Lysosomes function as the digestive system of a cell and are involved in macromolecular recycling, vesicle trafficking, metabolic reprogramming, and progrowth signaling. Although quality control of lysosome biogenesis is thought to be a potential target for cancer therapy, practical strategies have not been established. Here, we show that lysosomal membrane integrity supported by lysophagy, a selective autophagy for damaged lysosomes, is a promising therapeutic target for glioblastoma (GBM). In this study, we found that ifenprodil, an FDA‐approved drug with neuromodulatory activities, efficiently inhibited spheroid formation of patient‐derived GBM cells in a combination with autophagy inhibition. Ifenprodil increased intracellular Ca 2+ level, resulting in mitochondrial reactive oxygen species–mediated cytotoxicity. The ifenprodil‐induced Ca 2+ elevation was due to Ca 2+ release from lysosomes, but not endoplasmic reticulum, associated with galectin‐3 punctation as an indicator of lysosomal membrane damage. As the Ca 2+ release was enhanced by ATG5 deficiency, autophagy protected against lysosomal membrane damage. By comparative analysis of 765 FDA‐approved compounds, we identified another clinically available drug for central nervous system (CNS) diseases, amoxapine, in addition to ifenprodil. Both compounds promoted degradation of lysosomal membrane proteins, indicating a critical role of lysophagy in quality control of lysosomal membrane integrity. Importantly, a synergistic inhibitory effect of ifenprodil and chloroquine, a clinically available autophagy inhibitor, on spheroid formation was remarkable in GBM cells, but not in nontransformed neural progenitor cells. Finally, chloroquine dramatically enhanced effects of the compounds inducing lysosomal membrane damage in a patient‐derived xenograft model. These data demonstrate a therapeutic advantage of targeting lysosomal membrane integrity in GBM.

Lysosome, as the digestive system of the cell, participated in numerous cell biological processes, such as macromolecular degradation, cell adhesion/migration, and apoptosis by regulating metabolic status and pro-growth signaling. Therefore, lysosome could be a promising therapeutic target for cancer therapy, but it remains unclear how lysosome is involved in cancer malignancy. In this study, to investigate the roles of function lysosomes in the regulation of malignant status, we evaluated the proteolytic activity of lysosomes using BODIPY-dye conjugated BSA (DQ-BSA) in malignant gliomas characterized by high aggressivity, destructivity, and invasiveness, glioblastoma multiforme (GBM). While most GBM patient-derived cells showed higher lysosomal proteolytic activity than an immortalized human neural progenitor cell line derived from the embryonic brain, levels of lysosome activity positively co-related with sphere formation. Multi-omics analysis demonstrated that the level of lysosomal proteolytic activity was co-related with metabolic status, including activated mitochondria biogenesis and amino acid transport. The orthotopic xenograft model revealed that lysosome activity in vitro is a critical biomarker representing malignant phenotypes of GBM. We found that the MiT/TFE family, a master regulator of lysosomal biogenesis, controlled the malignant progression of GBM, including therapy resistance, indicating that quality control of lysosomes is a critical determinant for the malignant properties of gliomas. To develop a novel therapeutic approach for targeting lysosomal function, we performed screening of FDA-approved compounds and found that lysosomotropic agents, which induced galectin-3 punctation as an indicator of lysosomal membrane damage, efficiently suppressed tumor growth. Ifenprodil, a clinically available drug that acts as a lysosomotropic agent, increased intracellular Ca2+ levels, resulting in mitochondrial reactive oxygen species-mediated cytotoxicity. Furthermore, we found that the efficacy of ifenprodil was remarkably enhanced by metabolic modifications, such as amino acid restriction, in vitro and in vivo. Collectively, these data indicate that lysosomes as signaling hubs for metabolic conditions play critical roles in the regulation of the malignant status of GBM patients. Our findings propose that lysosomal biogenesis is a promising target to develop a novel therapeutic approach for GBM. Citation Format: Yongwei Jing, Masahiko Kobayashi, Atsushi Hirao. Therapeutic advantage of targeting lysosome as signaling hub for metabolic conditions in malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB258.