RUNX1-ETO RAPIDLY REPROGRAMS THE EPIGENETIC LANDSCAPE IN HUMAN EMBRYONIC DEFINITIVE HAEMATOPOIETIC PROGENITORS

Abstract

Acute Myeloid Leukaemia results from the successive acquisition of multiple genetic aberrations. Therefore, the sole study of patient samples, which represent the end-point of this process, hinders the understanding of the effect of each individual mutation on reprogramming the epigenome. In order to dissect tumour development, we generated human Embryonic Stem Cell lines carrying an inducible version of RUNX1-ETO – the product of the t(8;21) translocation – which we subsequently differentiated into haematopoietic cells. This mutation is observed in utero and in cells of long-term remission patients, where it consists a reservoir for relapse. When induced at physiological levels at early embryonic stages, RUNX1-ETO disrupts vasculogenesis and prevents blood formation. However, induction in already formed progenitors (i) blocks differentiation at an immature stage, (ii) reduces colony-forming capacity, (iii) induces a reversible cell-cycle block caused by the down-regulation of haematopoietic, cell cycle and replication genes, and (iv) induces the up-regulation of multiple signalling genes. Genome wide chromatin immunoprecipitation (ChIP) and ATAC-Seq analyses show that RUNX1-ETO displaces RUNX1 from a subset of binding sites, resulting in reduced chromatin accessibility and gene silencing. RUNX1-ETO induction dramatically alters gene expression with some changes appearing to be irreversible. Single cell gene expression analyses show that induction of RUNX1-ETO in the CD45+CD34+RUNX1C+ population results in the emergence of a new cell population unrelated to normal cells that may represent the pre-leukaemic clone. Our data are consistent with the idea that RUNX1-ETO establishes a precondition for leukaemic transformation by maintaining a reservoir of quiescent pre-leukaemic progenitors with susceptibility to expand upon acquisition of an additional oncogenic event.