Genome-wide CRISPR screen reveals an uncharacterized spliceosome regulator as new candidate immunotherapy target

Cancer immune evasion is orchestrated by tumor-intrinsic molecular constraints that remain incompletely defined. Here, we performed an in vivo genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function screen to catalogue gene regulatory determinants of immune evasion in cancer cells. We identify C9ORF50 as a novel splicing regulator whose inhibition profoundly sensitizes cancer to immune surveillance. Integrated multi-omics profiling reveals this intrinsically disordered protein exhibits liquid-liquid phase separation properties and forms nuclear condensates that colocalize with spliceosome components. Genetic ablation correlates with intron retention in multiple spliceosome components and cytoplasmic accumulation of double-stranded RNA, which is associated with type I interferon activation and enhances chemokine-mediated T cell recruitment. As a result, C9ORF50 inhibition amplifies tumor cell immunogenicity, enhancing T cell infiltration in poorly infiltrated tumors. Clinically, elevated C9ORF50 expression correlates with poor survival and diminished lymphoid infiltration across malignancies. Therapeutic targeting of C9ORF50 using RNA interference enhances T cell infiltration and suppresses tumor growth. Our work identifies C9ORF50 as a candidate therapeutic target that modulates RNA splicing and tumor immunity, suggesting splicing regulation as a potential strategy to enhance immunotherapy responses.