Inhibition of V-ATPase function drives apoptosis via GCN1/GCN2 kinase signaling

Natural products are a rich source of bioactive molecules that have served both as templates for drug discovery and as tools to uncover fundamental biological processes. While characterizing the pro-apoptotic activity of the cyanobacterial metabolite Nostatin A, we identified vacuolar-type H-ATPase (V-ATPase) as its molecular target and uncovered an unexpected signalling response preceding cell death initiation. V-ATPase inhibition rapidly activates the integrated stress response (ISR) through engagement of the GCN1/GCN2 kinase module, indicative of ribosomal collisions and translational shutdown. This response is conserved across established V-ATPase inhibitors, including bafilomycin A1, but not with compounds disrupting lysosomal function by other means. Mechanistically, V-ATPase inhibition depletes the pro-survival protein MCL-1 resulting in BAX/BAK-dependent mitochondrial apoptosis. Loss of MCL-1 creates a vulnerability that renders cells dependent on co-expressed BCL-2 family proteins, enabling potent synergy with the BH3 mimetics ABT-737 or venetoclax. Taken together, our results reveal a therapeutically exploitable vulnerability in V-ATPase-reliant or MCL-1 dependent cancers.