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

The {beta}-catenin destruction complex (BDC) is a central node in WNT/{beta}-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/{beta}-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 {beta}-catenin that regulates its binding to TCF/LEF transcription factors and demonstrate that the AXIN1-{beta}-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 {beta}-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/{beta}-catenin signaling mechanisms in health and disease, while revealing strategies for therapeutic intervention in WNT-driven cancers.