Chemical Genomic Screen Identifies Ionophores as Modulators of Notch1 in T-ALL

Although in recent years intensive chemotherapy regimens have improved outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL), side effects and relapse-rates remain high. An alternative therapeutic approach would be to target constitutively active mutated Notch1. Notch1, a trans-membrane receptor that acts as a transcription factor following proteolytic activation by the γ-secretase complex, harbors gain of function mutations in over 50% of T-ALL. Early clinical trials attempting to modulate Notch1 with γ-secretase inhibitors (GSI) were closed secondary to gastrointestinal toxicity, suggesting that alternative approaches to inhibiting Notch1 signaling are needed. Transcription factor complexes are difficult to target pharmacologically with conventional high-throughput screening approaches. We therefore sought to target Notch1 with a new gene expressionbased approach to small molecule library screening. Signatures for the Notch1 on versus off states were defined using microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a GSI (Notch1 off). We adapted this 32-gene signature to our gene expression-based high-throughput screening (GE-HTS) assay, which uses ligation-mediated amplification (LMA) and a Luminex bead-based detection system. As proof of principle, we confirmed that an shRNA against Notch1 induced the Notch1 off signature in DND41 cells (a T-ALL cell line bearing a mutated Notch1 allele). In order to facilitate future clinical translation, we screened a collection of 4,500 bioactive compounds highly enriched for FDA-approved drugs in DND41 cells. Twenty-eight compounds that induced the Notch1 off signature were identified as top hits. These compounds were tested in a secondary screen in a 2-fold dilution series in two GSI sensitive cell lines, DND41 and KOPTK1, and one GSI resistant cell line, PF382. Ion channel modulators emerged as the top class of compounds confirmed to induce the Notch1 off signature across all cell lines. We next focused on two representative compounds, the ionophores salinomycin and ionomycin. Similar to GSI, both of these molecules induced cell cycle arrest and markedly decreased levels of the activated intracellular portion of Notch (ICN). Both salinomycin and ionomycin, as well as numerous other ionophores that scored as inhibitors in the screen, are known to increase cytosolic calcium. These studies are consistent with a role for calcium in the activation of oncogenic forms of Notch1 and suggest a potential role for ion channel modulators in the treatment of T-ALL.