Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is a major global health burden, ranking sixth in incidence and third in cancer-related mortality. Despite therapeutic advances, treatment options for advanced liver disease and HCC are limited and strategies to prevent HCC development are lacking. To address the urgent need for chemopreventive strategies, we identified Aripiprazole, an oral atypical antipsychotic, as a candidate for HCC chemoprevention. Transcriptomic analyses of clinical liver tissues showed an association of Aripiprazole target gene expression with fibrotic liver diseases severity. In a rat model of MASH-induced HCC induced by choline-deficient L-amino acid-defined and high-fat diet, Aripiprazole prevented liver disease progression toward HCC development by modulating fibrogenesis, inflammation, and immunity-related pathways. Moreover, Aripiprazole exhibits an antifibrotic effect and reverses the high-risk status of the prognosis liver signature (PLS), a signature associated with disease progression and HCC risk in patients, in multiple human liver cell-based models. Mechanistic studies demonstrated that Aripiprazole exerts antifibrogenic, and anti-proliferative effects via suppression of TGF-β, NF-kB, cMet-ERK, and PI3K/AKT signaling in liver epithelial cells and myofibroblasts. Finaly, perturbation studies in patient-derived cell lines and patient-derived tumorspheroid system showed that Aripiprazole has a direct anticancer effect, by modulating the tumor microenvironment and suppressing tumor cell survival and invasion. Collectively, these findings suggest that treatment with aripiprazole is a clinically relevant approach for HCC chemoprevention. Citation Format: Nevena Slović, Sumit Mishra, Subhojit Paul, Marine A. Oudot, Frank Jühling, Hiroaki Kanzaki, Julien Moehlin, Margaux Denos, Anouk Charlot, Sarah C. Durand, Courtney Katz, Cloé Gadenne, Emanuele Felli, Joachim Lupberger, Emilie Crouchet, Yujin Hoshida, Thomas F. Baumert. Chemoprevention of hepatocellular carcinoma by next-generation antipsychotic aripiprazole [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 943.

Background and Aims: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is a major global health burden, ranking sixth in incidence and third in cancer-related mortality. Despite therapeutic advances, treatment options for advanced liver disease and HCC are limited, and strategies to prevent HCC development are lacking. To address the urgent need for preventive strategies, we identified aripiprazole, an oral atypical antipsychotic, as a candidate for HCC chemoprevention. Approach and Results: Analyses of clinical liver tissues showed that aripiprazole targets are expressed in different liver cell compartments, including fibroblasts, macrophages, and epithelial cancer cells, and that target gene expression is associated with fibrotic liver diseases and HCC. In a rat model of MASH-induced HCC induced by choline-deficient L-amino acid-defined high-fat diet, aripiprazole prevents liver disease progression and HCC development by modulating fibrogenesis-related pathways and inflammation. Mechanistically, aripiprazole exerts antifibrogenic and anti-inflammatory effects by modulating the phenotype of liver fibroblasts and macrophages. Moreover, perturbation studies in cancer cell models showed that aripiprazole prevents tumor initiation and reduces cell proliferation via inhibition of the cMET and ERK pathways and perturbation of mitochondrial functions. Finally, treatment of patient-derived tumor spheroids demonstrated that aripiprazole modulates immune responses in the tumor microenvironment. Conclusions: Collectively, these findings suggest that treatment with aripiprazole is a clinically relevant approach for HCC chemoprevention.

Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system that has recently risen in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein expressed in tight junctions and exposed on the cell surface in liver fibrosis and cancer. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of tissues of patients with CCA, we show that CLDN1 expression is up-regulated in cancer cells and is associated with stemness and cell fate. Genetic gain-of-function studies in CCA orthotopic in vivo mouse models showed decreased survival and enhanced tumor growth, unraveling a functional role of CLDN1 as an oncogenic driver. Targeting exposed nonjunctional CLDN1 using highly specific CLDN1 monoclonal antibodies (mAbs) inhibited tumor growth across CCA CDX and PDX mouse models and patient-derived CCA organoids, including tumors with medium or low CLDN1 expression. Moreover, antibody treatment inhibited tumor cell migration, invasion, and extrahepatic metastasis. Mechanistically, targeting exposed cell surface CLDN1 on CCA tumors using mAbs inhibited Notch1 and TROP2/STAT3 signaling pathways, resulting in decreased cancer cell stemness and epithelial-to-mesenchymal transition. Loss-of-function studies using CRISPR-Cas9 and RNAi combined with rescue and confocal imaging studies confirmed the functional and mechanistic role of these pathways. In conclusion, these results uncover CLDN1 as a previously undiscovered CCA driver and therapeutic target, paving the way for the clinical development of CLDN1 mAbs to improve the dismal outcome of patients with advanced CCA.

