Mutational scanning reveals substrate-assisted autoregulation of the WNT destruction complex.

The β-catenin destruction complex (BDC) regulates WNT-β-catenin signaling and is a prime therapeutic target in colorectal cancer, yet its biochemical complexity has hindered mechanistic understanding. We mapped the sequence-function landscape of the BDC using tiled base editor screens across its components CTNNB1, AXIN1, APC and GSK3B. Amongst ~150 previously unreported mutations that affected WNT signaling, we discovered gain-of-function and separation-of-function alleles that reveal mechanisms of complex assembly, including a β-catenin region regulating TCF/LEF transcription factor binding. Critically, we found that the AXIN1-β-catenin interface controls signaling flux through the oncogenic BDC found in APC-mutant cancers. In cells expressing truncated APC, β-catenin itself scaffolds BDC assembly, establishing a substrate-assisted autoregulatory mechanism. This architecture represents an unexploited therapeutic vulnerability: strengthening the AXIN1-β-catenin interaction restores destruction complex function and impairs the growth of colorectal cancer cells. Our mutational resource provides a foundation for mechanistic understanding and therapeutic targeting of the WNT pathway.