Abstract 4259: Peroxiredoxin 3 is a key regulator of mesothelioma progression and a target for therapeutic intervention

Abstract Tumor cells generate increased levels of reactive oxygen species (ROS) that promote tumor cell growth. To survive under elevated ROS levels, tumor cells must increase antioxidant expression, activity, and reliance on ROS scavenging pathways, including the mitochondrial matrix hydrogen peroxide (H2O2) scavenging enzyme peroxiredoxin 3 (PRX3). PRX3 expression is elevated in many cancers, including mesothelioma, and is associated with increased cell proliferation, resistance to apoptosis and chemotherapy. PRX3 is a novel molecular target of the first-in-human covalent inhibitor RSO-021. Recent phase 1 data demonstrated a favorable RSO-021 safety profile with early efficacy in patients with pleural mesothelioma, warranting transition to the ongoing phase 2 portion of the MITOPE trial (NCT05278975). RSO-021 inactivates PRX3 by covalently crosslinking active site cysteines, leading to increased levels of mitochondrial ROS, resulting in tumor cell death. In contrast normal cells remain unaffected by the same treatment. Tumor cells are vulnerable to pharmacological PRX3 inhibition; however, the specific role of PRX3 in tumor biology, particularly in mesothelioma, remains unclear. To investigate this, two PRX3 knockout (PRX3 KO) human mesothelioma cell lines were generated using CRISPR/Cas9 and compared to a mock CRISPR/Cas9 control cell line. In vitro characterization of the PRX3 KO clones showed that proliferation was significantly reduced, suggesting that PRX3 plays a critical role in promoting tumor cell growth. Assays using HyPer7, a genetically encoded H2O2-responsive fluorescent biosensor targeted specifically to the mitochondrial matrix, revealed a significant increase in HyPer7 oxidation in the mitochondrial matrix of PRX3 KO cells compared to control cells. Additionally, measurement of mitochondrial respiration by the Seahorse XF96 Cell Mito Stress Test showed PRX3 KO cells have a decrease in basal and maximal oxygen consumption further highlighting the role PRX3 plays in mitochondrial function. When injected into SCID mice PRX3 KO cells did not develop tumors, while the control mice presented with significant tumor burden over a four-week period. RNA-sequencing of PRX3 KO cells showed reduced expression of epithelial-to-mesenchymal transition (EMT) associated genes and TGFB signaling genes compared to controls. This correlates with RNA-sequencing run on 22 patient-derived primary mesothelioma explants following treatment with RSO-021. Results showed that the most sensitive explants had a reduction in EMT gene expression, a key signature associated with tumor cell migration and metastasis. Our results show that PRX3 supports mesothelioma tumor progression and survival through regulating redox metabolism and signaling while maintaining gene expression signatures supporting tumor aggressiveness, highlighting PRX3 as an important therapeutic target in oncology. Citation Format: Victoria Gibson, Jan Rogel, Joanna Dzialo, Terri Messier, Aida Habibovic, Charlotte Poile, Apostolos Nakas, Kudzayi Kutywayo, Aleksandra Bzura, George Naumov, Dean Fennell, Brian Cunniff. Peroxiredoxin 3 is a key regulator of mesothelioma progression and a target for therapeutic intervention [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 4259.