Patients with cholangiocarcinoma (CCA) have poor prognosis. Current cisplatin-based first-line chemotherapy offers limited survival benefits. Cisplatin induces single-strand DNA breaks, activating DNA repair mechanisms that diminish its effectiveness. Here, we present the design, chemical synthesis, and therapeutic evaluation of a new generation of chemotherapeutic agents (Aurkines) with unique polyelectrophilic properties. These agents cause a high frequency of double-strand DNA breaks, bypassing DNA repair, and promoting cancer cell death. Two novel compounds, Aurkine 16 and Aurkine 18, were designed and evaluated for their antitumor effects in both naïve and cisplatin-resistant CCA cells, cancer-associated fibroblasts (CAFs), healthy cholangiocytes, and in vivo models. Aurkines effectively induced double-strand DNA breaks, leading to increased DNA damage and elevated levels of reactive oxygen species, resulting in greater cytotoxicity than cisplatin in CCA cells. Phosphoproteomic and molecular analysis revealed that cisplatin activates DNA repair pathways, while Aurkines primarily induce apoptosis. Importantly, Aurkines also triggered apoptosis in cisplatin-resistant CCA cells and CAFs without harming healthy cholangiocytes. Additionally, Aurkines demonstrated cytotoxicity in other cisplatin-resistant cancers, such as breast and ovarian cancer. This tumor selectivity results from reduced uptake, increased efflux, and compact chromatin structure in normal cells, limiting Aurkine-DNA interactions. In vivo, Aurkines inhibited the growth of subcutaneous naïve and cisplatin-resistant CCA tumors, as well as orthotopic tumors in immunocompetent mice promoting antitumor immune cell recruitment, without any adverse events. Transport studies revealed that Aurkines were selectively taken up by OCT1, OCT3, CTR1, and OATP1A2, whereas only CTR1 transported cisplatin. Aurkines represent promising therapeutic drugs for both naïve and cisplatin-resistant cancers due to their unique polyelectrophilic properties and selective targeting of malignant cells. This study introduces a novel therapeutic strategy designed to induce frequent double-strand DNA breaks selectively in both naïve and cisplatin-resistant cancer cells, without evident toxic side effects at therapeutic doses. This approach may settle the basis for new strategies to overcome the critical challenge of drug resistance in cancer treatment, and has the potential to be a breakthrough not only for the treatment of biliary tumors but also for other cancers.

Background and Aims: Cholangiocarcinoma (CCA) includes a diverse group of biliary malignancies with poor prognosis. Alterations in post-translational modifications contribute to disrupted protein dynamics, cellular disturbances, and disease. This study investigates the role of protein SUMOylation in cholangiocarcinogenesis and its potential as a therapeutic target. Approach and Results: Analysis of CCA tumors from 4 patient cohorts and CCA cell lines, revealed increased expression of the SUMOylation machinery genes SUMO activating enzyme 1 ( SAE1 ) and the SUMO conjugating enzyme UBE2I , regardless of the tumor’s molecular profile, resulting in elevated levels of SUMO1-conjugated proteins. Higher SAE1 and UBE2I levels were both indicative of unfavorable clinical outcomes. Deregulated SUMOylated proteins in CCA, mostly linked to cell proliferation, survival, and homeostasis, were identified through immunoprecipitation and mass spectrometry. Genetic ( UBE2I -knockdown) and pharmacological (ML792 and S-adenosyl-methionine) inhibition of SUMOylation effectively suppressed tumorigenesis in subcutaneous and oncogene-driven CCA models, reducing the presence of cancer-associated fibroblasts and increasing the recruitment of antitumor immune cells. In vitro, targeting SUMOylation induced CCA cell death and reduced cell proliferation, colony formation, and spheroid growth. Importantly, ML792 and S-adenosyl-methionine did not adversely affect normal human cholangiocytes. Moreover, co-culture of wild-type or UBE2I -knockdown CCA cells with cancer-associated fibroblasts revealed that depleting SUMOylation in CCA cells impaired cancer-associated fibroblast cell growth and altered their protein secretome, ultimately disrupting CCA growth through a regulatory feedback loop. Conclusions: Aberrant SUMOylation drives CCA progression by enhancing cell survival, proliferation, and shaping the tumor microenvironment. Targeting SUMOylation shows potential in inhibiting CCA growth, representing a promising therapeutic strategy.

Ursodeoxycholic acid (UDCA) is widely used to treat cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), yet its molecular mechanisms remain unclear. This study investigated the impact of long-term UDCA therapy on circulating levels of the microRNAs miR-34a and miR-506, which are implicated in PBC pathogenesis, and explored associated changes in inflammatory markers and signaling pathways. Serum samples from patients with PBC and PSC were collected before and after UDCA treatment and analyzed for miRNA expression as well as levels of TREM-2 and sCD163. In vitro studies using human cholangiocytes and lipopolysaccharide (LPS) stimulation assessed changes in the expression of miR-34a, TREM-2, and ADAM17. The results showed that the baseline levels of miR-34a and miR-506 were significantly elevated in PBC patients compared to controls and were significantly reduced after UDCA therapy in PBC but not in PSC. UDCA also decreased serum levels of TREM-2 and sCD163. In vitro, it suppressed the LPS-induced expression of miR-34a and ADAM17 while enhancing TREM-2 expression. Single-cell RNA sequencing of liver tissue and immunofluorescence staining confirmed TREM-2 expression in cholangiocytes. These findings suggest that UDCA modulates key inflammatory pathways and miRNAs in PBC, providing mechanistic insights into its therapeutic effect

Background and Aim: Polycystic liver disease (PLD) is a hereditary disorder characterized by the progressive development and enlargement of intrahepatic biliary cysts, which can lead to significant morbidity. Liver transplantation remains the only curative treatment. Mutations in endoplasmic reticulum (ER)-related genes contribute to ER stress in cystic cholangiocytes, promoting disease progression. Given the functional interplay between ER and mitochondria, we investigated mitochondrial dynamics and metabolism in cystic cholangiocytes from both PLD patients and Pkhd1 ^mut rats to identify novel therapeutic targets. Approach and Results: Cystic cholangiocytes exhibited increased mitochondrial mass, membrane potential, and superoxide levels, along with enhanced bioenergetic capacity and ATP production compared with normal cholangiocytes. These alterations were linked to the upregulation of electron transport chain protein complexes. Metabolic reprogramming involved enhanced oxidation of glucose, glutamine, and/or fatty acids, as well as increased de novo cholesterol synthesis and accumulation. Chronic treatment with pravastatin, a cholesterol synthesis inhibitor, significantly reduced hepatomegaly, cyst volume, and fibrosis in Pkhd1 ^mut rats. It also normalized mitochondrial hyperactivity and reduced the proliferation of cystic cholangiocytes in culture, effects that were similarly observed with other statins such as atorvastatin and simvastatin. Importantly, a case-control study in PLD patients showed that statin use was associated with reduced liver growth, further supporting its potential therapeutic role. Conclusions: Mitochondrial and metabolic dysregulation are central to the pathogenesis of PLD. Targeting cholesterol metabolism with statins represents a promising therapeutic strategy to slow disease progression and reduce cyst burden.