PGE2-mediated NK cell reprogramming drives acquired immunotherapy resistance in lung adenocarcinoma

Background: Acquired resistance limits the durability of programmed cell death protein-1 (PD-1) blockade in lung adenocarcinoma, yet the tumor-intrinsic programs and immune circuits that drive acquired resistance relapse remain poorly defined. The purpose of this study was to identify tumor-intrinsic mediators of acquired resistance and determine how they remodel antitumor immunity. Methods: An orthotopic bioluminescence-tracked Lewis lung carcinoma (LLC1) lung adenocarcinoma model was established in immunocompetent mice treated with anti-PD-1. Tumor-intrinsic regulators were identified by an in vivo genome-wide CRISPR loss-of-function screen and validated using inducible tetracycline-off knockdown. Prostaglandin E2 (PGE2) signaling was interrogated through tumor-cell Ptgs2 knockdown/deletion, 16,16-dimethyl PGE2 administration, selective EP2/EP4 antagonists, and celecoxib treatment. Natural killer (NK)-cell function was analyzed by flow cytometry, immunofluorescence, RNA sequencing, cAMP measurement, calcium flux assays, mouse and human NK-cell co-culture cytotoxicity assays, and NK-cell adoptive transfer. Celecoxib was used to evaluate the therapeutic potential of pharmacologic PGE2 blockade in vivo. Public immunotherapy datasets were analyzed to assess the clinical relevance of PTGS2. Results: The orthotopic LLC1 model captured key features of heterogeneous anti-PD-1 responses, including relapse after initial regression. The CRISPR screen identified Ptgs2 as a key driver of acquired resistance. Tumor-derived PGE2 progressively increased in resistant tumors, and its genetic silencing overcame resistance by restoring NK-cell infiltration and function. Mechanistically, PGE2 signaled through EP2/EP4 receptors to elevate cAMP and induce CREM, thereby suppressing NK-cell cytotoxicity and cytokine production. This axis was validated in human NK cells. Pharmacologic inhibition of cyclooxygenase-2 with celecoxib reversed acquired resistance, an effect abrogated by NK-cell depletion. Conclusions: Tumor-derived PGE2 is an important contributor to acquired resistance to PD-1 blockade in lung adenocarcinoma. Therapeutic disruption of the EP2/EP4-cAMP-CREM axis restores NK-cell function and overcomes acquired resistance.