Llano, Juan Pablo Navarro Garcia de

Abstract Glioblastoma (GBM) remains the most lethal primary brain cancer due to its remarkable metabolic plasticity and therapeutic resistance. Here, we identify cholesterol dependency as a therapeutically exploitable vulnerability in GBM using two FDA-approved drugs: the H1 histamine antagonist clemastine and the retinoid X receptor agonist bexarotene. Combined treatment induces potent synergistic anti-tumor activity across patient-derived glioma models, suppressing proliferation, stemness, and survival at sub-IC50 concentrations. Mechanistically, this therapy disrupts cholesterol biosynthesis, transport, and homeostasis, triggering endoplasmic reticulum stress and activation of the unfolded protein response, ultimately leading to autophagy and apoptotic cell death. Orthotopic patient-derived glioma models recapitulate these mechanisms in vivo, where local intracranial administration significantly reduces tumor progression and prolongs survival using fourfold lower doses than systemic intraperitoneal delivery. Single-cell RNA sequencing revealed activation of regeneration and plasticity programs, accompanied by immune microenvironment remodeling and enhanced inflammatory signaling. Importantly, syngeneic models preserved immune cell composition, supporting future integration with immunotherapeutic strategies. Together, these findings establish cholesterol dysregulation–induced metabolic collapse as a promising therapeutic approach for GBM.

Abstract Glioblastoma is an aggressive brain cancer with poor prognosis. Recent evidence implicates metabolic reprogramming, including the hexosamine biosynthetic pathway (HBP) and pentose phosphate pathway (PPP), as critical for tumor survival, therapy resistance, and progression. HBP inhibition with a proprietary small molecule (G2Bz) reduced stemness and migration and increased apoptosis of GBM in vitro. HBP inhibition was concomitant with an accumulation of metabolites in the Pentose Phosphate Pathway (PPP). Therefore, we further inhibited HBP with G2Bz and/or PPP with 6-aminonicotinamide (6-AN) in primary patient-derived GBM lines and evaluated additive or synergistic effects. Ultrastructural and functional changes were examined by electron microscopy (EM), high content imaging of endoplasmic reticulum (ER) and lipid droplets (LD), extracellular-flux analysis, and lipid profiling. Synergism was validated in an orthotopic model receiving lipoparticle-encapsulated G2Bz via intranasal delivery and/or intraperitoneal 6-AN; tumor burden was monitored via bioluminescence. Dual inhibition of HBP/PPP revealed additive or synergistic anti-proliferative effect. EM displayed severe ultrastructural cell damage with mitochondrial disorganization, ER dilation, and cytoskeletal remodeling. These changes coincided with increased ROS generation and reduced spare respiratory capacity suggesting higher sensitivity and lower ability to fight stress. Reduced ER–mitochondria contacts accompanied by increased expression of BiP and ER expansion suggest an unfolded protein response. Loss of LDs were associated with downregulation of lipid biosynthesis regulator SREBF1 and an increase in free fatty acids with a rise in lipid peroxidation. Reduced mitochondria–LD contacts correlate with lower endogenous fatty acid oxidation and a shift to exogenous fatty acid utilization. Dual inhibition in-vivo, suppressed tumor growth compared to control. These findings support that dual inhibition of HBP/PPP synergistically disrupts tumor metabolism, promoting organelle disorganization and lipid remodeling and highlight metabolic co-targeting as a strategy to impair GBM metabolism.