Classon, Peyton

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.

Abstract INTRODUCTION: 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 BRCA1-associated 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 & 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 oeverload (MitoFerro green) and cell death (CellTiter-Glo). Phospholipidomics and proteomics were performed in isogenic cells by LC-MS/MS. Isogenic liver orthotopic CCA murine models 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. Strikingly, 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 BAP1-deficient CCA cells, restored ferroptosis sensitivity Notably, TLCD1 is significantly upregulated in human BAP1-mutant CCA as compared to WT tumors. We identified TLCD1 in the endoplasmic reticulum, Golgi and by mitochondrial outer membrane. In syngeneic liver orthotopic CCA murine models, engineered EpCAM-aptamer-coated lactosomes carrying GPX4 siRNA selectively targeted CCA cells, inducing ferroptosis and robustly suppressing tumor growth without systemic toxicity. Lastly, BAP1-mutant patient-derived xenografts (PDXs) and organoids were resistant to ferroptosis inducers, as compared to WT controls. CONCLUSIONS: Our findings reveal that BAP1 loss rewires mitochondrial phospholipid metabolism thus conferring CCA resistance to ferroptosis, identifying TLCD1 as a targetable metabolic vulnerability in BAP1-mutant CCA. Citation Format: Yu Zhao, Peyton Classon, Danielle Marie Carlson, Jayla Millender, Irene K. Yan, Sumera Ilyas, Rory L. Smoot, Gregory J. Gores, TUSHAR PATEL, Davide Povero. BAP1 loss confers ferroptosis resistance to cholangiocarcinoma via TLCD1-mediated membrane phospholipid remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2433.

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal epithelial cell malignancy of the hepatic biliary tract. Efficacy of current standard-of-care therapy is limited. Ferroptosis, an iron-catalyzed, caspase-independent cell death driven by phospholipid peroxidation, has emerged as a novel therapeutic target. Our goal is to develop a tumor-selective therapeutic approach targeting ferroptosis in CCA using siRNA-based nanotherapeutics. Materials & Methods: Milk-derived nanovesicles (lactosomes) coated with EpCAM aptamer and packaged with siRNA against Glutathione Peroxidase 4 (siGPX4-lactosomes) and Ferroptosis Suppressor Protein 1 (siFSP1-lactosomes), were used to induce ferroptosis in murine CCA cells (FAC) and in syngeneic liver orthotopic CCA-bearing mice. In vitro, lactosome uptake and efficacy were assessed by fluorescence-based, cell-viability and molecular biology assays. In vivo, target engagement, tissue distribution, safety and efficacy were assessed. Results: EpCAM (epithelial cell adhesion molecule expressed on CCA cells)-coated lactosomes showed robust uptake by FAC cells in a EpCAM-dependent manner. EpCAM knockdown via siRNA reduced lactosome uptake, confirming aptamer selectivity. Dose-dependent siGPX4/siFSP1-lactosome treatment led to GPX4/FSP1 knockdown and induced ferroptotic cell death in up to 60% of FAC cells after 72 hours (p<0.001 vs. siNC). In CCA-bearing mice, intravenous administration of siRNA lactosomes was well-tolerated with no liver or systemic toxicity observed and demonstrated robust tumor distribution compared to non-tumorous tissues. Efficacy studies demonstrated that siGPX4/siFSP1-lactosomes significantly reduced intratumor Gpx4 and FSP1 mRNA expression by 40-60% and induced significant upregulation of ferroptosis markers Ptgs2 and Acsl4 (p<0.01 vs. siNC). Ferroptosis in siRNA-lactosome treated tumors was confirmed by immunohistochemistry for phospholipid peroxidation markers oxPAPC and 4-HNE. Additionally, proliferation marker Ki67 was significantly downregulated in siGPX4/siFSP1-lactosome treated tumors (p<0.05 vs. siNC), indicating ferroptosis-mediated tumor suppression. Importantly, siGPX4/siFSP1-lactosomes reduced tumor burden and expression of CCA markers CK19/7 in treated mice, as compared to control mice. Conclusions: siRNA-based nanotherapeutics targeting CCA can effectively and safely induce ferroptosis in vitro and in vivo, representing a potential therapeutic strategy for CCA treatment. Citation Format: Peyton Classon, Danielle Marie Carlson, Jayla Millender, Irene Yan, Sumera Ilyas, Rory L. Smoot, TUSHAR PATEL, Gregory J. Gores, Davide Povero. Targeting cholangiocarcinoma with ferroptosis-inducing siRNA-based nanotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5664.

Our study examined healthy human MASH and MASH-associated hepatocellular carcinoma (MASH-HCC) livers using bulk and scRNA sequencing, spatial transcriptomics, and immunohistochemistry. We found that ACSLs displayed differential and spatially heterogeneous expression. ACSL4 was abundant in tumor tissues, whereas ACSL5 was elevated in noncancerous MASH tissues. ACSL4 was mainly found in immune cells like natural killer cells and natural killer T cells in murine MASH-HCC, suggesting its role in tumor immune microenvironment modulation.

ABSTRACT Metabolic-associated steatohepatitis-driven hepatocellular carcinoma (MASH-HCC) incidence is rapidly rising worldwide. Lipid metabolic reprogramming is a hallmark of solid tumors to satisfy cancer high metabolic demand. However, it may confer sensitivity to ferroptosis, a cell death mode driven by iron-dependent lipid peroxidation. In this report, we describe the lipid metabolic landscape in MASH-HCC and characterize long chain acyl-CoA synthetases (ACSLs), a family of enzymes involved in synthesis of cellular lipids. Bulk RNA-sequencing, single-cell RNA-sequencing, spatial transcriptomics and immunohistochemistry analyses of human MASH-HCC were integrated to identify differentially expressed lipid metabolism genes. Ferroptosis in vitro was assessed in human HCC cell lines. A characterization of ACSLs was also conducted at the single-cell level in a diet-induced experimental murine model of MASH-HCC. Our analysis revealed that in human MASH-HCC, ACSLs exhibit a heterogeneous expression, with ACSL4 notably enriched in tumor tissues, contrasting with ACSL5 upregulation in non-cancerous MASH. We identified a unique lipid metabolic gene signature of MASH-HCC, which included genes associated with ferroptosis vulnerability. In vitro , ACSL4 upregulation was associated with increased ferroptosis sensitivity in human HCC cell lines. Lastly, single-cell RNA-sequencing revealed elevated ACSL4 expression in immune cells in a murine MASH-HCC model, suggesting a role of ACSL4 in shaping the tumor immune microenvironment. Overall, this report offers new insights into lipid metabolic landscape and ferroptosis sensitivity for novel MASH-HCC treatments. GRAPHICAL ABSTRACT

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 440.