Abstract
The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. No MYC specific inhibitors are currently in clinical trials. Here, we show two distinct recombinant mini proteins act as Myc inhibitors and have cellular activity. The first mini protein is Omomyc, a well characterized Myc inhibitor that has been ectopically expressed in cells. Recombinant Omomyc is cell penetrant, reduces cell proliferation of tumor cells in a MYC dependent manner and reduces the expression of MYC target genes. Chromatin immunoprecipitation (CHIP) demonstrated that recombinant Omomyc can bind to specific E box sequences in the promoters of MYC target genes, and that Omomyc can reduce the ability of MYC to bind these sequences. Proximity ligation assay (PLA) results showed that after Omomyc treatment, Omomyc/Max and Omomyc/Omomyc dimers were present in the nucleus, while Myc/Omomyc and Myc/Max dimers were present at lower amounts only in the cytoplasm. Double chromatin immunoprecipitation (ReCHIP) experiments revealed that Omomyc binds DNA in the presence of Max, but not Myc, with the binding of an Omomyc/Max heterodimer replacing the binding of Myc/Max heterodimers, suggesting that Omomyc performs the function of the MXD and MNT proteins, which are cellular antagonists to MYC. The formation of Myc/Max and Omomyc/Max heterodimer formation occurs co-translationally, where Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions where ribosomes are intact. Omomyc displays poor pharmacokinetic properties that lead to a short half-life in plasma, with a rapid distribution to tissues, mainly the liver and kidneys. As a potential alternative to Omomyc, we designed an alternative mini-protein derived from the MXD1 protein, a cellular antagonist to MYC, referred to as Mad1. Mad1 is ten-fold more potent than Omomyc in cells and is cell penetrant, with Mad1 localizing to the nucleus upon treatment of cells. Mad1 binds to Max in cells, preventing the interaction between Myc and Max, and depleting cellular levels of Myc by promoting Myc’s proteosomal degradation. Mad1 also interacts with UBF, like Mxd1, and co-localizes with UBF to the nucleolus. Mad1 is capable of repressing transcription of Myc mediated target genes than Omomyc and can bind to E-Box DNA present in the promoters of Myc target gens, as well as to the promoters of rRNA genes as determined by Chromatin Immunprecitation (CHIP) assays. Taken together, our data suggest that the Omomyc has two distinct mechanisms of action. One is to bind DNA as either a homodimer and repress MYC transcriptional activity. The second is to inhibit MYC transcriptional activity by binding DNA as a heterodimer with Max, which is akin to the mechanism of action of cellular antagonists of Myc. The recombinant Mad1 mini protein is more potent than Omomyc, due to its mimicking the function of MXD1. Though Omomyc demonstrates poor pharmacokinetic properties that lead to a short half-life in plasma, understanding both Omomyc’s and Mad1’s mechanisms of action can provide insight for the development of a MYC inhibitor for the treatment of cancer.