In vivo CRISPR screening identifies LTA4H as a target for potentiating immunotherapy efficacy via the LTB4-neutrophil axis.

Despite the clinical success of PD-1 blockade, therapeutic resistance remains a major barrier. Using an in vivo CRISPR-Cas9 screen with a metabolism-focused sgRNA library in MC38 and B16 syngeneic tumor models, we identified leukotriene A4 hydrolase (LTA4H) as a candidate regulator of PD-1 resistance. Lta4h ablation was associated with enhanced PD-1 responsiveness, increased CD8+ T and NK-cell infiltration and function, and reduced myeloid accumulation. Single-cell and spatial transcriptomic analyses showed that LTA4H is expressed in both malignant and myeloid compartments, with LTA4H-high tumor regions associated with myeloid-enriched niches and TGF-β related signaling. Mechanistically, hypoxia-induced HIF1α upregulated Lta4h in tumor cells, supporting tumor cells as one relevant source of LTA4H/LTB4. LTA4H-LTB4 signaling was linked to myeloid remodeling and TGF-β1 production in LTB4-conditioned neutrophils. Pharmacological blockade of LTB4 receptor signaling with the BLT1 antagonist CP-105696 improved tumor control in combination with PD-1 blockade. These findings identify intratumoral LTA4H-LTB4 signaling as a metabolic-immune pathway associated with suppressive myeloid remodeling and support further investigation of LTB4 receptor blockade to improve PD-1 blockade responses.