Abstract 350: Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery

Abstract Prohibitins are ubiquitously expressed within the cell, but they are mainly located at the inner mitochondrial membrane. These proteins are closely associated with tumour progression, invasion, and apoptotic resistance, being overexpressed in various primary tumours such as lymphoma and endometrial cancer. Notably, we have developed a novel synthetic molecule, fluorizoline, which induces apoptosis by selectively targeting PHBs in different cancer cell lines and primary samples from various haematological neoplasias. We have previously described that fluorizoline-induced apoptosis is mediated through the activation of the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, concretely, by the ATF4-CHOP-NOXA axis. In a recent study, we identified compensatory mechanisms for four ISR-related kinases. Importantly, HRI emerged as the primary kinase responsible for activating this pathway and promoting apoptosis, suggesting that mitochondrial stress could lead to this activation of the ISR.Here, we investigate the OMA1-DELE1-HRI molecular pathway responsible for sensing mitochondrial stress and activating the ISR cancer cell lines. Firstly, we demonstrate that fluorizoline treatment triggers the cleavage of large DELE1 (L-DELE1) to its cleaved form (S-DELE1) through western blot analysis, and its subsequent sorting to the cytosol as confirmed by confocal analysis. Using DELE1 KO lines generated by CRISPR/Cas9 technology, we demonstrate that DELE1 is involved in the activation of the ISR and apoptosis induced by fluorizoline. However, although we cannot rule out OMA1 involvement, fluorizoline-induced ISR activation does not strongly depend on OMA1. Interestingly, our data suggests that targeting PHBs, either by fluorizoline or by their downregulation, could result in mitochondrial protein import machinery impairment. This impairment may potentially be the cause of DELE1 accumulation in the cytosol, leading to the subsequent activation of the ISR by HRI upon fluorizoline treatment. Citation Format: Ismael Sánchez-Vera, Nekane Maritorena-Hualde, Max-Hinderk Schuler, Ana M- Cosialls, Rodolfo Lavilla, Gabriel Pons, Lucas T Jae, Daniel Iglesias-Serret, Joan Gil. Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 350.