, immunotherapy is hindered by CCA's desmoplastic tumor microenvironment, known for its inflammatory and immunosuppressive characteristics, often termed a "cold tumor". Previously, we reported that Poly(I:C), a TLR3 agonist, synergizes with a Smac mimetic, an IAP antagonist, to trigger necroptosis when caspase activity is blocked. In this study, we aimed to evaluate rational combination strategies to advance Poly(I:C)-based necroptosis immunotherapy in preclinical models. Transcriptomic analysis of public CCA patient datasets revealed that low expression of TLR3, accompanied by low IFNG or IRF1 expression, is significantly associated with poorer prognosis. These findings highlight the clinical relevance of the IFN-γ/IRF1/TLR3 axis and support the development of therapies targeting this pathway. We further found that IFN-γ treatment increases TLR3 expression. Importantly, we demonstrated that IFN-γ could enhance Poly(I:C)-induced necroptosis and immunogenic cell death (ICD) when IAPs and caspases are inhibited by Smac mimetic and the pan-caspase inhibitor Z-VAD-FMK in human CCA cell lines. CU-CPT 4a, a highly selective TLR3 inhibitor, partially reduced this cell death. This indicates that the effect is mediated in part through TLR3. The combined effects were shown to depend on RIPK1/RIPK3/MLKL-induced necroptosis. Additionally, the dying cells released HMGB1, a marker of ICD, and their conditioned medium promoted dendritic cell maturation (CD80, CD86, HLA-DR), which is critical for antigen presentation and T-cell priming. From a personalized medicine perspective, TLR3, IFNG, and IRF1 expression levels may serve as predictive biomarkers to guide patient selection for TLR3 agonist-based therapies. These findings could advance TLR3 ligand, Poly(I:C)-based necroptosis cancer immunotherapy, alone or with immune checkpoint inhibitors, for CCA and possibly other cancers.

Necroptosis is a form of regulated cell death that depends on the receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). The molecular mechanisms underlying distinct instances of necroptosis have only recently begun to emerge. In the present study, we characterized RABGEF1 as a positive regulator of RIPK1/RIPK3 activation in vitro. Based on the overexpression and knockdown experiments, we determined that RABGEF1 accelerated the phosphorylation of RIPK1 and promoted necrosome formation in L929 cells. The pro-necrotic effect of RABGEF1 is associated with its E3 ubiquitin ligase activity and guanine nucleotide exchange factor (GEF) activity. We further confirmed that RABGEF1 interacts with cIAP1 protein by inhibiting its function and plays a regulatory role in necroptosis, which can be abolished by treatment with the antagonist Smac mimetic (SM)-164. In conclusion, our study highlights a potential and novel role of RABGEF1 in promoting TNF-induced cell necrosis.