Abstract B026: tmCLIC1 as a Key to Metformin’s Action: Connecting Molecular Insights to Glioblastoma Therapy

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor sustained by glioblastoma stem cells (GSCs), whose metabolic plasticity contributes to tumor progression and therapeutic resistance. Targeting tumor-specific membrane proteins that support this plasticity represents a promising strategy. Chloride Intracellular Channel 1 (CLIC1), particularly its transmembrane form (tmCLIC1), is selectively enriched on GSC membranes and largely absent in healthy cells, making it an attractive therapeutic target. Notably, tmCLIC1 is inhibited by the antidiabetic drug metformin, although the molecular basis of this interaction and its clinical relevance remain unclear due to the high drug concentrations required. Here, we investigated the structural and functional determinants of metformin binding to tmCLIC1 and explored strategies to enhance its therapeutic potential in GBM. Molecular dynamics simulations identified arginine 29 (R29) as a critical residue stabilizing a negatively charged binding pocket involving aspartate 76 (D76) and glutamate 81 (E81). Electrophysiological analyses in GSCs expressing CLIC1 mutants demonstrated that metformin fully inhibits wild-type tmCLIC1 activity, whereas R29 mutations abolish drug sensitivity, and D76A or E81A mutations partially reduce it. An R29M substitution confirmed that the positive charge at R29 is essential for metformin binding. Structural studies are underway, with crystals of the CLIC1–metformin complex and the R29A mutant successfully obtained for X-ray analysis. Functional assays revealed that metformin binding induces tmCLIC1 internalization and promotes intracellular and mitochondrial accumulation of the drug, as demonstrated by immunofluorescence and NanoSIMS imaging. Short-term exposure to metformin resulted in sustained reductions in GSC proliferation and mitochondrial activity, while prolonged low-dose treatment effectively inhibited tmCLIC1 currents and cell proliferation, recapitulating the effects of acute high-dose exposure. Together, these findings define R29 as a key molecular determinant of metformin–tmCLIC1 interaction and reveal a mechanism by which tmCLIC1 mediates metformin uptake and antitumor activity in GSCs. Importantly, they support continuous low-dose metformin administration as a feasible therapeutic strategy, opening new avenues for tmCLIC1-targeted, low-toxicity treatments for glioblastoma. Citation Format: Guido Rey, Francesca Cianci, Francesco Emanuele. Caridi, Sara Torabi, Michele Mazzanti. tmCLIC1 as a Key to Metformin’s Action: Connecting Molecular Insights to Glioblastoma Therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B026.