Development of ERG-enhancer fluorescent reporter system to decipher functional heterogeneity in human leukemia

Acute leukemia is an aggressive blood malignancy with low survival rates. High expression of stem-like programs in leukemias predicts poor prognosis and assumed to act aberrantly in phenotypically heterogeneous and incompletely understood Leukemia Stem Cells (LSCs). A lack of suitable tools to isolate LSCs based on their stemness precludes their comprehensive examination.

We hypothesized that tagging of the endogenous stemness-regulatory regions could generate a reporter for the putative leukemia stemness-state. 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 cellular heterogeneity in several leukemia cell lines. Remarkably, ERG+85^high cells exhibited increased resistance to chemotherapy and irradiation relative to their ERG+85^neg counterparts. Moreover, ERG+85^high fraction regenerated original cellular heterogeneity and was enriched for LSCs. Transcriptomic analysis of ERG+85^high cells uncovered distinct fingerprints associated with hematopoietic stem cell (HSC) identity, β-catenin pathway and relapse. Furthermore, we discovered that USP9X deubiquitinase and ERG form a positive feedback loop that reinforces ERG+85-associated stemness and implicated USP9X in leukemogenesis.

We propose that utilization of this novel research tool can decipher crucial determinants of LSCs and provide the foundation for their targeting.