Definition of a Small Core Transcriptional Circuit Regulated by AML1-ETO [RNA-seq]

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. Utilizing CRISPR-based genome editing, we inserted a degron tag into the endogenous AML1-ETO locus of Kasumi-1 cells, as well as overexpressed a degradable AML1-ETO protein in CD34+ human cord blood cells. This allowed rapid AML1-ETO protein degradation upon addition of a proteolysis targeting chimera (PROTAC). Furthermore, by combining rapid degradation with nascent transcript analysis (PRO-seq), RNA-seq and Cut&Run, we have defined the core AML1-ETO regulatory network. De-repression of this small set of genes set off a cascade of transcriptional events resulting in myeloid differentiation. Overall design: RNA-seq was performed using biological replicates. For Kasumi-1 cell samples, parental cells as well as untreated AML1-ETO-FKB12F36V cells served as negative controls. Cells were harvested at 2, 4, 8, and 24 hr post-treatment with dTAG-47 to induce AML1-ETO-FKBP12F36V degradation. For CD34+ human cord blood cultures expressing AML1-ETO-FKBP12F36V, RNA-seq was performed on biological replicates treated with dTAG-47 for 4 hours and compared with DMSO-treated control samples.