PI3K/mTOR Inhibitor Induces Context-Dependent Apoptosis and Methuosis in Cancer Cells

<b>Background/Objectives:</b> Targeting the PI3K/mTOR pathway is a promising strategy in cancer therapy, but its efficacy is often limited by apoptosis resistance. This study investigates the dual PI3K/mTOR inhibitor YYN-37, exploring its capacity to induce context-dependent cell death, particularly the non-apoptotic process of methuosis. <b>Methods:</b> We examined the effects of YYN-37 on HCT-116 and SJSA-1 cancer cell lines using cell viability assays, Western blot, and fluorescent tracers. Cell death mechanisms were probed with pathway-specific inhibitors. The role of VPS34 was assessed through kinase activity assays, siRNA-mediated knockdown, and rescue experiments with the agonist leucine. Proteomic profiling and an in vivo SJSA-1 xenograft model in BALB/c nude mice were utilized to evaluate broader mechanisms and anti-tumor efficacy. <b>Results:</b> YYN-37 induced caspase-3-dependent apoptosis in HCT-116 cells. In contrast, it triggered a reversible, cytoplasmic vacuolization in SJSA-1 cells, identified as methuosis. This vacuolization originated from endocytic pathways and was inhibited by EIPA and Baf-A1. YYN-37 directly inhibited VPS34 (IC₅₀ = 2.73 nM), and its knockdown replicated the vacuolization, which was conversely reversed by the VPS34 agonist leucine, confirming VPS34-dependency. Proteomics revealed lysosomal dysfunction in SJSA-1 cells and cell cycle alterations in HCT-116 cells. In vivo, YYN-37 treatment resulted in a 72.71% tumor growth inhibition, with histology confirming methuosis-like vacuolization. <b>Conclusions:</b> YYN-37 exerts potent, context-dependent anti-tumor effects by inducing apoptosis in HCT-116 cells and VPS34-mediated methuosis in SJSA-1 cells. This work establishes methuosis induction as a viable therapeutic strategy for apoptosis-resistant cancers and highlights VPS34 inhibition as a promising mechanism of action.