Overcoming ELDR-mediated cancer cell survival in vitro and in vivo via sphingomyelin-driven TRPML1 inactivation and TFEB suppression through lysosomal HSP70 targeting

Lysosomal adaptation through the endo-lysosomal damage response (ELDR) enables cancer cells to evade lysosome-targeted therapies. Here, we identified the flavonoid compound V8 as a first-in-class ELDR disruptor that eliminated cancer cells by sabotaging lysosomal resilience. Mechanistically, V8 bound lysosomal HSP70 via hydrogen bonding, destabilizing its interaction with bis(monoacylglycero)phosphate (BMP) and triggering pathological sphingomyelin (SM) accumulation. SM overload allosterically inhibited TRPML1, blocking calcineurin PPP3CB activation and subsequent TFEB dephosphorylation. This dual perturbation: enhanced lysophagy and failed TFEB-driven biogenesis drove catastrophic lysosomal bankruptcy. Crucially, V8 bypassed canonical ELDR activation: unlike lysosomotropic agent LLOMe, it induced global membrane remodeling rather than focal perforations, avoiding Ca²⁺-dependent endosomal sorting complexes required for transport (ESCRT) repair. Genetic validation using HSP70-knockout and point mutation models confirmed target specificity, while SM synthase inhibition rescued TRPML1 activity and mitigated apoptosis. Tumor-selective efficacy arose from malignant cells' heightened SM dependency and lysosomal HSP70 reliance, sparing normal counterparts. Our work established HSP70-BMP-ASM axis disruption as a strategy to subvert lysosomal homeostasis, providing a blueprint for next-generation lysosome-targeting agents that exploit lipid-mediated channelopathy to sensitize cancer cells to lysosomal damage.