Chromosome 21-Encoded miR-125b and Its Role in the Development of Myeloid Leukemia in Children with Down’s Syndrome.

Children with Down’s syndrome (DS) are at high risk to develop transient leukemia (TL) or acute myeloid leukemia (ML-DS), primarily the megakaryoblastic subtype (AMKL). Recently, acquired mutations of the hematopoietic transcription factor GATA1 - encoded on chromosome X - have been identified in leukemic blasts from virtually all patients with ML-DS or TL leading to the expression of a shorter isoform of GATA1 (GATA1s). During embryonic/fetal development mice carrying the GATA1s mutation show a hyperproliferation of a transient population of yolk sac/fetal liver megakaryocytic progenitors. However, GATA1s mice as well as GATA1s mice bred to mouse DS models (Ts65Dn and Ts1Cje) have normal adult hematopoiesis and do not develop leukemia. Hence, the factors on chromosome 21 that predispose to TL or ML-DS in cooperation with oncogenic GATA1s remain largely unknown. To date, four miRNAs (miR-99a, miR-125b-2, miR-155 and let-7c) are known to be encoded on chromosome 21. Three out of four miRNAs (miR-99a, miR-125b-2, let-7c) are not trisomic in the DS mouse model. To elucidate their role in the development of TL and ML-DS, we first measured their expression level in sorted cells from patient samples [ML-DS (n = 4), TL (n = 4), non-DS AMKL (n = 4)], and in megakaryocytes from healthy donors (n = 3) by qRT-PCR. The expression of miR-125b was clearly elevated in ML-DS (10-fold), TL (5-fold) and non-DS AMKL (3-fold) compared to normal megakaryocytes (p_{patients vs. control} <0.01). Using Gene Set Enrichment Analysis (GSEA), we confirmed the downregulation of putative target genes of miR-125b - predicted by Targetscan and Pictar - in the gene expression profile of ML-DS in comparison to non-DS AMKL. By retroviral overexpression using the Mouse Stem Cell Virus (MSCV), we could show that ectopic expression of miR-125b markedly enhanced proliferation of myeloid progenitor cells (MPC, CD34⁺CD33⁺) (MPC_miR-125b) in vitro. In colony-forming assays, the number of MPC_miR-125b derived colonies exceeded the number of MPC_vector derived colonies (empty vector) by 4-fold (χ² < 0.01). All MPC_miR-125b derived colonies contained a mixture of immature cells (CFU-GEMM) indicating an affected differentiation. This could also be confirmed by immunophenotyping of MPC cultured in liquid medium containing various cytokines (percentage CD34⁺CD33⁺ 26% vs 9%). In GATA1s fetal liver progenitors retroviral overexpression of miR-125b-2 could further enhance the proliferation rate and mediate the formation of large megakaryocytic colonies. These data support the hypothesis that miR-125b is involved in development of trisomy 21 associated leukemia by mediating the proliferation of MPC while repressing their differentiation. Therefore, miR-125b might be potential target in the therapy of ML-DS.