The Action Mechanism of the Myc Inhibitor Termed Omomyc May Give Clues on How to Target Myc for Cancer Therapy Mauro Savino 1 , Daniela Annibali1, Nicoletta Carucci1, Emilia Favuzzi1, Michael D. Cole2¤a, Gerard I. Evan3¤b, Laura Soucek3,4, Sergio Nasi1* 1 Consiglio Nazionale delle Ricerche - Istituto di Biologia e Patologia Molecolari (CNR – IBPM), Dipartimento di Biologia e Biotecnologie, Universita` Sapienza, Roma, Italia, 2 Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America, 3 Department of Pathology, University of California San Francisco (UCSF), San Francisco, California, United States of America, 4 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain Abstract Recent evidence points to Myc – a multifaceted bHLHZip transcription factor deregulated in the majority of human cancers – as a priority target for therapy. How to target Myc is less clear, given its involvement in a variety of key functions in healthy cells. Here we report on the action mechanism of the Myc interfering molecule termed Omomyc, which demonstrated astounding therapeutic efficacy in transgenic mouse cancer models in vivo. Omomyc action is different from the one that can be obtained by gene knockout or RNA interference, approaches designed to block all functions of a gene product. This molecule – instead – appears to cause an edge-specific perturbation that destroys some protein interactions of the Myc node and keeps others intact, with the result of reshaping the Myc transcriptome. Omomyc selectively targets Myc protein interactions: it binds c- and N-Myc, Max and Miz-1, but does not bind Mad or select HLH proteins. Specifically, it prevents Myc binding to promoter E-boxes and transactivation of target genes while retaining Miz-1 dependent binding to promoters and transrepression. This is accompanied by broad epigenetic changes such as decreased acetylation and increased methylation at H3 lysine 9. In the presence of Omomyc, the Myc interactome is channeled to repression and its activity appears to switch from a pro-oncogenic to a tumor suppressive one. Given the extraordinary therapeutic impact of Omomyc in animal models, these data suggest that successfully targeting Myc for cancer therapy might require a similar twofold action, in order to prevent Myc/Max binding to E-boxes and, at the same time, keep repressing genes that would be repressed by Myc. Citation: Savino M, Annibali D, Carucci N, Favuzzi E, Cole MD, et al. (2011) The Action Mechanism of the Myc Inhibitor Termed Omomyc May Give Clues on How to Target Myc for Cancer Therapy. PLoS ONE 6(7): e22284. doi:10.1371/journal.pone.0022284 Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France Received November 2, 2010; Accepted June 23, 2011; Published July 21, 2011 Copyright: 2011 Savino 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: This work was supported by research grants from AIRC, ASI, MIUR and Fondazione Guido Berlucchi (SN), a Bear Necessities Pediatric Cancer Foundation grant (LS), a CNR short term mobility fellowship (SN) and by CNR - Sapienza University PhD fellowships (MS, DA). 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: sergio.nasi@uniroma1.it ¤a Current address: Dartmouth Medical School, Lebanon, New Hampshire, United States of America ¤b Current address: Department of Biochemistry, Cambridge University, Cambridge, United Kingdom Introduction Myc transcriptional regulators – c-, N- and L-Myc – are basic, helix-loop-helix, leucine zipper (bHLHZip) proteins that bind to an array of genomic sites to either induce or repress transcription of genes important for cell growth, metabolism and differentiation [1–4]. These factors – especially c-Myc and N-Myc – are deregulated in the majority of human cancers. This is usually not due to Myc gene mutation but results from upstream mutations affecting other oncogenes or tumor suppressors. Myc activity thus appears to be required for development and maintenance of the majority of tumors, even when initiated by other causes. In tumors induced by Myc upregulation in transgenic mice, even brief Myc de-activation triggers tumor regression accompanied by growth arrest, differentiation, and collapse of the tumor vascular system [5]. Myc operates within a highly interconnected interactome network and a possible strategy for targeting Myc oncogenic function is dominant interference of Myc protein interactions. In this respect, Myc oligomerization domain – the bHLHZip region – proved to be capable of dominantly inhibiting Myc transforming ability in rat embryo fibroblast cells [6,7]. This domain mediates the direct interaction with Max and sequence specific binding to specific consensus sequences – the E boxes – in promoters of activated target genes. The bHLHZip domain is also involved in the interaction with other partners – such as Miz-1 – that mediate transcriptional repression by Myc [4,8]. To interfere with Myc protein-protein interactions, we developed a dominant negative molecule by introducing four selected mutations in the bHLHZip region of human c-Myc. The resulting 90 amino acid miniprotein – termed Omomyc for its capacity to form homodimers – is able to inhibit c-Myc/ Max association, to affect transcriptional activation by c-Myc, and to enhance c-Myc dependent apoptosis in tissue culture cells [9,10]. It was shown to prevent c-Myc induced papillomatosis in vivo without affecting tissue homeostasis [11] PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22284