Close association between the MLL fusion gene and Plzf/PLZF expression in HSCs. (A) Reporter assays using luciferase driven by the mouse Plzf and human HOXA9 promoters, respectively. RLU, relative light units. (B) Relative binding of MLL-ENL (detected by an anti-FLAG antibody) to Hoxa9, Plzf, and Hoxc8 (used as a negative control) promoter regions in the Tg LT-HSCs immortalized by conditional expression of FLAG-tagged MLL-ENL. (C) GSEA of MLL-rearranged AML samples (GSE17855) showing that a gene set upregulated in HSCs³⁵  was enriched in clinical samples expressing high levels of PLZF compared with those expressing low levels of PLZF. (D) GSEA of normal mouse KSL and GMP cells (GSE10627) showing that a gene set (MLL-PZ-H) representing the MLL-rearranged AML samples with high PLZF expression was enriched in KSL cells compared with GMP cells. (C-D) GSEAs were performed with Signal2Noise metric. NES, normalized enrichment score. (E) A proposed model of the leukemogenesis by endogenous-like expression of MLL-ENL. In contrast to retroviral transduction leading to strong expression, relatively weak but endogenous-like expression of MLL-ENL selectively transforms LT-HSCs into leukemic initiating cells (LICs), at least partly through upregulation of Plzf. The bar graphs show the mean ± SD of 3 independent experiments.