Transcription factor p53 induces cell cycle arrest and apoptosis in response to DNA damage 1 and cellular stress, 2 thereby playing a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase hDM2 controls p53 levels through a direct binding interaction that neutralizes p53 transactivation activity, exports nuclear p53, and targets it for degradation via the ubiquitylation-proteasomal pathway. 3,4 Loss of p53 activity, either by deletion, mutation, or hDM2 overexpression, is the most common defect in human cancer. 5 Tumors with preserved expression of wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize native p53. In this context, hDM2 targeting has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. 6 Supporting Information Available: Complete ref 16h, peptide characterization, experimentals, and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

Summary Cancer cells neutralize p53 by deletion, mutation, proteasomal degradation, or sequestration to achieve a pathologic survival advantage. Targeting the E3 ubiquitin ligase HDM2 can lead to a therapeutic surge in p53 levels. However, the efficacy of HDM2 inhibition can be compromised by overexpression of HDMX, an HDM2 homologue that binds and sequesters p53. Here we report that a stapled p53 helix preferentially targets HDMX, blocks the formation of inhibitory p53-HDMX complexes, induces p53-dependent transcriptional upregulation, and thereby overcomes HDMX-mediated cancer resistance in vitro and in vivo. Importantly, our analysis of p53 interaction dynamics provides a blueprint for reactivating the p53 pathway in cancer by matching HDM2, HDMX, or dual inhibitors to the appropriate cellular context.