Modulating AML1-ETO with Signature-Based Small Molecule Library Screening.

We sought to develop a strategy to pharmacologically modulate AML1-ETO, the most common gene rearrangement associated with acute myeloblastic leukemia (AML). Like many other oncogenic transcription factors associated with the acute leukemias, AML1-ETO has been considered undruggable. In principle, these well-characterized somatic mutations identified in the acute leukemias represent unique, tumor-specific therapeutic targets. In practice, acute leukemia therapy continues to focus on nonspecific cytotoxic agents. In order to address this challenge, we developed a genomic, signature-based small molecule library screening approach, Gene Expression-based High-throughput Screening (GE-HTS). This approach uses gene expression signatures as surrogates for different biological states in a small molecule library screen. We focused our initial efforts on identifying modulators of AML1-ETO. First, a 25-gene signature for AML1-ETO abrogation was defined by transcriptional profiling of t(8;21) Kasumi-1 cells with and without AML1-ETO-directed RNA interference and with a U937 inducible model of AML1-ETO. The signature was confirmed in microarray data from t(8;21)-containing primary patient leukemias (p < 0.001). Next, the ability of 2,600 FDA-approved drugs and bioactive agents to induce this abrogation signature was evaluated by ligation mediated amplification (LMA) and bead-based fluorescence detection. The screen identified 16 hits confirmed on repeat testing, including seven corticosteroids. Five hits were selected for further study based on their chemical diversity, ability to induce a robust expression signature, and potential for clinical translation: 5-aza-deoxycytidine, floxuridine, methotrexate, methylprednisolone, and pyrimethamine. Next, whole genome effects of these compounds with microarray-based expression profiling were evaluated. Using Gene Set Enrichment Analysis (GSEA), we determined that all five compounds induce both whole genome effects consistent with AML1-ETO knockdown and a whole genome program consistent with neutrophil maturation. In a subset of these compounds, we also see changes in cell surface markers and morphological features consistent with myeloid maturation. Intriguingly, two of the hits are well-characterized DNA methyltransferase inhibitors, and two of the hits are dihydrofolate reductase inhibitors that increase S-adenosylhomocysteine, an inhibitor of methyltransferases. Thus, reversal of AML1-ETO-mediated gene silencing by demethylation may overcome its repressive effects. This application of knockdown-derived expression signatures to small molecule library screening should enable the targeting of nearly any oncogenic transcription factor.