Identification of Novel p53 Pathway Activating Small- Molecule Compounds Reveals Unexpected Similarities with Known Therapeutic Agents Karita Peltonen1, Laureen Colis2, Hester Liu2, Sari Ja¨ a¨ maa1,3, Henna M. Moore1, Juulia Enba¨ ck4, Pirjo Laakkonen 4 , Anne Vaahtokari5, Richard J. Jones2, Taija M. af Ha¨ llstro¨ m1, Marikki Laiho1,2,5* 1 Molecular Cancer Biology Program and Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland, 2 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Laboratory Division, University of Helsinki, Helsinki, Finland, 4 Molecular Cancer Biology Program and Institute of Biomedicine, University of Helsinki, Helsinki, Finland, 5 Molecular Imaging Unit, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Abstract Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents. Citation: Peltonen K, Colis L, Liu H, Ja¨a¨maa S, Moore HM, et al. (2010) Identification of Novel p53 Pathway Activating Small-Molecule Compounds Reveals Unexpected Similarities with Known Therapeutic Agents. PLoS ONE 5(9): e12996. doi:10.1371/journal.pone.0012996 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received July 14, 2010; Accepted September 4, 2010; Published September 27, 2010 Copyright: 2010 Peltonen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for M.L. was from the Academy of Finland Center of Excellence Program, Finnish Cancer Organization, Foundation for the Finnish Cancer Institute, Biocentrum Helsinki and Sigrid Juselius Foundation. K.P. received support from the Finnish Cultural Foundation and the Biomedicum Helsinki Foundation. H.M.M. and J.E. were supported by the Helsinki Biomedical Graduate School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mlaiho1@jhmi.edu Introduction p53 is a key activator of cellular cascades governing cell life and death [1,2]. It is activated in response to both physiological and non-physiological stresses such as oxidative, viral, oncogenic and genotoxic stress, and hypoxia [1–3]. During tumor evolution, the p53 gene, TP53, is frequently mutated, yet, over 50% of human tumors harbor the wild type gene [3,4]. The p53 pathway has been an attractive target for therapeutic manipulation. It has been proposed that the activation of p53 pathway enhances tumor cell killing [1–3,5]. Genetic evidence, both in human and mice, definitively show the essential tumor suppressive activity of TP53 [4,6,7]. More recent, switchable p53 expression models in mouse demonstrate that activation of p53 expression leads to regression of several tumor types by invoking apoptosis, senescence and the cellular innate immunity [8–10]. The current p53-related experimental therapeutic arsenal can be classified to those with known mechanisms of action (e.g. Hdm2 inhibitors) and to drugs that have demonstrated activation of either wild type (wt) or mutant p53 cells but no or poorly understood mechanisms of action. These have arisen through targeted drug design (Hdm2-inhibitors like nutlin-3 and MI-219) or through screens for wt and mutant p53 activating small- molecule compounds [11–19]. Pilot studies using Hdm2 inhibitory compounds show remarkable in vivo anti-tumor effects without side effects [13,17,19,20]. We have established that nutlin-3 is the first and highly effective agent inducing B-cell lymphoma (Kaposi’s sarcoma herpes virus (KSHV) infected pleural effusion lymphoma) cell killing both in vitro and in vivo mouse models [20]. Thus, based on these studies, inactivation of the p53 pathway by the KSHV virus lies in the pathogenesis of this incurable malignancy. Furthermore, these studies provide an indication that depending on the context (i.e. genetic composition and inherent dysfunctional pathways) of the tumor, activation of the p53 pathway can launch a cytotoxic response. Mechanisms of action of the p53 pathway affecting drugs, with the exception of the Hdm2 inhibitors, are largely unresolved [19]. p53, structurally, is not easily amenable for targeting by small- molecule compounds. Many of the drugs identified to activate either wild-type or mutant p53 function arose from screening protocols using p53 sequence-specific binding and consequent reporter activation [19]. These unbiased screens have likely PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12996