SUMMARY

Gallbladder cancer (GBC) is a highly aggressive malignancy with dismal outcomes. To dissect its molecular characteristics and identify potential therapeutic avenues, we performed proteogenomic characterization of 195 tumors and 135 adjacent non-cancerous gallbladder tissues. Integrative analyses highlighted TP53 and ELF3 mutations as key drivers disrupting signaling and metabolism. ErbB2 amplification, a pivotal genomic event, was associated with reduced canonical PI3K/AKT and RAS/MAPK/ERK signaling yet enhanced proliferative activity. We discovered potential gain-of-function mutations in ErbB2 and ErbB3 predicted to enhance ErbB2-ErbB3 heterodimer activity. ACAT1 and PHGDH were identified as metabolic drivers of GBC liver invasion. Integrated molecular and immune subtyping delineated four distinct multi-omics and immune microenvironment subtypes, each carrying prognostic and therapeutic relevance. Although rare, neuroendocrine GBC was separately characterized, revealing MEIS1 as a potential regulator of neuroendocrine- like features. Together, this study establishes a proteogenomic landscape of GBC, providing biological insights and guiding future translational efforts.