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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.

SLAS Discovery : Advancing Life Sciences R & D
|June 23, 2020
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Summary

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.

Keywords:
Notch inhibitorRBPJ-DNA interactionsT-ALLauranofinflow cytometry-based high-throughput screening

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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.