Abstract 6919: Targeting CLIC3 as a novel strategy to inhibit metastasis in breast cancer

Abstract Women with metastatic breast cancer have a 5-year survival rate of only 28%. This study aims to investigate the role of CLIC3 (chloride intracellular channel 3) in breast cancer metastasis and explore its potential as a therapeutic target. CLIC3, secreted by cancer cells and cancer-associated fibroblasts, promotes tumor invasion and angiogenesis through its oxidoreductase activity that inhibits TGM2 (transglutaminase-2). High CLIC3 levels correlate with poor prognosis across all breast cancer subtypes. CLIC3 was knocked down in metastatic breast cancer cells, which were evaluated for cell migration and invasion using Boyden chambers, non-adherent growth in soft agar, and tumor formation and metastasis in mice. Small molecule inhibitors of CLIC3 activity were identified using an in silico screen of >10 million commercially available small molecules and 94 compounds were tested as possible CLIC3 inhibitors (CLIC3i) using recombinant CLIC3. We synthesized over 70 analogs of the top inhibitor for structure-activity relationship analysis, with the most promising inhibitors also assessed for CLIC3 binding using thermal shift assays and migration in scratch assays. CLIC3 knockdown significantly reduced cell migration, invasion, and growth in soft agar, effects rescued by shRNA-insensitive CLIC3. In vivo, CLIC3 knockdown decreased tumor growth and completely blocked lung metastasis, underscoring its role in the colonization and growth of macroscopic metastases. Four promising compounds with mid-μM IC50 values for CLIC3 activity inhibition were identified (E5, D1, A5, D5). Among them, HA-22-31 emerged as a lead compound (IC50 = 1.7 μM), demonstrating direct binding to CLIC3 and significant inhibition of migration. In conclusion, CLIC3 enhances metastasis in breast cancer through its enzymatic activity. These findings suggest that further development of small molecule CLIC3 inhibitors may represent a promising new strategy for targeting metastatic breast cancer. Citation Format: Paul Mellor, Stephanie Kendall, Shari Smith, Jonas Olsen, Henok Sahile, Brent Page, Anurag Saxena, Deborah Anderson. Targeting CLIC3 as a novel strategy to inhibit metastasis in breast cancer [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 6919.