Tumor-intrinsic metabolic pathways essential for tumorigenesis and resistance to anti-PD1 in oncogenic Kras-driven lung adenocarcinoma.

Immune checkpoint blockade (ICB) targeting PD-1 has transformed cancer therapy, yet many tumors display primary or acquired resistance. Metabolic interactions within the tumor microenvironment are increasingly recognized as key modulators of anti-tumor immunity. To identify tumor-intrinsic metabolic pathways that contribute to resistance to anti-PD1 therapy, we performed an in vivo CRISPR-based negative selection screen using a metabolism-focused sgRNA library in an oncogenic Kras and p53 loss-driven murine lung adenocarcinoma cell line. The IgG control arm revealed essential metabolic dependencies for in vivo tumor growth, including the TCA cycle, electron transport chain, antioxidant pathways, one carbon metabolism and de novo lipogenesis. Differential analysis of anti-PD1–treated tumors uncovered metabolic genes whose loss sensitized cancer cells to PD-1 blockade, highlighting pathways in antigen presentation, metabolite transport, arachidonic acid metabolism, and peroxisomal function. Secondary subpooled screens across multiple lung cancer cell lines validated the prominence of these pathways. Although Pla2g4a emerged as a top candidate across Kras-driven tumors, genetic deletion of this enzyme individually did not enhance responsiveness to PD-1 therapy. Overall, this work defines metabolic vulnerabilities that contribute to primary tumor growth in vivo, while providing a resource for selecting tumor-intrinsic metabolic targets that need further validation for combination strategies with PD-1 blockade.