Targeting Ferroptosis in Cholangiocarcinoma with siRNA-based Nanovesicles - PROJECT SUMMARY ABSTRACT Cholangiocarcinoma (CCA) is a highly lethal malignancy of the liver and biliary tract, with incidence and mortality rates steadily increasing worldwide. Despite advancements in cytotoxic therapy, the 5-year survival rate remains below 10% due to high rates of tumor recurrence, drug resistance, and toxicity. This highlights an incomplete understanding of the mechanisms regulating CCA cell death. Ferroptosis, an iron-dependent and regulated mode of cell death driven by phospholipid peroxidation, has emerged as a promising alternative for inducing cell death in tumors resistant to conventional therapies. However, the therapeutic benefits of ferroptosis in CCA are poorly defined, and its induction in vivo faces challenges like poor selectivity, off-target effects, and acquired ferroptosis resistance. Resistance to cell death often results from inactivating mutations in tumor suppressors, such as BRCA1-associated protein 1 (BAP1), which is mutated in up to 25% of CCAs and associated with ferroptosis resistance in renal cell carcinoma. This proposal aims to elucidate the therapeutic benefits of ferroptosis in both sensitive and resistant CCA cells in vitro and in vivo. Preliminary data show that BAP1 inactivation or downregulation confers resistance to ferroptosis in CCA cells, while ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibitors in mice bearing BAP1 wild-type CCA reduces tumor growth. Additionally, EpCAM- aptamer coated nanoparticles packaged with siRNA against GPX4 efficiently target CCA cells, demonstrating tumor enrichment and good tolerability. Our central hypothesis is that ferroptosis provides a novel therapeutic opportunity in both BAP1 wild-type and BAP1-mutant CCA cells, maximizing cytotoxic effects. To investigate this hypothesis, we have outlined two specific aims: 1) evaluate the therapeutic efficacy of EpCAM-aptamer coated nanoparticles with siRNA against GPX4 in inducing ferroptosis in BAP1 wild-type CCA; 2) identify genes mediating ferroptosis resistance in BAP1-mutant CCA cells using a genome-wide CRISPR/Cas9 loss-of-function screen. The proposed research will uncover new targets underlying ferroptosis resistance in BAP1-mutant CCA, revealing metabolic vulnerabilities for future therapeutic interventions. Our approach includes EpCAM-aptamer coated nanovesicles for selective targeting, CRISPR knockout libraries, and sophisticated cellular and animal models to examine ferroptosis and tumor characterization.
