A Novel Flow Cytometric Assay to Identify Inhibitors of RBPJ-DNA Interactions

Authors

Robert J. Lake, Department of Internal Medicine, Division of Molecular Medicine, Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA
Mark K. Haynes, Department of Pathology, Program in Cancer Therapeutics, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USACenter for Molecular Discovery, University of New Mexico Health Science Center, Albuquerque, NM, USA
Kostiantyn Dreval, Department of Internal Medicine, Division of Molecular Medicine, Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA
Rabeya Bilkis, Department of Internal Medicine, Division of Molecular Medicine, Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA
Larry A. Sklar, Department of Pathology, Program in Cancer Therapeutics, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA Center for Molecular Discovery, University of New Mexico Health Science Center, Albuquerque, NM, USA
Hua-Ying Fan, Department of Internal Medicine, Division of Molecular Medicine, Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA

Document Type

Article

Publication Date

9-1-2020

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.

Recommended Citation

Lake RJ, Haynes MK, Dreval K, Bilkis R, Sklar LA, Fan HY. A Novel Flow Cytometric Assay to Identify Inhibitors of RBPJ-DNA Interactions. SLAS Discov. 2020 Sep;25(8):895-905. doi: 10.1177/2472555220932552. Epub 2020 Jun 22. PMID: 32567455; PMCID: PMC7442708.