Abstract Malignant cutaneous melanoma is a highly aggressive cancer responsible for the majority of skin cancer-related deaths with increasing prevalence world-wide. While early-stage melanoma can be efficiently treated by surgery, the treatment of advanced melanoma with immune checkpoint (ICT) therapy remains unsatisfactory with a large number of patients displaying intrinsic resistance or developing resistance over time. A key therapeutic challenge is tumour heterogeneity with melanoma cells existing in multiple states with differing proliferative and invasive capacities. The transmembrane protein Claudin-1 (CLDN1) exhibits a dual role as tight junction protein regulating epithelial barrier function and as a regulator of epithelial-to-mesenchymal transition (EMT), a hallmark of fibrotic disease and cancer. Analyses of public TCGA and TIDE patient datasets showed that CLDN1 is preferentially expressed in mesenchymal melanoma cells and correlates with poor survival of metastatic melanoma and of ICT treated patients. In accordance, CLDN1 expression is associated with an immune-depleted tumour microenvironment (TME). Using gain and loss-of-function studies in human melanoma spheroids and xenografts in syngeneic mouse models, we define two critical roles for CLDN1 in melanoma, a requirement for growth of mesenchymal melanoma cells and the ability to reprogram a TME depleted in cytotoxic CD8 T cells that promotes tumour growth. CLDN1 antibody targeting further restored an immune-infiltrated TME to inhibit tumour growth and cooperation with anti-PD-1 ICT. Collectively, our data show that CLDN1 plays an important functional role melanoma and is a candidate target for antibody-based therapies. Citation Format: Antonin Lallement, Frank Jühling, Zeina Nehme, Bujamin Vokshi, Guillaume Davidson, Emilie Crouchet, Thomas F. Baumert, Irwin Davidson. Claudin-1 modulates the melanoma immune microenvironment to promote tumor growth and is a candidate therapeutic target for advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB085.

DNA methylation is a critical epigenetic mark across numerous species, and identifying differentially methylated regions (DMRs) is essential for understanding genome regulation. Most existing DMR detection methods require predefined sample conditions, limiting the discovery of new epigenetic patterns, especially when group identities are unknown or uncertain, as is common in clinical settings. Additionally, only a very few approaches enable comparisons across multiple conditions. To address this significant gap, we present metilene3, a method for rapid, multi-condition DMR detection that operates in both supervised and unsupervised modes, using user-provided labels or autonomously clustering unlabeled samples. By segmenting the genome based on multiple pairwise methylation difference signals, metilene3 enables sample classification and DMR-anchored inference of epigenetic relationships. Using simulated and diverse human datasets, we show that metilene3 accurately detects DMRs, robustly clusters samples, and holds the potential to reveal new regulatory elements and sample stratifications. Specifically, in a pancreatic tissue dataset, metilene3 identifies DMRs enriched for key transcription factors involved in pancreatic cancer development, hinting towards an altered NFKB-NFAT regulatory program. Together, metilene3 provides a fast, interpretable framework for exploring heterogeneous methylomes and discovering epigenetic patterns across complex biological and clinical datasets.

