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

The RNF43 p.G659fs (RNF43^659mut) mutation occurs in 8% of colorectal cancers (CRCs) and is enriched in microsatellite instability-high (MSI-H) tumors. Although RNF43^659mut 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 RNF43^659mut 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 RNF43^659mut tumors, yet these cells displayed inhibitory phenotypes. Bulk RNA sequencing and flow cytometry of NK cells cocultured with RNF43^659mut isogenic lines and MSI-H patient-derived organoids demonstrated reduced functional NK subsets, decreased activation markers, increased inhibitory receptors, and impaired cytotoxicity. Mechanistically, RNF43^659mut-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.