Abstract The majority of the FDA-approved kinase inhibitors target ATP binding sites of kinases. Despite their activity against tumorigenesis, these therapies are often non-specific and are susceptible to resistance mechanisms. One method of enhancing the selectivity of these compounds and overcoming potential chemoresistance is to better identify novel compounds that are highly specific. In this study, we introduce one such strategy by identifying kinase inhibitors that target kinase-substrate interactions. c-MYC overexpression and deregulation has been implicated in the development and progression of a number of malignancies. We have previously identified that OCT4 binds to the MYC promoter/enhancer region to transcriptionally activate c-MYC. Novel OCT4 binding sites in the c-MYC promoter region were identified, and kinases (DNA-PKcs for the current study) that mediate the phosphorylation of specific amino acid residues of OCT4 were determined. We then identified the domains of DNA-PKcs that are critical to bind and phosphorylate OCT4. The goal of the current project is to develop an assay to identify the compounds that interferes the interaction between OCT4 and DNA-PKcs and to validate the identified "hits." In order to selectively inhibit the interaction of protein-kinase related to c-MYC transcriptional activation, we co-expressed the fragments of OCT4 required for c-MYC expression and the fragments DNA-PKcs required to activate OCT4 to 1) confirm the interaction between DNA-PKcs and OCT4, and 2) develop a cell-based assay that can screen novel compounds that prevent this interaction. After confirming the kinase-substrate interaction, we co-transduced the crucial fragments of DNA-PKcs and OCT4 tagged with luminescent probes in HEK 293FT cells. We then screened a chemical library of compounds to identify hits that inhibited luminescence, and thus our kinase-substrate protein interaction. In our screening assay, 67 compounds were found to inhibit the luminescence interaction between the tagged OCT4 and DNA-PKcs protein fragments. After the initial screening, we conducted a two-step hit validation for the 67 hits identified that consisted of: 1) the decrease in pOCT4S93 expression along with the reduction in c-MYC expression: 2) and the inhibition of binding between DNA-PKcs and OCT4 by co-immunoprecipitation. Additional experiments to validate the hits include kinase activity assays and immunoblotting to determine if the screened hits inhibited DNA-PKcs kinase activity. In conclusion, our screening assay and validation experiments identified 7 novel compounds that demonstrated: 1) impairment of DNA-PKcs-mediated phosphorylation of OCT4; and 2) inhibition of the DNA-PKcs-OCT4 kinase-substrate interaction. These newly identified compounds have the potential to oppose aberrant c-MYC expression and warrant further investigation in order to determine their activity in high c-MYC-expressing malignancies. Citation Format: Ismail S. Mohiuddin, Sung J. Wei, Inhyoung Yang, Gloria Martinez, Hwangeui Cho, Min H. Kang. A cell-based screening assay to identify novel kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4040.