Claudin-1 (CLDN1), a tight junction protein overexpressed and mis-localized in colorectal cancer (CRC), plays a critical role in tumor progression, stemness, and therapy resistance. Integrative analyses of bulk and single-cell transcriptomic datasets revealed that CLDN1 is enriched in stem-like CRC cells, increases during metastatic progression, and is associated with microsatellite stable disease. High CLDN1 expression correlates with epithelial-to-mesenchymal transition and activation of oncogenic pathways including AKT/mTOR, Myc, and NF-κB. Here, we evaluate the therapeutic efficacy of an investigational humanized monoclonal antibody (H3L3) directed against overexpressed non-junctional CLDN1 in preclinical models of CRC. In xenograft mouse models and patient-derived organoids (PDOs), CLDN1 mAb significantly reduced tumor growth in CLDN1-expressing tumors. Mechanistically, CLDN1 mAb disrupted CLDN1-mediated signaling, notably inhibiting the AKT/mTOR pathway. CRISPR-mediated CLDN1 knockout abolished H3L3 efficacy, confirming target specificity. Transcriptomic analysis of PDOs treated with H3L3 revealed broad suppression of stemness, oncogenic signaling, and hallmark cancer pathways. Single-cell analysis further demonstrated that targeting CLDN1 modulates cellular plasticity, driving stem-like tumor cells toward a more differentiated epithelial phenotype. Together, these findings establish anti-CLDN1 monoclonal antibodies as a promising therapeutic approach for CRC. The selective binding of CLDN1 mAb to tumor-associated exposed and overexpressed CLDN1, along with its capacity to inhibit key oncogenic pathways and reprogram cancer cell states including tumors with different mutations conferring resistance to standard of care, underscores its potential to improve treatment outcomes in patients with CLDN1-expressing CRC.

HCC is the third most common cause of cancer-related death and the leading cause of death among cirrhotic patients. While several treatments for HCC have been approved, chemopreventive strategies for HCC are lacking. Aiming to discover novel therapeutic targets, we combined genome wide transcriptomic analysis of liver tissues from patients with advanced liver disease and HCC and a cell-based system predicting liver disease progression and HCC risk. Computational analysis identified peroxiredoxin 2 (PRDX2) as a candidate driver of hepatocarcinogenesis. The role of PRDX2 in liver disease progression and HCC development was investigated by Prdx2 KO in a state-of-art CRIPSR/Cas9 mouse model for MASH-induced hepatocarcinogenesis. The mechanism of action was explored using RNA-Seq analyses of mouse liver tissues and validated by perturbation studies in human liver cell-based models. The translatability of our results was confirmed by targeting Prdx2 using GalNac siRNA in a mouse model of MASH, CDX mouse model, as well as perturbation studies in patient-derived HCC tumorspheroids. In vivo perturbation studies in mouse models for MASH driven hepatocarcinogenesis showed that Prdx2 KO improves steatosis and robuslty prevents HCC development. RNA-Seq analyses of mouse liver tissues with validation in human cells showed that loss of Prdx2 function suppresses oncogenic signaling (i. e. p38/MAPK, PI3K/AKT signaling) and improves metabolic liver functions (i.e cholesterol, fatty acid and bile acid metabolism) through activation of the AMPK pathways. Finally, PRDX2 loss-of-function studies in hepatoma cell-based models, a CDX mouse model and patient tumorspheroids unraveled that targeting PRDX2 sensitizes cancer cell to oxidative stress and triggers apoptosis. Our findings demonstrate an important functional role of PRDX2 in hepatocarcinogenesis. Targeting PRDX2 is a previously undiscovered therapeutic opportunity for prevention of MASH-induced HCC. Emilie Crouchet, Eugénie Schaeffer, Frank Jühling, Hussein El Saghire, Anouk Charlot, Naoto Fujiwara, Shija Zhu, Fahmida Akter Rasha, Julien Moehlin, Marine A. Oudot, Clara Ponsolles, Romain Martin, Nicolas Brignon, Sarah C. Durand, Marie Parnot, Nourdine Hamdane, Danijela Heide, Jenny Hetzer, Mathias Heikenwälder, Emanuele Felli, Patrick Pessaux, Joffrey Zoll, Nathalie Pochet, Laurent Mailly, Yujin Hoshida, Thomas F. Baumert, Catherine Schuster. Targeting peroxiredoxin 2 prevents hepatocellular carcinoma development in metabolic liver disease [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 2369.

KRAS-dependent acinar-to-ductal metaplasia (ADM) is a fundamental step in the development of pancreatic ductal adenocarcinoma (PDAC), but the involvement of cell death pathways remains unclear. Here, we show that key regulators of programmed cell death (PCD) become upregulated during KRAS-driven ADM, thereby priming transdifferentiated cells to death. Using transgenic mice and primary cell and organoid cultures, we show that transforming growth factor (TGF)-β-activated kinase 1 (TAK1), a kinase regulating cell survival and inflammatory pathways, prevents the elimination of transdifferentiated cells through receptor-interacting protein kinase 1 (RIPK1)-mediated apoptosis and necroptosis, enabling PDAC development. Accordingly, pharmacological inhibition of TAK1 induces PCD in patient-derived PDAC organoids. Importantly, cell death induction via TAK1 inhibition does not appear to elicit an overt injury-associated inflammatory response. Collectively, these findings suggest that TAK1 supports cellular plasticity by suppressing spontaneous PCD activation during ADM, representing a promising pharmacological target for the prevention and treatment of PDAC.

