Abdelrahman, Amro M

Abstract Background Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy with marked molecular heterogeneity, limited systemic treatment options, and poor prognosis. Chemoresistance and KRAS mutations remain major therapeutic challenges. Dysregulation of the Hippo pathway, particularly YAP/TAZ hyperactivation, drives CCA progression and resistance. TEAD, the essential transcriptional partner of YAP/TAZ, represents a promising target. IAG933 is a novel oral pan-TEAD inhibitor designed to disrupt YAP/TAZ–TEAD interactions. RMC-6236, a pan-KRAS inhibitor, has also gained attention. Combining TEAD and KRAS inhibition may overcome resistance and improve efficacy in CCA. Methods We evaluated IAG933 in human (HuCCT1, RBE) and murine (SB1, FAC) CCA cell lines. Cells were treated with IAG933 alone or in combination with GemCis or RMC-6236. Cell viability assays were used to determine IC50 values and calculate combination index (CI). TEAD activity was assessed by luciferase reporter and qRT-PCR of YAP/TAZ target genes (CTGF, CYR61, NUAK2). In vivo efficacy was evaluated in syngeneic orthotopic and patient-derived xenograft (PDX) models. Results IAG933 suppressed TEAD-dependent transcription (IC50≈500 nM) and downregulated YAP/TAZ target genes. Combination with GemCis or RMC-6236 produced synergistic effects (CI < 1) in all CCA lines. In vivo, IAG933 monotherapy suppressed tumor growth similarly to GemCis. Dual TEAD and KRAS inhibition achieved marked tumor regression and significantly delayed tumor regrowth after treatment cessation in KRAS-mutant PDX models. Conclusion IAG933 effectively suppresses YAP/TAZ–TEAD signaling and inhibits CCA growth. While the combination with GemCis showed limited additional benefit due to strong single-agent efficacy, synergy with RMC-6236 produced durable antitumor effects, particularly in run-out studies, delaying regrowth in KRAS-mutant models. These findings highlight IAG933 as a promising therapeutic candidate targeting the Hippo–TEAD pathway, with potential benefit in KRAS-mutant CCA.

Abstract Background Cholangiocarcinoma (CCA) is a deadly cancer of the hepatic bile duct epithelial cells. Loss-of-function mutations of epigenetic regulator ubiquitin C-terminal hydrolase deubiquitinase BRCA1associated protein 1 (BAP1) occur in 26-32% of human CCA and are associated with worse prognosis and resistance to cytotoxic therapy. The goal of this study is to identify how BAP1 loss promotes cell death evasion through metabolic rewiring. Materials and methods Isogenic wild-type and BAP1-deficient human and murine CCA cell lines were generated with shRNA/siRNA or CRISPR/Cas9 and were used to assess ferroptosis sensitivity by treatment with ferroptosis inducers. In vitro, ferroptosis was assessed by phospholipid peroxidation (C-11BODIPY), mitochondrial phospholipid peroxidation (MitoPeDPP, MitoCLox), iron overload (MitoFerro green) and cell death (CellTiter-Glo). Phospholipidomics and proteomics were performed in isogenic cells by LC-MS/MS. Syngeneic liver orthotopic CCA murine models and patient-derived xenografts (PDX) were used for in vivo pre-clinical studies. Results Our findings indicate that BAP1 loss confers CCA ferroptosis resistance. BAP1 loss protects CCA against mitochondrial iron overload, ROS accumulation, and phospholipid peroxidation. BAP1 loss protects from mitochondrial phospholipid peroxidation and cardiolipin oxidation. Unbiased phospholipidomic profiling revealed that BAP1 loss remodels membrane phospholipid composition, enriching for ferroptosis blocking monounsaturated phospholipids (MUFA-PLs) at the expense of ferroptosis-inducing polyunsaturated (PUFA-PLs). We found that BAP1 loss upregulates TLCD1, a phosphatidylethanolamine acyltransferase that incorporates MUFAs into membrane phospholipids, thereby stabilizing membranes against peroxidation. TLCD1 knockdown in ferroptosis-resistant BAP1-deficient CCA cells, restored ferroptosis sensitivity. Notably, TLCD1 is significantly upregulated in human BAP1-mutant CCA as compared to BAP1-expressing tumors. While not a mitochondrial protein, TLCD1 localizes in the ER and by the mitochondrial outer membrane, as demonstrated by our immunofluorescence analysis in human CCA cells. From a therapeutic perspective, we developed EpCAM (adhesion molecule highly expressed in CCA cells)-aptamer coated lactosomes packaged with siRNA against glutathione peroxidase 4 (GPX4), as a proof-of-concept strategy to safely induce ferroptosis selectively in CCA in vitro and in vivo. Administration of siGPX4-lactosomes in pre-clinical syngeneic liver orthotopic CCA murine models demonstrated a favorable safety profile, achieved robust intratumoral GPX4 knockdown (>50%), exhibited optimal biodistribution, induced significant tumor lipid peroxidation, and resulted in reduced tumor burden. Lastly, ferroptosis inducer JKE-1674, combined with gemcitabine/cisplatin (Gem/Cis) significantly blocked tumor growth of Gem/Cis-resistant patient-derived xenografts. Conclusions Our findings reveal that BAP1 loss reprograms mitochondrial phospholipid metabolism thus conferring CCA resistance to ferroptosis, identifying TLCD1 as a targetable metabolic vulnerability in BAP1-mutant CCA. Additionally, our findings support ferroptosis inducing compounds as promising therapeutic approaches for CCA.