Massively parallel interrogation of human functional variants modulating cancer immunosurveillance

SUMMARY Anti-PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has revolutionized clinical cancer treatment, while abnormal PD-L1 or HLA-I expression in patients can significantly impact the therapeutic efficacy. Somatic mutations in cancer cells that modulate these critical immune regulators are closely associated with tumor progression and ICB response. However, a systematic interpretation of cancer immune-related mutations is still lacking. Here, we harnessed the ABEmax system to establish a large-scale sgRNA library encompassing approximately 820,000 sgRNAs that target all feasible Serine/Threonine/Tyrosine residues across the human genome, which systematically unveiled thousands of novel mutations that decrease or augment PD-L1 or HLA-I expression. Notably, we revealed functional mutations that co-regulate PD-L1 and HLA-I expression, represented by the clinically relevant mutation SETD2_Y1666, and verified that it can benefit from immunotherapy in vivo . Our findings generate an unprecedented resource of functional residues regulating cancer immunosurveillance, meanwhile, offer valuable guidance for clinical diagnosis, ICB therapy, and the development of innovative drugs in cancer treatment.