ETO2-GLIS2 Controls Differentiation Arrest and Self-Renewal through Aberrant Enhancers Regulation in Pediatric Leukemia

Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain.

We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface.