The Action Mechanism of Omomyc Figure 6. Omomyc promotes Myc binding and transrepression of the p21 (CDKN1) promoter. A) Luciferase activity of the human p21 promoter reporter plasmid – p21Cip1-Luc – transfected in 293T cells together with FLAG-c-Myc and Omomyc expressing plasmids. Data were normalized by cotransfection of pRL-TK Renilla luciferase. The basal activity of the reporter construct was set to a value of 100. B) Quantitative ChIP assay of Myc and Omomyc binding to the p21 promoter region (p21cip1, grey bars). Top: FLAG-c-Myc binding in 293T cells transfected with the p21 reporter p21Cip1-Luc together with FLAG-c-Myc and Omomyc expressing plasmids. Bottom: FLAG-Omomyc binding in 293T cells transfected with the p21 reporter together with FLAG-Omomyc and c-Myc expressing plasmids. A region of the luciferase coding sequence (Luc; black bars) was used as control. Bars represent the percentage of input DNA immunoprecipitated, after background subtraction. ChIP values are expressed as % of input DNA. doi:10.1371/journal.pone.0022284.g006 proliferation and survival of Rat1 fibroblasts constitutively expressing – or not – c-Myc and Omomer [10] (Figure 9A). In the absence of tamoxifen, Rat1 cells expressing or not c-Myc and Omomer had similar growth and death rate. Rat1-control and Rat1-Omomer cells behaved similarly also upon tamoxifen treatment, which modestly slowed down proliferation of both. Rat1 cells co-expressing c-Myc and Omomer instead behaved differently from the other two cell types in the presence of tamoxifen: initially they grew similarly, but after two days their proliferation stopped two due to massive cell death (Figure 9A). Therefore, Omomyc appeared to only affect survival of Rat1 cells over-expressing c-Myc, in agreement with the hypothesis that activation of the cell death pathway requires Myc over-expression [57]. Finally, we measured proliferation and death of SH-SY5Y human neuroblastoma cells infected with Omomer producing or control lentiviruses (Figure 9B). Switching on Omomyc activity by tamoxifen in the neuroblastoma cells quickly inhibited growth and triggered death, whereas tamoxifen had no effect on cells infected with the control virus (Figure 9B). Omomer expression in lentivirus infected neuroblastoma cells was stronger than in Rat1-Omomer cells (not shown), suggesting that the greater sensitivity of the neuroblastoma cell line – as compared to Rat1 cells – to the action of Omomyc might reflect its higher expression level. Discussion The interest in Omomyc derives from its outstanding anti- tumor activity, unparalleled by other Myc inhibitory treatments [16]. This molecule selectively affects the Myc protein interaction network. On the basis of our results, we propose that Omomyc acts like an edge-specific perturbation of the network that produces opposite effects on the two arms of Myc activity: transactivation and transrepression of gene transcription. Edgetic perturbations of a protein network confer distinct functional consequences from node removal [58], achieved by technologies like gene knockout or RNAi. The finding that Omomyc can bind N-Myc as well as c- Myc and, presumably, all Myc family proteins suggests that it may be able to prevent cells from eluding inhibition of a single Myc protein by upregulating another Myc family member. Omomyc – by inhibiting Myc interaction with Max – suppresses binding to E- boxes and transactivation by Myc, whereas – by allowing Myc interaction with Miz-1 – it favors binding to promoters of repressed targets like p21 and transrepression. As a result, the Myc network action is channeled to transrepression. This conclusion is supported by the changes – in transcriptional response to serum stimulation and epigenetic marks – observed in fibroblast cells ectopically expressing Omomyc. Upon serum stimulation, many more genes were downregulated in the presence of Omomyc than PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22284