Transmembrane CLIC1 sustains EGFRvIII–STAT3 oncogenic signaling in Glioblastoma through interaction with OSMR

Oncostatin M receptor (OSMR) plays diverse and important roles in several human malignancies, including brain, breast, and pancreatic cancer ^1-4 . Glioblastoma (GB) is the most malignant genetically diverse brain tumour, with no cure. The most common genetic mutation in GB is a truncated active mutant of epidermal growth factor receptor (EGFR), the EGFRvIII. OSMR orchestrates a feedforward signaling mechanism with EGFRvIII and the signal transducer and activator of transcription 3 (STAT3), to drive GB progression ⁴ . Beyond EGFRvIII, OSMR promotes brain tumour stem cells (BTSCs) via upregulation of mitochondrial oxidative phosphorylation and contributes to therapy resistance ⁵ . The molecular mechanisms underlying the multifaceted roles of OSMR in different contexts are largely unclear. Here, we systematically mapped the OSMR interactome using Mammalian Membrane Two-Hybrid High-Throughput Screening (MaMTH-HTS). This unbiased approach led to the identification of OSMR-specific and OSMR/EGFRvIII-specific binding proteins, revealing context-dependent OSMR functions. Among a subset of common interactors, we uncovered chloride intracellular channel 1 (CLIC1) as a critical regulator of both OSMR-STAT3 signaling and the OSMR/EGFRvIII complex in GB. CLIC1 physically associates with both OSMR and EGFRvIII and plays a key role in EGFRvIII packaging into extracellular vesicles (EVs). Genetic deletion of CLIC1 disrupts the OSMR/EGFRvIII interaction, impairs STAT3 activation, reduces EGFRvIII EV content, and slows GB progression. Using whole-cell patch-clamp recordings and a monoclonal antibody that selectively targets transmembrane CLIC1 (tmCLIC1omab), we establish a distinct pharmacologically and biophysically tmCLIC-mediated current in GB indispensable for sustaining EGFRvIII/STAT3 signaling. Importantly, we show that OSMR is required for maintaining CLIC1-mediated ionic balance at the plasma membrane (PM). Our study uncovers a bidirectional cross-talk between OSMR and tmCLIC1 in GB, which is essential for fueling its malignant growth.