Abstract 3108: Imipridones ONC201, ONC206, and ONC212 promote immune-mediated cell death and anti-tumor activity in biliary tract cancer models <i>in vitro</i>

Abstract Background: Biliary tract cancers (BTCs) are an aggressive group of malignancies with 5-year survival rates of less than 2% in those diagnosed with metastatic disease. Chemoimmunotherapeutic approaches have limited efficacy highlighting an urgent need for novel approaches. Imipridones (Dordaviprone/ONC201, ONC206, and ONC212) have demonstrated promising antitumor activity in multiple preclinical cancer subtype models but remain underexplored in BTCs. ClpP agonist and DRD2/3 antagonist ONC201 exerts antitumor effects by upregulating TRAIL, activating the Integrated Stress Response (ISR), inhibiting AKT/ERK signaling, disrupting oxidative phosphorylation, and altering cancer cell metabolism. Second-generation imipridones (ONC206, ONC212) show enhanced potency relative to ONC201. To further investigate their therapeutic potential, we employed co-culture systems with natural killer (NK) cells to evaluate the immunomodulatory effects of these compounds. Additionally, the roles of ferroptosis, necroptosis, and autophagy modulation in sensitizing BTC cells to apoptosis and immune-mediated destruction were explored. Methods: Two BTC cell lines, RBE and HuCCT1, were tested for drug sensitivity and synergy using the CellTiter-Glo® luminescent cell viability assay. for imipridones (ONC201, ONC206, ONC212) and trametinib. Co-culture experiments involved RBE cells (blue CMAC dye) and NK-92MI cells (green CMFDA dye), treated with ONC201 (2.53 μM), ONC206 (917 nM), ONC212 (46.99 nM), and trametinib (5.94 μM). Live/dead fluorescent imaging was conducted at 0, 24, 48, and 72 hours using DAPI, FITC, and cherry red channels on the ImageXpress® system. Cell death pathways were analyzed in RBE and HuCCT1 cells treated with ONC201, ONC206, or ONC212 (100 μM) alone or combined with inhibitors (Ferrostatin-1, Z-VAD-FMK, Bafilomycin A1, Necrostatin-1s).Cell viability was measured after 72 hours with the CellTiter-Glo® Assay. Results: Imipridones and MEK inhibitors exhibit potent antineoplastic effects in BTC cell lines. In RBE cells, half maximal inhibitory concentrations (IC50) were 2.53 μM (ONC201), 917 nM (ONC206), 46.99 nM (ONC212), and 5.94 μM (trametinib), while in HuCCT1 cells, they were 1.97 μM (ONC201), 1.06 μM (ONC206), 38.95 nM (ONC212), and 3.71 μM (trametinib). Imipridones (ONC201 and ONC212) with trametinib showed synergy in HuCCT1 cells. ONC201 primarily results in cell death through apoptosis, and further analysis of alternative cell death pathways including ferroptosis, necroptosis, and autophagy is underway. RNA-seq studies are also ongoing. Imipridones enhanced NKcell-mediated tumor death. These findings underscore the therapeutic potential of imipridones in BTC, driven by immune-mediated signaling and offering promising prospects for future treatment strategies. Citation Format: Archana Saxena, Alexis J. Lannigan, Grace Sun, Jasper Chan, Jane Trainor, Lanlan Zhou, Varun V. Prabhu, Wafik S. El-Deiry, Alexander G. Raufi. Imipridones ONC201, ONC206, and ONC212 promote immune-mediated cell death and anti-tumor activity in biliary tract cancer models in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3108.