Targeting prohibitins activates the ISR through DELE1-HRI by impairing protein import into the mitochondrial matrix

Prohibitins (PHBs) are predominantly located at the inner mitochondrial membrane, displaying significant roles in tumor progression, invasion, and apoptotic resistance, often overexpressed in primary tumors. Importantly, we developed a synthetic molecule, fluorizoline, that induces apoptosis by selectively targeting PHBs in various cancer cell lines and primary samples from different hematological neoplasms. Fluorizoline induces apoptosis by activating the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, specifically via the ATF4-CHOP-NOXA axis. We identified compensatory mechanisms for four ISR-related kinases, with HRI emerging as the primary kinase responsible for the activation of the ISR and apoptosis induction, implicating mitochondrial stress in ISR activation. Here, we investigate the mitochondrial stress response signaling pathway responsible for activating HRI after targeting PHBs either by fluorizoline treatment or by PHBs downregulation in HeLa and HAP1 cancer cell lines. In this study, we describe how PHBs regulate the localization of the mitochondrial stress sensor DELE1, leading to ISR activation and apoptosis induction in HeLa and HAP1 cells. Our findings demonstrate that DELE1 promotes ISR activation upon fluorizoline treatment and PHBs downregulation. Although fluorizoline treatment activates the cleavage of long DELE1 (L-DELE1) to its cleaved form (S-DELE1), OMA1 was found to be dispensable for activating the ISR upon fluorizoline treatment. Furthermore, our findings indicate a potential impairment of the mitochondrial protein import machinery upon targeting PHBs, as the import of other mitochondrial proteins beyond DELE1 is also disrupted. These findings reveal a previously unknown physiological role of PHBs in preserving the mitochondrial protein import pre-sequence pathway, possibly due to the interaction between PHBs and DNAJC19. This novel insight underscores the potential of targeting PHBs, such as with fluorizoline, to overwhelm mitochondrial stress in cancer.