Pharmacological disruption of the Notch transcription factor complex

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance and resistance to cancer therapy. Several strategies, such as monoclonal antibodies (MAbs) against Notch ligands and receptors, as well as small molecule -secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Using a Notch signaling dependent co-culture system, we developed a high-throughput small molecule screening assay. Herein we report the discovery of an orally active small molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T-ALL and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest, thereby impairing proliferation. Notably, CB-103 has the ability to downregulate Notch signaling in GSI-resistant human tumor cell lines that harbor chromosomal translocations and rearrangements in Notch genes. CB-103 mimics Notch loss-of-function phenotypes in flies and mice, retarding tumor growth in xenograft models of breast cancer and patient-derived xenograft models of human leukemia, notably without causing the frequently observed dose-limiting intestinal toxicity induced by other Notch inhibitors. In summary, we describe a pharmacological strategy to interfere with Notch signaling by disrupting the transcriptional regulatory complex it assembles, with therapeutic potential for the treatment of human tumors. Newly synthesized RNA expression profile comparisons of sorted RPMI-8402 cells treated with CB-103 or DMSO control after 1h, 4h or 24h treatment.