GL-V9 disrupts the mitochondrial homeostasis and triggers the integrated stress response by promoting the binding of cytosolic MDM2 with NDUFS1 in colorectal cancer

• MDM2 plays a non-oncogenic potential in CRC by restricting mitochondrion localization of NDUFS1. • GL-V9 is a new MDM2 modulator and suppresses CRC independent of p53 status • GL-V9 facilitates the binding of MDM2 with NDUFS1, and disrupts the mitochondrial homeostasis. • Mitochondrial stress activates OMA1-DELE1 axis and induces ISR-triggered apoptosis. The oncogenic role of mouse double minute 2 (MDM2) is primarily attributed to its regulation of p53-dependent signaling cascades. The colorectal cancer (CRC) remains in the top five most prevalent and lethal cancers. P53 mutations are detected in 45–50% of CRC, leading to the failure of such MDM2 inhibitors in clinical trials. Small molecular compound GL-V9 targets MDM2 and leads a non-canonical function of MDM2 mediated anti-CRC effects. Interaction of MDM2 with NDUFS1 as well as the mitochondrial location of NDUFS1 were assessed by a pull-down assay and immunofluorescence analysis. The binding of GL-V9 to MDM2, was analyzed by molecular docking, cellular thermal shift assay (CESTA), surface plasmon resonance (SPR), GST-pulldown and amino acid mutations. Mitochondrial homeostasis was evaluated by mitochondrial membrane potential, mitochondrial superoxide, ATP generation and oxygen consumption rate. Different from MDM2 inhibitors, GL-V9 binds to the MDM2 amino-terminal domain (amino acids 1–101) and facilitates the interaction of MDM2 with NDUFS1 in cytoplasm through a p53-independent manner, instead of disruption of p53-MDM2 binding or the promotion of MDM2 protein degradation. This process additionally inhibits the formation of electron transport chain complex I and disrupts the mitochondrial homeostasis, which finally activates OMA1-DELE1 signaling axis and induces the integrated stress response (ISR)-triggered apoptosis. This study provides a novel candidate for CRC therapy with favorable safety profile. Importantly, the novelty mode of action by GL-V9, working as molecular glue for MDM2/NDUFS1, provides a new insight for targeting MDM2 regardless of p53 status.