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Updated: Nov 18, 2025
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and
Ismail S Mohiuddin1,2, Sung-Jen Wei1,2, In-Hyoung Yang1,2
1Cancer Center, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
Abstract:
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.
Insights
Researchers identified novel compounds targeting cancer-driving c-MYC activity. A new assay screened compounds that inhibit kinases (MAPKAPK2 and DNA-PKcs) phosphorylating OCT4, leading to c-MYC activation, yielding five promising cancer-killing drug candidates.
Area of Science:
- Molecular Oncology
- Drug Discovery
- Cancer Therapeutics
Background:
- Amplification-independent c-MYC overexpression is prevalent in numerous cancers.
- Targeting c-MYC activity presents therapeutic potential but has faced significant challenges.
- Identifying druggable targets to modulate c-MYC activity is crucial for cancer treatment.
Purpose of the Study:
- To identify novel druggable targets for modulating c-MYC activity in cancer.
- To develop a cell-based assay for identifying inhibitors of kinase-OCT4 interactions.
- To discover and validate lead compounds with anti-cancer properties.
Main Methods:
- Identified MAPKAPK2 (MK2) and DNA-PKcs as kinases phosphorylating OCT4 (Ser111 and Ser93, respectively) to activate c-MYC.
- Developed a novel cell-based luminescence assay to screen for inhibitors of MK2-OCT4 and DNA-PKcs-OCT4 interactions.
- Screened approximately 80,000 compounds, followed by validation of hits using specific antibodies for phosphorylated OCT4 (pOCT4).
Main Results:
- Identified 56 compounds inhibiting DNA-PKcs-OCT4 interaction and 65 compounds inhibiting MK2-OCT4 interaction.
- Validated hits confirmed effects on OCT4 phosphorylation and activation.
- Selected five candidate compounds (two from DNA-PKcs assay, three from MK2 assay) demonstrating significant cancer cell-killing ability in the nanomolar range.
Conclusions:
- A novel cell-based luminescence assay effectively identifies inhibitors of c-MYC transcriptional activation.
- Five validated compounds show potential as lead compounds for further development in cancer therapy.
- This approach offers a promising strategy for targeting c-MYC-driven cancers.