Resistance to immunotherapy is a major challenge in the treatment of solid tumors. Cholangiocarcinoma (CCA) exemplifies this challenge by poor outcome and very limited response to approved checkpoint inhibitors (CPI). Claudin-1 (CLDN1) is a transmembrane protein overexpressed in epithelial cancer cells and shown to be a mediator of carcinogenesis, invasion and metastasis. However, the functional role of CLDN1 for the tumor microenvironment (TME) is largely unknown. Using CCA as a model disease, we aimed to study the impact of CLDN1 for the TME and its potential role as a therapeutic target to address CPI resistance. CLDN1 expression and its association with TME was analyzed by spatial transcriptomics in CCA patient tissues. Immunocompetent orthotopic animal models of intrahepatic CCA were generated by hydrodynamic tail vein injection (HDTV) of proto-YAP/AKT or the KRAS/p19 oncogenes together with a CLDN1 expressing plasmid. Following tumor development within the liver, mice were treated with monoclonal antibodies (mAbs) targeting CLDN1 and/or PD-1. Spatial analyses of patient tissues revealed that CLDN1 was overexpressed in cancer cells and CLDN1 expression was associated with oncogenic signaling and reduced T cell infiltration. In orthotopic mouse models for CCA, CLDN1 gain-of-function studies showed enhanced tumor growth and decreased survival. Treatment with CLDN1 mAbs markedly and significantly reduced tumor burden and intratumoral fibrosis and resulted in increased intratumoral CD3+ and CD8+ T cell infiltration as well as modulation of peritumoral macrophage distribution. Gene expression analyses revealed down-regulation of pathways for fibrogenesis, carcinogenesis and up-regulation of pathways for T cell activation and proliferation. Whereas PD1 mAb single agent therapy did not show significant efficacy, the combination of PD-1 with CLDN1 mAbs markedly and significantly improved overall survival. CLDN1 plays a functional role in the TME by modulating tumor fibrosis and immune cell infiltration. Treatment with CLDN1 mAbs provides an opportunity to overcome resistance and improve the efficacy of CPIs by enhancing tumoral immune responses. Romain Desert, Zeina Nehme, Bocar Kane, Emilie Crouchet, Frank Jühling, Vikas Ranvir, Alberto Toso, Mathias Heikenwälder, Catherine Schuster, Thomas Baumert. Reversing resistance to checkpoint inhibitors by treatment with claudin-1 specific antibodies in an orthotopic in vivo model for cholangiocarcinoma [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 7295.

Treatment options for advanced liver disease and hepatocellular carcinoma (HCC) are limited, and strategies to prevent HCC development are lacking. Aiming to discover therapeutic targets, we combined genome-wide transcriptomic analysis of liver tissues from patients with advanced liver disease and HCC and a cell-based system predicting liver disease progression and HCC risk. Computational analysis predicted peroxiredoxin 2 (PRDX2) as a candidate gene mediating hepatocarcinogenesis and HCC risk. Analysis of tissues from patients with HCC confirmed a perturbed expression of PRDX2 in cancer. In vivo perturbation studies in mouse models for hepatocarcinogenesis driven by metabolic dysfunction-associated steatohepatitis showed that specific Prdx2 KO in hepatocytes improved metabolic liver functions, restored AMPK activity, and prevented HCC development by suppressing oncogenic signaling. Perturbation studies in HCC cell lines, a cell line-derived xenograft mouse model, and patient-derived HCC spheroids revealed that PRDX2 also mediates cancer initiation, cancer cell proliferation, and survival through its antioxidant activity. Targeting PRDX2 may therefore be a strategy to prevent HCC development in metabolic liver disease.

