Splicing-mediated control of hnRNPD isoform switching by SRSF2 drives PD-L1-dependent immune evasion in gallbladder cancer

Abstract

Gallbladder cancer (GBC), a lethal malignancy of the biliary tract, is associated with a poor clinical prognosis. Although chemo-immunotherapy combinations demonstrate preliminary efficacy, the molecular determinants of treatment response remain elusive. Emerging evidence implicates aberrant alternative splicing in modulating tumor immunity. Through an in vitro CRISPR/Cas9 screen, we identified SRSF2 as a key RNA-binding protein regulating PD-L1 expression. Intriguingly, SRSF2 does not directly bind PD-L1 mRNA. Multi-omics analyses (mRNA-seq, RIP-seq, and proteomics) revealed that SRSF2 induces exon skipping in hnRNPD, shifting isoform expression from full-length P45 to truncated P40. Functional studies established that P45—but not P40—binds to AU-rich elements in the PD-L1 3’-UTR to promote mRNA degradation. Leveraging this mechanism, we designed splice-switching antisense oligonucleotides (ASOs) that block SRSF2-mediated exon skipping, restoring P45 expression. This intervention effectively reduced PD-L1 levels and potentiated T-cell-mediated cytotoxicity in vitro and in vivo. These findings elucidate a splicing-centric mechanism of immune evasion and highlight the therapeutic potential of splicing modulation in cancer immunotherapy. Proposed model of the SRSF2-hnRNPD-PD-L1 axis in gallbladder cancer (GBC) immune evasion and its therapeutic targeting. Overexpression of SRSF2 drives hnRNPD exon skipping, shifting the isoform balance from the PD-L1-degrading P45 to the truncated P40. This transition stabilizes PD-L1 mRNA and facilitates tumor immune evasion. Conversely, therapeutic intervention with splice-switching ASOs blocks SRSF2-mediated alternative splicing, restores P45 expression, and effectively reactivates T-cell-mediated cytotoxicity against GBC cells. Proposed model of the SRSF2-hnRNPD-PD-L1 axis in gallbladder cancer (GBC) immune evasion and its therapeutic targeting. Overexpression of SRSF2 drives hnRNPD exon skipping, shifting the isoform balance from the PD-L1-degrading P45 to the truncated P40. This transition stabilizes PD-L1 mRNA and facilitates tumor immune evasion. Conversely, therapeutic intervention with splice-switching ASOs blocks SRSF2-mediated alternative splicing, restores P45 expression, and effectively reactivates T-cell-mediated cytotoxicity against GBC cells. Gallbladder cancer is a highly aggressive disease that often evades the body’s natural immune defense, making current treatments less effective. Our study aimed to understand the hidden mechanisms allowing these cancer cells to hide from immune attacks. Using laboratory tumor models and patient tissue samples, we investigated the internal genetic processes of these cancer cells. We discovered that a specific protein (SRSF2) acts like a rogue editor, altering the genetic instructions of another molecule (hnRNPD). This specific modification ultimately creates a “shield” (PD-L1) on the surface of the cancer cell, effectively blinding the immune system and allowing the tumor to grow unchecked. Understanding this specific genetic editing process is highly significant, as it provides a vital new target. In the future, blocking this pathway could strip away the cancer’s shield, potentially making existing immunotherapies much more effective for patients.