The p53 inactivation caused by aberrant expression of its major regulators (e.g., MDM2 and MDMX) contributes to the genesis of a large number of human cancers. Recent studies have shown that restoration of p53 activity by counteracting p53 repressors is a promising anticancer strategy. Although agents (e.g., nutlin3a) that disrupt MDM2-p53 interaction can inhibit tumor growth, they are less effective in cancer cells that express high levels of MDMX. MDMX binds to p53 and can repress the tumor suppressor function of p53 through inhibiting its trans-activation activity and/or destabilizing the protein. Here we report the identification of a benzofuroxan derivative [7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium, NSC207895] that could inhibit MDMX expression in cancer cells through a reporter-based drug screening. Treatments of MCF-7 cells with this small-molecule MDMX inhibitor activated p53, resulting in elevated expression of proapoptotic genes (e.g., PUMA, BAX, and PIG3). Importantly, this novel small-molecule p53 activator caused MCF-7 cells to undergo apoptosis and acted additively with nutlin-3a to activate p53 and decrease the viability of cancer cells. These results thus show that small molecules targeting MDMX expression would be of therapeutic benefits.

Aberrant gene expression is one of the driving forces for cancer progression and is considered an ideal target for chemical intervention. Although emerging bioluminescence reporter systems allow high-throughput searches for small molecules regulatory for gene expression, frequent silencing of reporter genes by epigenetic mechanisms hinders wide application of this drug discovery strategy. Here we report a novel system that directs the integration of a promoter-reporter construct to an open chromosomal location by Flp-mediated homologous recombination, thereby overcoming reporter-gene silencing. Using this system, we have screened more than 8000 compounds in the DIVERSet chemical library for repressors of a matrix metalloproteinase-9 (MMP-9) promoter and identified 5-methyl-2-(4-methylphenyl)-1H-benzimidazole (MPBD) inhibitory for MMP-9 gene expression. Consistent with this effect, MPBD inhibits MMP-9-dependent invasion of UMSCC-1 oral cancer cells, preosteoclast migration, and receptor activator of nuclear factor-B ligand-induced osteoclast activity over concentration ranges that repressed MMP-9 expression. Mechanistic studies indicated that MPBD antagonizes AP-1 function by inhibiting its transactivation activity. We conclude that the Flp-mediated homologous recombination system to direct reporter integration into open chromatin regions represents a novel strategy allowing for the development of high-throughput systems screening for lead compounds targeting aberrant gene expression in cancer.

P53 inactivation caused by aberrant expression of its major regulators(e.g, MDM2 and MDMX) contributes to 50% of human cancers. Recent studies have demonstrated that restoration of p53 activity by counteracting p53 repression is a promising anti-cancer strategy. Although agents(e.g., Nutlin-3a) disrupting the MDM2-p53 interaction can inhibit tumor growth, they are less effective in cancer cells with high MDMX expression levels. MDMX binds to p53 and serves as a major p53 antagonist by mainly repressing its transcriptional activity. Here we reported the identification of two samll molecules inhibitory for MDMX expression through high-throughput screening. Treatments of MCF-7 cells with these MDMX inhibitors dramatically increased the stability of p53, leading to elevated expression of proapoptotic genes(e.g., PUMA, Bax, and PIG3). These results argue for a recent notion that MDMX is a major regulator of the p53 stability. While they exerted limited effects on cell cycle progression, this new class of small-molecule p53 activitors induced MCF7 cells to undergo apoptosis. However, the MDMX inhibitors only modestly enhanced p53 activition induced by Nutlin-3a. We conclue that small molecules targeting MDMX expression would be of therapeutic benefits and be useful in dissecting the p53 network through chemical biological approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4528.