Overexpression of c-MYC protein without genomic amplification occurs in multiple cancers, including neuroblastoma and small cell lung cancer. In searching for the mechanisms of c-MYC protein overexpression, we demonstrated that the transcription factor, OCT4 mediates c-MYC transcriptional activation in progressive disease neuroblastoma. Subsequently, we identified two kinases, MAPKAPK2 (MK2) and DNA-PK, which are predicted to bind and phosphorylate OCT4 at S111 and S93 residues, respectively. Based on these novel observations, we developed a cell-based luminescence assay to screen and identify compounds that inhibit the interactions between MK2 and OCT4. By screening 79,671 compounds, we identified 65 compounds we designated as “hits”. Using a two-step validation of co-immunoprecipitation and pOCT4S111 detection, the compounds were further narrowed down to three for further studies. The three compounds were tested for their ability to inhibit kinase activity, in vitro cytotoxic activity, and anti-inflammatory activity. In conclusion, we developed a cell-based luminescence assay for the discovery of new agents targeting the c-MYC transcriptional activation pathway. Screening and subsequent validation identified a small number of compounds for further development.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the S111 and the S93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between DNA-PKcs and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction. Using a custom antibody specific for pOCT4S93, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Seven compounds were selected for the second step of validation, which focused on the interaction between kinase and substrate. After further characterization, we identified two compounds that significantly impaired the ability of DNA-PKcs to bind to and phosphorylate OCT4. The compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found two compounds that may function as lead compounds for further development.