The functional landscape of patient-derived RNF43 mutations predicts sensitivity to Wnt inhibition.

A subset of Wnt-addicted cancers are sensitive to targeted therapies that block Wnt secretion or receptor engagement. RNF43 loss-of-function mutations that increase cell surface Wnt receptor abundance cause sensitivity to Wnt inhibitors. However, it is not clear which of the clinically identified RNF43 mutations affect its function in vivo. Here we assayed 119 missense and 45 truncating RNF43 mutations found in human cancers using a combination of cell-based reporter assays, genome editing, flow cytometry, and immunofluorescence microscopy. Five common germline variants of RNF43 exhibited wild-type activity. Cancer-associated missense mutations in the RING ubiquitin ligase domain and a subset of mutations in the extracellular domain hyperactivated Wnt/β-catenin signaling through formation of inactive dimers with endogenous RNF43 or ZNRF3. RNF43 C-terminal truncation mutants including the common G659fs mutant were loss-of-function specifically when endogenous mutations were examined, unlike their behavior in transient transfection assays. PDX and cell lines with C-terminal truncations showed increased cell surface FZD and Wnt/β-catenin signaling and were responsive to PORCN inhibition in vivo, providing clear evidence of RNF43 impairment. Our study provides potential guidelines for patient assignment, as virtually all RNF43 nonsense and frameshift mutations including those in the C-terminal domain and a large number of patient-associated missense mutations in the RING domain and N-terminal region compromise its activity and therefore predict response to upstream Wnt inhibitors in cancers without microsatellite instability. This study expands the landscape of actionable RNF43 mutations, extending the benefit of these therapies to additional patients.