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A Novel Flow Cytometric Assay to Identify Inhibitors of RBPJ-DNA Interactions
Robert J Lake1, Mark K Haynes2,3, Kostiantyn Dreval1
1Department of Internal Medicine, Division of Molecular Medicine, Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA.
Abstract:
Notch signaling is often involved in cancer cell initiation and proliferation. Aberrant Notch activation underlies more than 50% of T-cell acute lymphoblastic leukemia (T-ALL); accordingly, chemicals disrupting Notch signaling are of potential to treat Notch-dependent cancer. Here, we developed a flow cytometry-based high-throughput assay to identify compounds that disrupt the interactions of DNA and RBPJ, the major downstream effector of Notch signaling. From 1492 compounds, we identified 18 compounds that disrupt RBPJ-DNA interactions in a dose-dependent manner. Cell-based assays further revealed that auranofin downregulates Notch-dependent transcription and decreases RBPJ-chromatin interactions in cells. Most strikingly, T-ALL cells that depend on Notch signaling for proliferation are more sensitive to auranofin treatment, supporting the notion that auranofin downregulates Notch signaling by disrupting RBPJ-DNA interaction. These results validate the feasibility of our assay scheme to screen for additional Notch inhibitors and provide a rationale to further test the use of auranofin in treating Notch-dependent cancer.
Insights
Researchers identified compounds disrupting Notch signaling, a key driver in T-cell acute lymphoblastic leukemia (T-ALL). Auranofin shows promise by downregulating Notch-dependent transcription and RBPJ-DNA interactions, offering a potential new cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Notch signaling is integral to cancer cell initiation and proliferation.
- Aberrant Notch activation is implicated in over 50% of T-cell acute lymphoblastic leukemia (T-ALL).
- Targeting Notch signaling presents a therapeutic strategy for Notch-dependent cancers.
Purpose of the Study:
- To develop a high-throughput assay for identifying compounds that disrupt RBPJ-DNA interactions, a critical step in Notch signaling.
- To screen a chemical library for inhibitors of RBPJ-DNA binding.
- To evaluate the efficacy of identified compounds, particularly auranofin, in cell-based models of T-ALL.
Main Methods:
- Development of a flow cytometry-based high-throughput screening assay.
- Screening of 1492 diverse chemical compounds for RBPJ-DNA interaction disruption.
- Cell-based assays to assess Notch-dependent transcription and RBPJ-chromatin interactions.
- Sensitivity assays using T-ALL cell lines dependent on Notch signaling.
Main Results:
- Identified 18 compounds that inhibit RBPJ-DNA interactions in a dose-dependent manner.
- Auranofin was found to downregulate Notch-dependent transcription and RBPJ-chromatin binding in cells.
- T-ALL cells reliant on Notch signaling exhibited increased sensitivity to auranofin treatment.
- The study validated the high-throughput assay for identifying novel Notch inhibitors.
Conclusions:
- The developed assay is effective for screening Notch signaling inhibitors.
- Auranofin demonstrates potential as a therapeutic agent for Notch-dependent cancers by disrupting RBPJ-DNA interactions.
- Further investigation into auranofin for treating Notch-dependent malignancies is warranted.
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