Pictilisib and nutrient stress synergize to induce methuosis via PI(4,5)P2-dependent macropinocytic dysregulation in cancer cells.

Aberrant phosphatidylinositol 3-kinase (PI3K) activation drives many cancers, but PI3K inhibitors like Pictilisib often induce cytostasis rather than cytotoxicity, limiting their therapeutic potential. Here we demonstrate that PI3K inhibition combined with nutrient stress triggers methuosis, a non-apoptotic form of programmed cell death characterized by dysregulated macropinosomes. This response occurs selectively in PI3K-aberrant cancer cells that maintain macropinocytic uptake despite PI3K inhibition. Methuosis-associated vacuoles originate from macropinosomes that retain endosomal markers but fail to undergo lysosomal fusion. Active macropinocytic uptake is essential for methuosis, as demonstrated by suppression with EIPA and Bafilomycin A1, whereas the AKT inhibitor MK2206 has no effect, establishing that direct PI3K inhibition, rather than AKT signaling, is required. Mechanistically, PI3K blockade prevents conversion of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) causing PI(4,5)P2 to accumulate on internalizing macropinosomal membranes. This aberrant PI(4,5)P2 enrichment impairs ion channel function across multiple channel families, disrupting intracellular osmotic balance. Ion dysregulation triggers aquaporin-1-mediated water influx, driving catastrophic vacuolar expansion and cell death. Although Pictilisib activates pro-survival autophagy, this fails to prevent methuosis-mediated cytotoxicity. In xenograft models, dietary restriction synergizes with Pictilisib to suppress tumor growth, correlating with pronounced intratumoral vacuolization. These findings reveal that combining PI3K inhibition with nutrient restriction converts cytostatic responses into methuosis-driven cytotoxicity via PI(4,5)P2-dependent macropinocytic dysregulation, providing a rational pharmacologic-dietary strategy to enhance PI3K-targeted cancer efficacy.