BACKGROUND & AIMS:Primary sclerosing cholangitis (PSC) is a cholangiopathy associated with a high risk of progression to end-stage liver disease and hepatobiliary cancer. Its pathogenesis remains poorly understood, and current clinical management offers limited therapeutic options, primarily liver transplantation. Claudin-1 (CLDN1), a transmembrane protein highly expressed in liver epithelial cells, plays a critical role in cell-cell communication and signaling. We aimed to investigate the functional role of CLDN1 as both a mediator and potential therapeutic target for PSC using patient cohorts alongside murine and patient-derived intervention models. METHODS:CLDN1 expression patterns and associated cellular phenotypes were analyzed in liver tissues from five PSC patient cohorts using single-cell RNA sequencing, spatial transcriptomics, and multiplex proteomics. Proof-of-concept studies employing CLDN1-specific monoclonal antibodies (mAbs) and genetic loss-of-function approaches were performed in state-of-the-art mouse models of PSC and cholangiopathies. Perturbation studies in human cell-based models were conducted to explore underlying mechanisms. RESULTS:In liver tissues from patients with PSC, CLDN1 expression was markedly upregulated and correlated with disease progression. Spatial transcriptomics and proteomics revealed elevated CLDN1 expression in diseased cholangiocytes and cholestatic periportal hepatocytes, accompanied by activation of pro-inflammatory and pro-fibrotic signaling pathways. Therapeutic administration of CLDN1-specific mAbs or genetic knockout improved liver function in PSC mouse models by reducing hepatobiliary fibrosis and cholestasis. Mechanistic studies indicated that mAb treatment inhibited pro-inflammatory and pro-fibrotic signaling in cholangiocytes and hepatocytes perturbed in PSC liver tissues. CONCLUSIONS:These findings demonstrate a functional role for CLDN1 in the pathogenesis of PSC and biliary fibrosis. In vivo proof-of-concept studies, combined with expression analyses in patients with PSC, support the clinical development of CLDN1-specific mAbs as a therapeutic strategy for PSC. IMPACT AND IMPLICATIONS:Primary sclerosing cholangitis (PSC) is a chronic fibrosing cholangiopathy with limited therapeutic options. We identified the cell surface protein Claudin-1 as a key mediator and potential therapeutic target for PSC. In patients, Claudin-1 expression correlates with disease stage and clinical outcomes. Conditional liver epithelial-specific Claudin-1 knockout in mice reduced liver injury, fibrosis, and cholestasis. Monoclonal antibodies targeting Claudin-1 inhibited fibrosis, cholestasis, and the ductular reaction across advanced PSC mouse models by suppressing pro-inflammatory and fibrogenic signaling. These preclinical findings support the clinical development of Claudin-1-specific antibodies for PSC treatment and have significant implications for physicians, researchers, and drug developers in the field of biliary disease.

Objective Impaired hepatic expression of protein tyrosine phosphatase delta (PTPRD) is associated with increased STAT3 transcriptional activity and reduced survival from hepatocellular carcinoma in patients with chronic hepatitis C virus infection. However, the PTPRD-expressing hepatic cell types, signalling pathways responsive to PTPRD and their role in non-viral liver disease are largely unknown.Methods We studied PTPRD expression in single-cell and bulk liver transcriptomic data from mice and humans, and established a Ptprd-deficient mouse model for metabolic dysfunction-associated steatohepatitis (MASH). Identified pathways were validated by perturbation studies in human hepatocytes and PTPRD substrates by pull-down assays. The clinical relevance was further explored in a cohort with metabolic disease by ranking patients according to PTPRD expression and analysing its association with metabolic disease markers.Results The analysis of individuals ranked according to PTPRD expression and Ptprd-deficient mice, showed that PTPRD levels were associated with hepatic glucose/lipid signalling and peroxisome function. Hepatic PTPRD expression is impaired in aetiologies of chronic liver diseases that are associated with metabolic disease. We further validated PTPRD as a STAT3 phosphatase in the liver, acting as a regulator of peroxisomal fatty acid metabolism. During MASH, low PTPRD led to increased liver steatosis in Ptprd+/− mice and a pronounced unfolded protein response, which impacts insulin signalling. Accordingly, silencing of PTPRD blunted insulin-induced AKT phosphorylation. Patients with obesity and low hepatic PTPRD expression exhibit increased levels of metabolic risk factors.Conclusion Our data revealed an important regulatory role of the hepatic PTPRD-STAT3 axis in maintaining glucose/lipid homeostasis, which is recapitulated in clinical manifestations of metabolic liver disease.