Development of ERG-Enhancer Fluorescent Reporter System to Decipher Functional Heterogeneity in Leukemia

Acute leukemia is an aggressive blood malignancy with low survival rates. High expression of hematopoietic stem-like programs in leukemia samples predicts poor prognosis. These programs are thought to act aberrantly in phenotypically heterogeneous and incompletely understood Leukemia Stem Cells (LSCs), which persist throughout chemotherapy and initiate relapse. A lack of suitable tools to isolate LSCs based on their stemness precludes their comprehensive study. In this study, we revealed that ERG+85 enhancer region is active in the most immature human hematopoietic cells and developed a fluorescent lentiviral reporter that faithfully recapitulate its endogenous activity. Using this novel reporter we revealed previously unrecognizable cellular heterogeneity in widely used leukemia cell lines. Remarkably, ERG+85high cells exhibited increased resistance to chemotherapy and irradiation relative to their ERG+85neg counterparts. Moreover, ERG+85high fraction regenerated original cellular heterogeneity and was enriched for LSCs. Transcriptomic and bioinformatics analysis of ERG+85high cells uncovered distinct transcriptional fingerprints associated with HSC identity, b-catenin pathway activation and AML relapse. Furthermore, we discovered that USP9X deubiquitinase and ERG form a positive feedback loop that reinforces ERG+85-associated stemness network and thus implicated USP9X in leukemogenesis. We propose that utilization of this novel and versatile research tool can decipher crucial determinants of LSCs and provide the foundation for their targeting.