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

The β–catenin destruction complex (BDC) is a central node in WNT/β–catenin signaling, governing embryonic development and adult tissue homeostasis. Although recognized as a prime therapeutic target in colorectal cancer (CRC) for three decades, its dynamic architecture and biochemical complexity have hindered mechanistic understanding. Here, we systematically mapped the sequence–function landscape of the BDC using tiled base editor screens across four endogenous components— CTNNB1 , AXIN1 , APC , and GSK3B . Validation studies identified ~150 previously unreported mutations across these genes that affected WNT/β–catenin signaling. In addition to known cancer-associated mutations, we discovered rare gain–of–function and separation–of–function alleles of AXIN1 and CTNNB1 that provide mechanistic insights into complex assembly and regulation. We describe a region in β–catenin that regulates its binding to TCF/LEF transcription factors and demonstrate that the AXIN1–β–catenin interface is critical for controlling signaling flux through the oncogenic BDC. Mechanistic studies revealed that assembly of the oncogenic BDC is scaffolded by its own substrate β–catenin, establishing an autoregulatory mechanism that represents an unexploited vulnerability in cancers harboring common APC truncations. Our comprehensive mutational resource provides a foundation for understanding WNT/β-catenin signaling mechanisms in health and disease, while revealing strategies for therapeutic intervention in WNT–driven cancers.