Gain of function mutations in Notch1, which encodes a signaling protein that is converted into a transcription factor upon activation, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Although inhibiting Notch1 activity represents a potential therapeutic opportunity, the discovery of new Notch1 pathway antagonists poses a difficult challenge. Traditional small molecule library screening approaches have not been amenable to modulating transcription factor abnormalities. In order to overcome this challenge, we applied Gene Expression-based High-throughput Screening (GE-HTS) to discover new Notch1 modulators. GE-HTS uses gene expression signatures as surrogates for different biological states. We derived a 32-gene Notch1 expression signature from genome-wide microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a Notch1 inactivating γ-secretase inhibitor (GSI; Notch off) and screened a small molecule library for compounds inducing the Notch1 off state in DND41 mutant Notch1 T-ALL cells. Among numerous ion flux modulators validated to induce the Notch1 off signature, one of the top hits was the FDA-approved calcium channel blocker, bepridil, used to treat patients with cardiac disease. In multiple mutant Notch1 T-ALL cell lines, bepridil induced the Notch1 off signature. We next confirmed that bepridil indeed targets Notch signaling by demonstrating its inhibitory effects on a Notch-sensitive luciferase reporter gene in heterologous U2OS cells expressing a mutated form of Notch1. Similar to the phenotypic effects of GSI, bepridil induced a G0/G1 cell cycle arrest, inhibited cellular viability, and decreased cell size in multiple T-ALL cell lines, including the GSI-resistant cell line PF382. Next, in order to confirm dependency of the induced phenotype on inhibition of Notch, we utilized the 8946 T-ALL cell line. This murine line depends on a doxycycline-repressible human c-myc transgene for growth and can be rescued from transgene withdrawal with activated Notch1, which upregulates the endogenous c-myc gene. In these cells, the phenotypic effect of bepridil on viability is also dependent on Notch1 inhibition because cells rescued from transgene withdrawal with activated Notch1 were more sensitive to the effects of the drug than were those cells still dependent on the c-myc transgene. Finally, we asked whether bepridil altered the level of active Notch1 protein in T-ALL cell lines. As with GSI, bepridil treatment results in decreased levels of intracellular Notch (ICN1). In contrast to GSI, however, bepridil treatment decreased levels of the furin-processed extracellular and transmembrane forms of Notch1 while the full length Notch1 precursor form accumulated upon bepridil treatment. One hypothesis is that by altering ER/Golgi compartment calcium, bepridil prevents the folding of newly synthesized Notch1 polypeptides, leading to its retention in the ER/Golgi and a failure to traffic to cellular compartments where receptor activation occurs. Consistent with this hypothesis co-localization studies in U2OS cell lines expressing the L1601P mutant Notch1 suggest retention of Notch1 in the ER/Golgi. An alternative hypothesis under investigation is that bepridil affects the activity of furin, a calcium-dependent protease that is required for processing of Notch receptors. In summary, we have identified an FDA-approved drug with Notch1 modulating activity in T-ALL by a mechanism unique from GSI. These studies have potential for rapid translation to clinical testing.
Ferrando:Merck, Pfizer: Research Funding.
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