Loss of p53's tumor-suppressive function, either via TP53 mutation or hyperactive p53 inhibitory proteins, is one of the most frequent events in the development of human cancer. Here, we describe a strategy of pharmacologically inhibiting iASPP, a negative regulator of p53, to restore wild-type p53's tumor-suppressive function. iASPP knockdown in the colon cancer cell line HCT116 efficiently promoted p53's transcriptional activity and induced p53-dependent cell death, suggesting a key role for iASPP in silencing p53 in this cell line. Screening of a preclinical and clinical drug library using isogenic HCT116 cell models revealed that cyclin-dependent kinase 9 (CDK9) inhibitors preferentially inhibit p53+/+, rather than p53-/-, cells. Mechanistically, CDK9 inhibitors downregulated iASPP at the transcriptional level. This downregulation was dose- and time-dependent. CDK9 inhibitors further showed synergistic effects in killing p53+/+ HCT116 cells when combined with the MDM2 inhibitor Nutlin-3. In a large TCGA pan-cancer cohort, iASPP overexpression predicted poor overall survival (OS) in wild-type p53 patients, with worse OS observed when MDM2 was simultaneously overexpressed. Our study identifies CDK9 inhibitors as p53-reactivating agents, and proposes a strategy to treat cancer by efficiently reactivating p53 via the concurrent inhibition of iASPP and MDM2.,Copyright © 2019 Elsevier Inc. All rights reserved.