Necroptosis, a regulated form of necrosis, has emerged as a novel therapeutic strategy that could enhance cancer immunotherapy. However, its role in tumorigenesis is still debated because recent studies have reported both anti- and pro-tumoral effects. Here, we aimed to systematically evaluate the associations between tumor necroptosis (mixed lineage kinase domain-like protein, MLKL; phosphorylated MLKL, pMLKL; and receptor-interacting protein kinase 1–receptor-interacting protein kinase 3, RIPK1–RIPK3 interaction) and tumor-infiltrating immune cells (CD8+ and FOXp3+ T cells and CD163+ M2 macrophages) and tumor PD-L1 by immunohistochemistry in 88 cholangiocarcinoma (CCA) patients who had undergone surgical resection. Their associations with clinicopathological characteristics, survival data, and prognosis were evaluated. MLKL was found to be an unfavorable prognostic factor (p-value = 0.023, HR = 2.070) and was inversely correlated with a clinically favorable immune cell signature (high CD8+/high FOXp3+/low CD163+). Both pMLKL and RIPK1–RIPK3 interaction were detected in CCA primary tissues. In contrast to MLKL, pMLKL status was significantly positively correlated with a favorable immune signature (high CD8+/high FOXp3+/low CD163+) and PD-L1 expression. Patients with high pMLKL-positive staining were significantly associated with an increased abundance of CD8+ T cell intratumoral infiltration (p-value = 0.006). Patients with high pMLKL and PD-L1 expressions had a longer overall survival (OS). The results from in vitro experiments showed that necroptosis activation in an RMCCA-1 human CCA cell line selectively promoted proinflammatory cytokine and chemokine expression. Jurkat T cells stimulated with necroptotic RMCCA-1-derived conditioned medium promoted PD-L1 expression in RMCCA-1. Our findings demonstrated the differential associations of necroptosis activation (pMLKL) and MLKL with a clinically favorable immune signature and survival rates and highlighted a novel therapeutic possibility for combining a necroptosis-based therapeutic approach with immune checkpoint inhibitors for more efficient treatment of CCA patients.

Excessive DNA damage triggers various types of programmed cell death (PCD), yet the regulatory mechanism of DNA damage-induced cell death is not fully understood. Here, we report that PANoptosis, a coordinated PCD pathway, including pyroptosis, apoptosis and necroptosis, is activated by DNA damage. The Z-DNA binding protein 1 (ZBP1) is the apical sensor of PANoptosis and essential for PANoptosome assembly in response to DNA damage. We find endogenous retroviruses (ERVs) are activated by DNA damage and act as ligands for ZBP1 to trigger PANoptosis. By using ZBP1 knock-out and knock-in mice disrupting ZBP1 nucleic acid-binding activity, we demonstrate that ZBP1-mediated PANoptosis contributes to the toxic effects of chemotherapeutic drugs, which is dependent on ZBP1 nucleic acid-binding activity. We found that ZBP1 expression is downregulated in tumor tissue. Furthermore, in colorectal cancer patients, dsRNA is induced by chemotherapy and sensed by ZBP1 in normal colonic tissues, suggesting ZBP1-mediated PANoptosis is activated by chemotherapy in normal tissues. Our findings indicate that ZBP1-mediated PANoptosis is activated by DNA damage and contributes to the toxic side effects of DNA-damage-based chemotherapy. These data suggest that ZBP1 could be a promising therapeutic target to alleviate chemotherapy-related side effects.

Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-α (TNF-α) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKL-dependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-α mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-α and gemcitabine induced RIPK1/RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore, our findings have pivotal implications for designing a novel necroptosis-based therapeutic strategy for CCA patients.

Cholangiocarcinoma (CCA) is an aggressive cancer associated with a very poor prognosis and low survival rates, primarily due to late-stage diagnosis and low response rates to conventional chemotherapy. Therefore, there is an urgent need to identify effective therapeutic strategies that can improve patient outcomes. Flavonoids, such as quercetin and kaempferol, are naturally occurring compounds that have attracted significant attention for their potential in cancer therapy by targeting multiple genes. In this study, we employed network pharmacology and bioinformatic analysis to identify potential targets of quercetin and kaempferol. The results revealed that the target genes of these flavonoids were enriched in G2/M-related genes, and higher expression of G2/M signature genes was significantly associated with shorter survival in CCA patients. Furthermore, in vitro experiments using CCA cells demonstrated that quercetin or kaempferol induced cell-cycle arrest in the G2/M phase. Additionally, when combined with a Smac mimetic LCL-161, an IAP antagonist, quercetin or kaempferol synergistically induced RIPK1/RIPK3/MLKL-mediated necroptosis in CCA cells while sparing non-tumor cholangiocyte cells. These findings shed light on an innovative therapeutic combination of flavonoids, particularly quercetin and kaempferol, with Smac mimetics, suggesting great promise as a necroptosis-based approach for treating CCA and potentially other types of cancer.

Cholangiocarcinoma (CCA) is a bile duct malignancy. Our previous comprehensive analysis showed that ferroptosis-related genes can stratify CCA patients into low-risk and high-risk groups based on survival time. Here, we explored the role of ferroptosis in CCA by analyzing mRNA expression in CCA patients from public databases. We identified acyl-CoA synthetase long chain family member 3 (ACSL3) as a potential ferroptosis suppressor in high-risk CCA patients. Using a panel of CCA cell lines, we confirmed ACSL3 upregulation in CCA cell lines associated with high-risk CCA, correlating this with resistance to the ferroptosis inducer RSL3. Lipidomic analysis revealed increased monounsaturated fatty acid (MUFA)-containing phospholipids in resistant cell lines. ACSL3 silencing sensitized these cells to RSL3. Resistance to ferroptosis was also dependent on exogenous MUFAs and was enhanced by lipid droplet biogenesis inhibition. These findings highlight ACSL3 as a promising target for therapeutic strategies aimed at overcoming ferroptosis resistance in CCA.

Necroptosis and ferroptosis are two distinct forms of necrotic-like cell death in terms of their morphological features and regulatory mechanisms. These two types of cell death can coexist in disease and contribute to pathological processes. Inhibition of both necroptosis and ferroptosis has been shown to enhance therapeutic effects in treating complex necrosis-related diseases. However, targeting both necroptosis and ferroptosis by a single compound can be challenging, as these two forms of cell death involve distinct molecular pathways. In this study, we discovered that KW-2449, a previously described necroptosis inhibitor, also prevented ferroptosis both in vitro and in vivo. Mechanistically, KW-2449 inhibited ferroptosis by targeting the autophagy pathway. We further identified that KW-2449 functioned as a ULK1 (Unc-51-like kinase 1) inhibitor to block ULK1 kinase activity in autophagy. Remarkably, we found that Necrostatin-1, a classic necroptosis inhibitor that has been shown to prevent ferroptosis, also targets the autophagy pathway to suppress ferroptosis. This study provides the first understanding of how necroptosis inhibitors can prevent ferroptosis and suggests that autophagy is a targetable pathway for necroptosis inhibitors to prevent ferroptosis. Therefore, the identification and design of pharmaceutical molecules that target the autophagy pathway from necroptosis inhibitors is a promising strategy to develop dual inhibitors of necroptosis and ferroptosis in clinical application.