Category: Assay Development and Screening

1246-B - A Differential Phenotypic HTS for Inhibitors of Notch Dependent Tumor Cells

Monday, February 5, 2018
5:00 PM - 6:00 PM

The Notch signaling pathway regulates proliferation, differentiation, survival and angiogenesis. Deregulation of Notch signaling is frequently observed in many cancers through mutations and amplification of the components of the Notch signaling pathway, which can also contribute to chemoresistance. Notch is one of the most commonly activated signaling pathways in cancers, with more than 50% Notch activating mutations in T-cell Acute Lymphatic Leukemia (T-ALL). Current approaches to target Notch signaling are focused on inhibitors of g-Secretase (GSIs), which cleaves the activated Notch receptor to propagate downstream signaling. However, the efficacy of GSI therapy of Notch dependent cancers is limited by lack of substrate specificity of g-secretase, on-target side effects, as well as resistance formation.

In order to identify alternative options to inhibit Notch dependent cancer cell lines, we have developed a phenotypic screening cascade for identification of inhibitors with differential activities on Notch dependent versus Notch independent T-ALL cell lines.

For selection of Notch dependent and independent cells, we tested 42 tumor cell lines from  different tissue origins for both Notch target gene expression as well as  sensitivity to GSIs. The Notch dependent T-ALL cell lines RPMI-8402 and MOLT-14, as well as the Notch independent cell lines Sup-T11 were chosen to be screened against our internal screening collection of 200k compounds, including diverse and focused compounds collections, as well as 5k substances with known bioactivities and natural product fractions from marine invertebrate organisms (provided by Jeanette H. Andersen, University of Tromsø, Norway). Hits with selectivity for the two Notch dependent versus the independent cell line were further profiled in an extended panel of Notch dependent and independent cells. Finally, hits with the most promising profiles in the extended cell panel were tested for inhibition of Notch target gene expression in RPMI-8402 cells.

The final hit list includes known inhibitors of Notch dependent T-ALL cell lines, validating the assay principle. Furthermore, published compounds with so far unknown activity on Notch dependent cell lines were identified, as well as novel substances with unknown activity on Notch. Target deconvolution and Hit-to-Lead optimization of the frontrunner hits is ongoing.  

Jan E. Eickhoff

Head of Screening
Lead Discovery Center GmbH
Dortmund, Nordrhein-Westfalen, Germany

Head of Assay development and Screening at the Lead Discovery Center in DOrtmund, Germany. Member of SLAS Europe.