Proteogenomic profiling of gallbladder cancer for identification of distinct biology and subtype-specific therapeutic vulnerabilities.

e16263 Background: Gallbladder cancer (GBC) is an aggressive malignancy accounting for 80-95% of biliary tract cancers, with a 5-year survival rate of less than 10%. Limited molecular characterization and therapeutic options highlight the need for comprehensive multi-omics analysis to uncover subtype-specific vulnerabilities. Methods: Integrated proteogenomic profiling (whole-exome sequencing, genomics, RNA sequencing, label-free proteomics, phosphoproteomics, and lactylomics) was performed on 53 pairs of treatment-naïve primary GBC tumors and matched adjacent normal tissues. Somatic mutations, copy number alterations (CNAs), and post-translational modifications were analyzed. Molecular subtyping was conducted via unsupervised non-negative matrix factorization (NMF) of multi-omics data. Functional validation included in vitro experiments (CCK8, Western blot, comet assay, immunofluorescence) to assess the role of PARP1 lactylation in proliferation and chemoresistance. Results: A total of 10,201 nonsynonymous mutations were identified, with TP53 (62%) and TTN (29%) as the most frequently mutated genes. CNA analysis revealed high-prevalence 17q12 amplifications (19.2% of cases) and 18q21.2 deletions (40% of cases). Three distinct multi-omics subtypes (NMF1, NMF2, NMF3) were defined: NMF1 (metabolic signature, enriched 17q12 amplification/ERBB2/ERBB3 overexpression), NMF2 (EMT/stromal expansion, immune-suppressive microenvironment with TGF-β pathway activation), and NMF3 (high proliferation, DNA repair/G2M checkpoint activation, elevated Ki67 IHC score and multi-gene proliferation scores). Global hyperlactylation was a hallmark of GBC tumors, with PARP1 K654 lactylation (K654la) significantly upregulated in NMF3 (log2 fold-change = 1.8, p = 2.3e-06). Functional experiments confirmed that PARP1 K654la enhanced ADP-ribosylation activity (1.5-fold higher than wild-type, p < 0.01), promoted cell proliferation (K654T mutant vs. WT: relative proliferation = 1.32 at 72h, p < 0.05), and conferred resistance to cisplatin (IC50 = 3.2 μM vs. 1.8 μM in K654R mutant, p < 0.01) and olaparib. Additionally, 18q21.2 deletion was associated with immune exclusion (reduced CD4+/CD8+ T cells and NK cells) via HNF4G upregulation. Conclusions: This comprehensive proteogenomic study delineates novel molecular subtypes of GBC and identifies PARP1 K654 lactylation as a key mediator of proliferation and chemoresistance in the high-proliferation subtype. The findings provide a foundational resource for developing personalized treatment strategies, including targeting ERBB2 for NMF1, TGF-β/immune checkpoint combinations for NMF2, and PARP1 K654 lactylation or CDK inhibitors for NMF3.