EphB4 inhibition defines a druggable synthetic-lethal vulnerability in MYC-driven triple-negative breast cancer.

The MYC oncoprotein drives aggressive tumor behavior across many cancer types, yet its intrinsically disordered structure has limited direct pharmacologic targeting. Building on our previous kinome-wide CRISPR screen, we identify the receptor tyrosine kinase EphB4 as a druggable synthetic-lethal vulnerability in MYC-driven cancers. Genetic ablation or pharmacologic inhibition of EphB4 selectively triggers robust apoptosis in MYC-activated normal cells and MYC-high triple-negative breast cancer (TNBC) cell lines, while sparing MYC-low counterparts. This apoptotic response is Bcl-2-sensitive and p53-independent, overcoming a major resistance barrier in TNBC. In vivo, EphB4 inhibition markedly suppresses MYC-driven tumor growth. Notably, co-targeting EphB4 and Bcl-2 with ABT-199 yields synergistic apoptosis and induces tumor regression in TNBC models. Mechanistically, EphB4 inhibition leads to the selective transcriptional repression of PSMB5, the β5 catalytic subunit of the proteasome, resulting in the impairment of proteasome activity and the induction of MYC-dependent apoptotic stress. This establishes an unexpected link between EphB4 signaling and proteostasis maintenance, a heightened dependency in MYC-overexpressing cells due to their elevated biosynthetic load. Targeting PSMB5 transcription, rather than its catalytic active site, also provides a potential strategy to circumvent or delay resistance to conventional proteasome inhibitors. Together, these findings define the EphB4-PSMB5 axis as a mechanistically distinct and therapeutically actionable vulnerability in MYC-high TNBC, positioning EphB4 inhibition as a promising approach to treat MYC-driven cancers.