ERG enhancer derived regulatory sequences define “stem-like” cells in human leukemia

Adult acute myeloid leukemia (AML) is a rapidly progressing blood cancer, with low survival rates. This is due to inexplicable intrinsic resistance mechanisms that act in the rare and highly regenerative Leukemia Stem Cells (LSCs) which persist in the course of chemotherapy and initiate leukemia relapse. A lack of tools to directly isolate viable human LSCs precludes their comprehensive study and efficient targeting. To that end, we developed a lentiviral reporter system that pinpoints LSCs by exploiting a stem-like genetic program driven by the combinatorial binding of the heptad transcription factors (SCL/TAL1, LMO2, LYL1, ERG, FLI1, RUNX1, GATA2) aberrantly activated in a wide-range of leukemias. To generate a reporter, we cloned the binding sites of the heptad from the endogenous ERG+85 stem cell enhancer upstream of the Blue Fluorescent Protein (BFP). Using a broad panel of human leukemia cell lines we revealed highly heterogeneous reporter activity. Remarkably, high reporter activity cells (BFPhigh) demonstrated increased clonogenicity and resistance to chemotherapy and irradiation relative to their BFPlow counterparts. Moreover, BFPhigh cells' growth was selectively arrested by the retinoic acid treatment, reinforcing the distinct transcriptional status of these cells. Importantly, we revealed BFPhigh cells as a minor population in primary AML samples. Currently, we use transcriptomics, proteomics and chemogenomics approaches to systematically characterize regulators that drive a stem-like program in BFPhigh leukemic cells. We hope that the utilization of this novel and versatile research tool will identify crucial determinants of leukemia resistance that might be used as prognostic markers, but most importantly, will provide the foundation for their targeting and thus also their cure.