suggesting a capacity of targeting tumor cells without damaging the normal tissue. Despite its pervasive role in human cancer, Myc met with considerable skepticism as a therapeutic target since its requirement for proliferation and maintenance of adult stem cell compartments raised concern about the toxicity of Myc inhibition for healthy tissues [12,13]. In part thanks to work on Omomyc the potential of Myc as a therapeutic target is now established. Many doubts about selectivity for tumors have been dispelled by studies showing that transiently inhibiting Myc in skin, intestinal epithelium and other tissues does not dramatically alter tissue homeostasis [11,14,15]. The efficacy and safety of Myc targeting a` la Omomyc has been conclusively demonstrated by reversible expression of Omomyc in transgenic models [16,17]. Systemic expression of Omomyc attenuated proliferation in rapidly dividing tissues, but this was well-tolerated; tissue homeostasis was maintained, no apoptosis was observed in the normal tissues and all side effects were readily reversed following Omomyc removal. In Ras-driven lung tumors, the impact of Omomyc was impressive. Mice continuously expressing Omomyc failed to develop lung adenocarcinoma. In mice that had previously developed advanced cancer, induction of Omomyc triggered tumor regression which was accompanied by reduced proliferation and increased apoptosis of the tumor tissue [16]. An analogous anticancer impact was found in a simian virus 40 (SV40)-driven pancreatic islet tumor model [17], in breast cancer (G. Evan, personal communication) and glioma (in preparation). So, manipulating Myc function similarly to Omomyc might have the potential of an effective anticancer strategy for various tumor types. In view of the striking properties of this molecule, it is extremely relevant to elucidate its mechanism of action. Omomyc biological effects are simply the results of tout court Myc function ablation, as it would occur with Myc gene or mRNA knockouts? Clearly, to explain the remarkable properties of Omomyc it is important to understand whether they result from selective targeting of the Myc interactome and how they impact on the Myc activated and repressed targets. These issues are addressed in the present work. Our data indicate that Omomyc does not cause a global inhibition of Myc function but acts as an edge-specific perturbation of the Myc interactome, channeling its activity towards transrepres- sion. This may be key to its success as an anticancer agent. Results Omomyc selectively targets the Myc interactome Direct physical interaction with the bHLHZip protein Max is crucial to Myc function: the Myc/Max complex binds DNA – recognizing E-boxes – and works as a transcriptional activator [18]. Omomyc is able to homodimerize, to form heterodimers with c- Myc and Max proteins, and to interfere with c-Myc/Max complex formation and binding to E-boxes in vitro [9,10]. To better address Omomyc selectivity we investigated its capacity to bind N-Myc – which shares substantial functional redundancy with c-Myc and has important roles in tumor formation in the nervous system –, Mad – a strictly related bHLHZip factor that dimerizes with Max, binds E- boxes and acts as transcriptional repressor [4] –, Heb and Id1 – two HLH proteins representative of a large family of transcriptional regulators implicated in developmental processes [19]. To assess the capacity to bind N-Myc we performed immunoprecipitations on 293T cells ectopically expressing Omomyc fused to the oestrogen receptor ERTM – Omomer [10] – together with FLAG tagged c- or N-Myc. We found that Omomyc bound to N-Myc similarly to c- Myc (Figure 1A), in agreement with the virtual identity of the bHLHZip domain amino acid sequences of Myc family proteins. To assess binding to Max, Mad and the two HLH proteins Heb (an The Action Mechanism of Omomyc E protein) and Id1 we performed pull-down assays with GST-linked Max, Mad, Heb and Id1 on extracts of 293T cells ectopically expressing FLAG-tagged Omomyc. Whereas binding to Max was strong as previously reported [9], Omomyc binding to Mad was barely visible and binding to Heb and Id1 was undetectable (Figure 1B). The faint signal in the GST-Mad pull-down is likely due to the very high levels of FLAG-Omomyc expression in the transfected cells and not reflective of physiological interaction between the two proteins. In summary, we found that Omomyc binding specificity for Max and Mad proteins was the same as Myc. Similarly to Myc, Omomyc does not appear to interact with HLH proteins and therefore does not act by disrupting HLH protein networks crucial for differentiation control [20–22]. We then asked wether Omomyc interacted with Hypoxia-inducible factor 1-alpha (Hif-1a), a protein that functions as a master transcriptional regulator of the adaptive response to hypoxia, plays an essential role in tumor angiogenesis and shows a considerable interplay with Myc in the regulation of multiple glycolytic genes [23–25]. Hif-1a contains a bHLH domain and antagonizes Myc by binding to Max [24]. To investigate Omomyc binding to Hif-1a we performed immunoprecipitations on 293T cells ectopically expressing FLAG tagged Omomyc together with Hif-1a. We found (Fig. 1C) that Omomyc does not bind Hif-1a, while Max does as reported. Altogether, these experiments clearly indicate that Omomyc is Myc- Max-Mad network specific and – within this network – selectively affects Myc/Max dimerization, required for Myc binding to E- boxes and transactivation of a large number of genes. A key aspect of Myc function is transrepression of numerous genes involved in growth control, differentiation and tumor suppression [4]. This activity does not appear to implicate direct Myc binding to E-boxes. Myc is recruited to promoters of repressed genes only indirectly, upon interaction with proteins that directly bind to such promoters. The best known of them is Miz-1, a zinc finger protein involved in Myc dependent repression of cell cycle inhibitors – p15-INK4b (Cyclin-dependent kinase 4 inhibitor B) and p21(CDKN1: cyclin-dependent kinase inhibitor 1) – and in Myc dependent apoptosis in response to growth factor withdrawal [26–29]. Presumably in complex with Max, Myc contacts Miz-1 N-terminal region through the amino acid sites D394 and S405 [28] that are located in the HLH region. As these sites are not mutated in Omomyc, we hypothesized that Omomyc retained the capability to bind Miz-1. We tested this possibility via immuno- precipitation on 293T cells ectopically expressing Omomer and Miz-1. Figure 1D demonstrates that Omomyc directly interacts with Miz-1. Endogenous Miz-1 was reported to accumulate in the cytoplasm of cells, with a minor fraction in the nucleus; Myc overexpression triggers nuclear translocation of Miz-1 and sequestration in discrete subnuclear foci [30]. To investigate the interaction and intracellular localization of Omomyc, Omomyc/ Miz-1 and Omomyc/c-Myc complexes, we transfected 293T cells with FLAG tagged Omomyc or c-Myc expressing plasmids together with plasmids expressing untagged Miz-1 and c-Myc, and detected protein localization by immunofluorescence with anti FLAG, c-Myc, and Miz-1 antibodies (Figure 2). In cells transfected with single expression plasmids, Miz-1 was mostly (about 90%) localized in the cytoplasm as previously reported [30], Myc was present exclusively in the nucleus as expected, and Omomyc was largely in the nucleus (about 85%) with a minor part in the cytoplasm (Figure 2). Omomyc lacks the nuclear localization signal of Myc proteins: it may enter the nucleus due to its small size or upon dimerization with endogenous Myc or Max. When co- transfected, most Myc proteins co-localized with Omomyc in the nucleus; a fraction of Omomyc remained in the cytoplasm - not associated to Myc - likely due to a higher level of expression. PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22284