RNF43 p.G659fs leads to natural killer cell dysfunction in MSI-high colorectal cancer through PI3K/AKT/mTOR signaling and HLA-E up-regulation.

The RNF43 p.G659fs (RNF43659mut) mutation occurs in 8% of colorectal cancers (CRCs) and is enriched in microsatellite instability-high (MSI-H) tumors. Although RNF43659mut promotes tumor growth through phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) activation independent of WNT signaling, its immunologic effects are poorly defined. We show that RNF43659mut shapes an immunosuppressive natural killer (NK) cell landscape in MSI-H CRC. Single-cell RNA sequencing and multiplex immunohistochemistry revealed increased NK cell infiltration in RNF43659mut tumors, yet these cells displayed inhibitory phenotypes. Bulk RNA sequencing and flow cytometry of NK cells cocultured with RNF43659mut isogenic lines and MSI-H patient-derived organoids demonstrated reduced functional NK subsets, decreased activation markers, increased inhibitory receptors, and impaired cytotoxicity. Mechanistically, RNF43659mut-mediated PI3K/AKT activation induced HLA-E expression, suppressing NK cell function. Pharmacological or small interfering RNA-mediated PI3K/AKT inhibition restored NK cell activity and enhanced tumor cell killing. Spatial analysis of MSI-H patient tumors revealed close proximity between NKG2A+NK cells and HLA-E+ tumor cells, which were themselves adjacent to PI3K/AKT-activated tumor cells, highlighting a localized immunosuppressive niche. These findings uncover an immune evasion mechanism in MSI-H CRC, implicating the PI3K/AKT-HLA-E/NKG2A axis as a promising therapeutic target to overcome immunotherapy resistance.