Eukaryotic elongation factor 2 kinase (eEF2K) has been considered as a putative target for cancer therapy
however, the underlying mechanisms of eEF2K in triple-negative breast cancer (TNBC) progression remain to be fully elucidated. In this study, it is shown that eEF2K is highly expressed in TNBC and is associated with poor prognosis. In vitro, in vivo, and patient-derived organoid experiments demonstrate that knockdown of eEF2K significantly impedes progression of TNBC. Proteomic analysis and confirmation experiments reveal that eEF2K positively regulates the mRNA and protein expressions of sex-determining region Y-box 8 (SOX8). Mechanistically, eEF2K binds to and phosphorylates aurora kinase A (AURKA) at S391, a newly identified phosphorylation site critical for maintaining AURKA protein stability and kinase activity. Moreover, the compound C1, a molecular glue to degrade eEF2K, is optimized by designing and synthesizing its derivatives using reasonable structure-based optimization approach. The new compound C4 shows better ability to degrade eEF2K and stronger anti-cancer activity than C1. These findings not only uncover the pivotal role of the eEF2K/AURKA/SOX8 axis in TNBC progression, but also provide a promising lead compound for developing novel drug for treatment of TNBC.