Members of the Mir-99/100~125 Tricistrons Cooperatively Induce a Pre-Leukemic Myeloproliferative Disorder

MicroRNAs (miRNAs) reflect the best studied class of regulatory non-coding RNAs (ncRNAs), which control genetic networks with key cellular functions. In vertebrate genomes, a significant number of miRNA genes are located closely adjacent to each other in miRNA polycistrons. The mature miRNAs of the three human miR-99/100~125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are processed from one single transcript and are highly expressed in acute promyelocytic leukemia (APL). Expression profiling by qPCR in sorted murine hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), megakaryocytic erythroid progenitors (MEPs) and granulocytic monocytic progenitors (GMPs) revealed high expression levels of miR-99/100 and miR-125 family members in HSCs and CMPs. However, the consequences of the coordinated expression of the miRNAs belonging to different seed families on self-renewal and proliferation of HSCs and myeloid progenitors and their contribution to the pathogenesis of APL are not well understood.

To elucidate the genetic interactive network of miR-99/100~125 miRNAs and the role of each individual miRNA within this network, we generated a set of eight different constructs covering any permutation of miRNA family members from the two miR-99/100~125 clusters on hsa11 and hsa21 (miR-99a, miR-125b-2, let-7c, miR-99a/let-7c, miR-100/miR-125b-1, let-7a-2/miR-125b-1, miR-100/let-7a-2/miR-125b-1 and miR-99a/let-7c/miR-125b-2). Lentiviral overexpression of these constructs in human hematopoietic stem and progenitor cells (HSPCs) resulted in a significant reduction of monocytic/macrophage colony-forming units (CFU-M; 2.2-2.8-fold, p≤0.05) and granulocytic CFU-Gs (2-4.3-fold, p≤0.05) in methylcellulose-based CFU assays exclusively for miR-125-containing bi-/tricistronic constructs (miR-100/miR-125b-1, let-7a-2/miR-125b-1, miR-100/let-7a-2/miR-125b-1 and miR-99a/let-7c/miR-125b-2), but not for the single miRNA expression constructs. Accordingly, during myelomonocytic differentiation HSPCs transduced with those miR-125-containing bi-/tricistronic constructs gave rise to a major population of monomorphic, non-adherent cells devoid of granulocytic and monocytic markers, which was not present in single miRNA-transduced cells. In murine isogenic transplantation experiments (N=105), only the combined miRNA expression of miR-125b with let-7 and/or miR-99/100 led to the expansion and retention of immature Gr-1(low)/Mac-1(+)/B220(-) cells in the bone marrow (1.6-1.8fold; p≤0.01). Accordingly, either the CMP or GMP compartment of transplanted mice was expanded in miR-125-containing bi-/tricistronic constructs (CMPs 1.6-fold in let-7a-2/miR-125b-1, GMPs 1.8-1.9-fold in miR-100/miR-125b-1 and tricistrons; p≤0.01;), but not upon single miRNA overexpression (1.1-1.3-fold; p≥0.1).

Global gene expression profiling of human HSPCs transduced with the eight miRNA constructs revealed a core expression signature commonly regulated  by the four miR-125b-containing bi-/ tricistronic constructs (367 genes upregulated [>1.5-fold]; 417 genes downregulated). Strikingly, this core signature is enriched for genes with concordant expression in leukemic stem cells (LSCs) and HSCs (FDR q≤1.8x10-14). The genes of the core signature were not or only modestly affected in the context of the single miRNAs.

 Thus, the miR-99/100~125 tricistron miRNAs form an interaction network, wherein the combined activity of miR-125b with let-7 and/or miR-99/100 family members converged to induce a stem cell signature creating a synthetic phenotype. The synthetic phenotype can only be observed in the combination of two or all three miRNAs but not for each miRNA alone, and is generated by miR-99/100 and/or let-7 altering the hematologically dominant miR-125 phenotype. This interactive network might explain the genomic miR-99/100~125 clustering and reveals a novel cooperative mode to induce self-renewal and a differentiation block in myeloid progenitor cells, predisposing them to leukemic transformation in APL.