Effect of therapeutic targeting of xCT on USP15/SMAD-driven gemcitabine resistance in KRAS-mutant pancreatic cancer.

763 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, characterized by poor prognosis and limited response to chemotherapy. The efficacy of gemcitabine, the first line treatment for PDAC, is frequently limited by chemoresistance. In addition, KRAS mutations, which are prevalent in 90% of PDAC, further promote therapeutic resistance. In pancreatic cancer, KRAS mutations upregulate antioxidant pathways, including the glutamine/cysteine transporter SLC7A11, to promote tumor cell survival under oxidative stress. However, the molecular mechanisms that regulate gemcitabine resistance, especially in KRAS mutations, remain poorly understood. Methods: We performed transcriptomic profiling of gemcitabine resistant (GR) and gemcitabine sensitive (GS) PDAC cells and identified significant overexpression of SLC7A11 in GR cells. Functional downstream studies were conducted following siRNA-mediated knockdown of SLC7A11 in GR KRAS wild-type and KRAS-mutant PDAC cells. Effects of SLC7A11 knockdown were assessed through cell viability assays, apoptosis, and EMT analyses. Lipid ROS generation was measured by FACS. Gene expressions of downstream pathways, particularly in the SMAD pathway, were investigated via RNA sequencing and western blot analysis. Results: Silencing SLC7A11 in chemoresistant PDAC cells significantly decreased the GSH/GSSG ratio and elevated oxidative stress. Also, suppression of SLC7A11 reduced tumorigenic traits, including decreased migration and invasion and increased apoptosis. We found that the effects of the knockdown were more pronounced in KRAS-mutant than in KRAS wild-type cells, indicating mutation-specific dependency. RNA sequencing analysis revealed a significant downregulation of SMAD signaling, including notable reduction in SMAD2/3 phosphorylation in KRAS-mutant cells. Mechanistically, SLC7A11 upregulates a deubiquitinating enzyme (DUB) that stabilizes receptor-regulated SMADs(R-SMADs), enhancing SMAD signaling in GR PDAC. Subcutaneous xenograft experiments revealed that silencing SLC7A11 significantly impaired tumor growth and enhanced gemcitabine sensitivity. Conclusions: Our findings highlight a novel mechanism in which SLC7A11 contributes to gemcitabine resistance in KRAS-mutant PDAC by modulating redox balance and promoting SMAD signaling. Targeting SLC7A11 may represent a promising translational strategy to overcome chemoresistance in KRAS-driven pancreatic cancer.