the miR-99∼125 Polycistrons Promote Leukemogenesis in a Cell-Context Dependent Manner by Shifting the Balance Between TGFβ- and Wnt-Signaling

Abstract 109

Three human miR-99∼125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are located within or next to long non-coding RNA (lncRNA) host genes on distinct chromosomal regions. In this study, we investigated the integrative function of these three polycistron-miRNAs. Expression profiling in sorted leukemic blasts indicated an enrichment of mature miR-99∼125 miRNAs in acute megakaryoblastic leukemia (AMKL) and promyelocytic leukemia (APL). We demonstrated that intronic miR-99∼125 miRNAs are produced from one primary transcript with their lncRNA host genes and that the leukemogenic transcription factor and stem cell regulator HOXA10 acts as an upstream transcriptional activator. Therefore, we assessed the consequences of concerted activation of miR-125b, miR-99/100 and let-7 in the megakaryocytic and myelomonocytic system.

During megakaryocytic in vitro differentiation of hematopoietic stem and progenitor cells (HSPCs), lentiviral overexpression of the tricistron (miR-99a/let-7c/miR-125b-2) or miR-125b-2 alone increased proliferation and survival of megakaryocytic cells. Megakaryocytic/erythroid progenitors (MEP) were expanded upon tricistron and miR-125b-2 overexpression, and not by miR-99a and/or let-7c overexpression. In an unbiased triple-transduction RGB-competition assay using distinct color-marked (red/green/blue) monocistronic miRNA constructs, miR-125b-2 alone or in combination with miR-99a or let-7 conferred a selective growth advantage to murine and human HSPCs. In murine transplantation experiments, only combined expression of all three cluster-miRNAs led to the expansion and retention of an immature Gr-1(low)/Mac-1(+)/B220(-) cell population in the bone marrow. Similarly when the miR-99a/let-7c/miR-125b-2 tricistron-transduced HSPCs differentiated along the myelomonocytic lineage (liquid culture and methylcellulose colony-forming assay), we observed a major cell population of monomorphic, non-adherent cells devoid of granulocytic and monocytic markers, which was not present in the miR-99a-, let-7c- or miR-125b-2 single transduced cells.

Global gene expression profiling followed by qRT-PCR-validation of transduced HSPCs identified high confidence targets commonly or individually downregulated by the three miRNAs. Among these targets, we found TGFβ pathway agonists and Wnt antagonists to be enriched. miR-125b and -even stronger- the tricistron decreased the reporter activity of a SMAD response element in 293T cells treated with TGFβ and increased the activity of a TCF response element in synergy with a Wnt-activator (GSK3-inibitor). Western blotting and luciferase reporter assays demonstrated cooperative targeting of TGFBR1, ALK7, BMPR1a, BMPR2, SMAD2, SMAD4 and APC by the tricistron. Strikingly, inhibition of miR-125b by a lentiviral decoy sensitized TGFβ-resistant AMKL-cell lines (CMK and CMY) to TGFβ-mediated apoptosis and proliferation arrest. Conversely, overexpression of miR-125b or the tricistron rendered MV4:11 and NB4 cells resistance to TGFβ. miR-125b or the tricistron-transduced HSPCs displayed a marked increase in cell viability and cells in S-Phase upon TGFβ-treatment during megakaryocytic differentiation. The application of a TGFβ-inhibitor phenocopied the effects of miR-125b or the tricistron during megakaryocytic differentiation. These effects were not further enhanced by combined Wnt activation. In contrast, recapitulating the effects of the polycistron in the myelomonocytic lineage required Wnt-activation.

Collectively we deciphered the functional linkage of miR-99/100, let-7 and miR-125, which are produced from one primary transcript with their lncRNA host genes and are regulated by the stem cell regulator HOXA10. Our study creates a microRNA interaction network, wherein the concerted action of the three miRNAs converges at the TGFβ pathway by a combinatorial block of this pathway. During myelomonocytic commitment only the combination of all polycistron-miRNAs enhanced Wnt signaling to inhibit differentiation. These synthetic phenotypes form an epistatic circuit regulating stemness and developmental fate.