The DNA-dependent protein kinase (DNA-PK) plays an instrumental role in the overall survival and proliferation of cells. As a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, DNA-PK is best known as a mediator of the cellular response to DNA damage. In this context, DNA-PK has emerged as an intriguing therapeutic target in the treatment of a variety of cancers, especially when used in conjunction with genotoxic chemotherapy or ionizing radiation. Beyond the DNA damage response, DNA-PK activity is necessary for multiple cellular functions, including the regulation of transcription, progression of the cell cycle, and in the maintenance of telomeres. Here, we review what is currently known about DNA-PK regarding its structure and established roles in DNA repair. We also discuss its lesser-known functions, the pharmacotherapies inhibiting its function in DNA repair, and its potential as a therapeutic target in a broader context.

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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 (MK2) and the DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs), which phosphorylate the Ser111 and the Ser93 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 these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction between DNA‐PKcs and OCT4, and 65 hits inhibiting the MK2–OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Using a two‐step method for validation, we identified two candidate compounds from the DNA‐PKcs assay and three from the MK2 assay. All five 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 five compounds that may function as lead compounds for further development.

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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.

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c-MYC overexpression plays an important role in tumorigenesis as well as in the progression of tumors. Historically, direct targeting strategies have been challenging due to c-MYC being a transcription factor. Our observation demonstrated that MAPKAPK2 as one of the two major protein kinases involved in binding and phosphorylating OCT4 at serine 111 residue to induce c-MYC activation in progressive disease neuroblastoma. In targeting this novel pathway of c-MYC activation, we considered two options: direct inhibition of MAPKAPK2 or inhibition of MAPKAPK2-OCT4 protein-protein interaction. Given the limitations of direct targeting kinases, namely resistance and off-target effect, we pursued the inhibition of the protein-protein interaction. The purpose of this study is to introduce a novel cell-based assay for compound screening and validation steps to identify compounds that inhibit OCT4-MAPKAPK2 interaction. First, OCT4 (NTD and POUs: the domains critical for c-MYC activation) and MAPKAPK2 were exogenously expressed by a single vector with CMV promoter using a lentiviral vector system in NCI-H82, a small cell lung cancer cell line. Upon the optimization of the assay, we screened approximately 100,000 compounds to identify “hits” by statistically analyzing the reduction in luminescence. Seventy-six compounds were found to inhibit the interaction of MAPKAPK2-OCT4. Subsequently, we have used a two-step hit validation for the 76 compounds: 1) the decrease in pOCT4S111 expression along with the reduction in c-MYC expression by immunoblotting: 2) and the inhibition of binding between MAPKAPK2 and OCT4 by co-immunoprecipitation. The validation step further narrowed down the hits to three compounds that reduced the phosphorylation of OCT4 at S111 and inhibited the MAPKAPK2-OCT4 interaction. To verify the compounds interfering the MAPKAPK2-OCT4 interaction, we used in vitro assay using recombinant human MAPKAPK2 kinase and OCT4 and all three compounds inhibited phosphorylation of OCT4S111. These all compounds also tested minimally affected ATP-dependent MAPKAPK2 kinase relative to the known MAPKAPK2 inhibitor (PF3644022). In conclusion, our screening assay and validation experiments was successfully implemented in identifying hits and the validation step confirmed three novel compounds that demonstrated: 1) impairment of MAPKAPK2-mediated phosphorylation of OCT4; and 2) inhibition of the MAPKAPK2-OCT4 kinase-substrate interaction. These compounds may be further optimized to minimize systemic toxicities and improve the anti-cancer activity for the treatment of neuroblastoma or small cell lung cancer with high c-MYC expression. Citation Format: Inhyoung Yang, Sung-Jen Wei, Ismail Mohiuddin, Gloria M. Martinez, Min H. Kang. A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4 [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 705.

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