RNF43 p.G659fs activates the PI3K/AKT-HLA-E axis to drive NK cell dysfunction in MSI-high colorectal cancer 2257795

Abstract

Abstract Introduction The RNF43 p.G659fs (RNF43659mut) mutation is a recurrent alteration in ∼8% of colorectal cancers (CRCs), enriched in microsatellite instability-high (MSI-H) tumors. While this mutation activates the PI3K/AKT pathway independent of WNT signaling, its immunologic impact remains undefined. Given the central role of PI3K/AKT/mTOR signaling in immune evasion, we investigated how RNF43659mut influences natural killer (NK) cell function in MSI-H CRC. Methods Single-cell RNA sequencing and multiplex immunohistochemistry of MSI-H CRC tumors were integrated with in vitro coculture assays using RNF43659mut isogenic lines and MSI-H patient derived organoids (PDOs). NK cell phenotypes were assessed by bulk RNA sequencing and flow cytometry, while pharmacologic and siRNA-based inhibition probed PI3K/AKT pathway dependence. Spatial analyses mapped PI3K/AKT activation, HLA-E expression, and NK cell localization within patient tumors. Results RNF43659mut tumors exhibited increased NK cell infiltration but marked functional exhaustion, characterized by reduced CD56dim/CD56bright subsets, lower activation markers (CD16, NKG2D), and elevated inhibitory receptors (NKG2A, TIM3, KIR3DL1). Mechanistically, RNF43659mut driven PI3K/AKT activation upregulated HLA-E, leading to suppression of NK cytotoxicity. PI3K/AKT blockade using inhibitors or siRNA restored NK activity and enhanced tumor cell killing in cocultures. Spatial profiling revealed NKG2A+ NK cells juxtaposed to HLA-E+, PI3K/AKT-activated tumor cells, indicating a localized immunosuppressive niche. Conclusion RNF43659mut establishes an immune evasive microenvironment in MSI-H CRC through PI3K/AKT-dependent HLA-E induction and NK cell dysfunction. Targeting the PI3K/AKT-HLA-E/NKG2A axis represents a promising strategy to reverse immune suppression and improve immunotherapy response in MSI-H colorectal cancer. Funding Source NA Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)