Methylene blue modulates G3BP1-associated condensation and attenuates Kras-driven pancreatic preneoplastic lesions

Abstract Stress granules (SGs) are dynamic biomolecular condensates implicated in pancreatic ductal adenocarcinoma (PDAC), with G3BP1/2 serving as key regulators of their assembly. We combined structure-based screening of FDA-approved drugs with cellular and biochemical assays and identified methylene blue (MB) as a modulator of SG assembly. MB suppressed SG accumulation across multiple PDAC cell lines under arsenite (AS)-induced stress, nutrient deprivation, and heat shock without reducing the abundance of major SG-associated proteins. In reconstituted systems, MB inhibited G3BP1 condensation with or without added RNA and showed a nonsignificant trend toward lower G3BP1-Caprin1 co-immunoprecipitation in cells. Molecular docking and molecular dynamics simulations predicted a potential MB interaction site within the G3BP1 NTF2-like (NTF2L) domain. The G3BP1 V11A/F15A/F33A/F124A mutant retained basal condensate-forming capacity but showed reduced responsiveness to MB compared with wild-type G3BP1, implicating this region in MB-mediated regulation. In Kras-driven KC mice, chronic oral MB administration was associated with reduced pancreatic G3BP1-positive puncta, increased acinar preservation, and decreased preneoplastic lesion burden, epithelial proliferation, and stromal remodeling, without overt abnormalities in the evaluated endpoints. Together, these findings identify MB as a small-molecule probe that modulates G3BP1 condensation dynamics and demonstrate that chronic MB treatment is associated with the attenuation of early Kras driven pancreatic preneoplastic lesion progression in vivo.