Hedgehog/GLI1 regulates EMT and cancer stem cell properties via the GLI1-Bmi1 axis in diffuse large B-cell lymphoma

R-CHOP-treated diffuse large B-cell lymphoma (DLBCL) shows heterogeneous outcomes. High-risk relapsed/refractory (R/R) patients, especially activated B-cell (ABC) and double-/triple-hit subtypes, have poor prognosis, highlighting an urgent need to uncover the underlying aggressive molecular mechanisms. Epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties drive its invasion, metastasis, and drug resistance. This study investigated the regulatory role and mechanism of Hedgehog (Hh) pathway key factor GLI1 in DLBCL. Immunohistochemistry detected GLI1, Bmi1, and SOX2 expression in DLBCL tissues. DLBCL cell lines (HBL-1, SUDHL-2) were treated with pan-GLI inhibitor GANT61, or transfected with GLI1 overexpression/silencing plasmids combined with Bmi1 intervention. CCK-8, flow cytometry, RT-qPCR, Western blot, Co-IP, tumor sphere, and immunofluorescence assays were performed. In vivo xenograft experiments were conducted to verify GANT61's anti-tumor effect. GLI1 was aberrantly activated in DLBCL tissues and positively correlated with Bmi1/SOX2. GANT61 inhibited DLBCL cell proliferation, induced G0/G1 arrest and apoptosis, reversed EMT, and reduced CSC-related molecules. Co-IP confirmed GLI1-Bmi1 interaction. GLI1 overexpression enhanced proliferation, EMT, and CSC properties, while GLI1 silencing exerted opposite effects. Bmi1 inhibitor reversed GLI1's pro-tumor effects, and Bmi1 agonist partially restored GLI1 silencing-induced inhibition. In vivo, GANT61 significantly reduced tumor volume/weight in HBL-1 xenografts with good tolerability. GLI1 modulates EMT-like plasticity and stem cell traits in DLBCL via direct protein interaction with Bmi1, confirming a functional regulatory connection between these two molecules that forms the core Hh-GLI1-Bmi1 regulatory pathway. Targeting this axis provides a novel therapeutic strategy for DLBCL. Key words: Hedgehog signaling pathway; Diffuse large B-cell lymphoma (DLBCL); Epithelial-mesenchymal transition (EMT); Cancer stem cell properties; GLI1-Bmi1 axis; GANT61; Xenograft model.