MicroRNAs in Normal and Malignant Myelopoiesis

__abstract__ Hematopoiesis is the lifelong continuous process in which hematopoietic stem and progenitor cells (HSPCs) proliferate and differentiate towards mature blood cells. Hematopoiesis is tightly controlled by a network of growth factors and the hematopoietic niche in the bone marrow (BM). This ensures the balanced blood cell production under homeostatic conditions and allows for transient elevation of specific blood cell types production in response to infections or bleeding 1. In mammalian organisms, long-term hematopoietic stem cells (LT-HSCs) reside in the BM and have self-renewal capacity over the lifespan of the organism 2,3. The estimated amount of LT-HSCs is approximately 0.007% of all hematopoietic cells in the BM 4. LT-HSCs give rise to short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs) (Figure 1). These cells have the potential to differentiate into all the different hematopoietic cell types but have less self-renewal capacity 4. Together, LT-HCSs, ST-HCSs and MPPs constitute 0.05% of mouse BM cells 2. MPPs differentiate into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) (Figure 1). Subsequently, CLPs differentiate into B-cell and T-cell lineages. CMPs first develop into more specified myeloid progenitors, which are megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs). Granulocytes, monocytes and macrophages arise from GMPs, whereas MEPs differentiate towards erythrocytes and thrombocytes (platelets) (Figure 1). The process of differentiation of HSPCs towards mature myeloid cells is referred as myelopoiesis. In adult mammalian organisms, myelopoiesis occurs in the BM. Disruption of the balance between cell proliferation, differentiation and cell death leads to different hematopoietic disorders, e.g., leukemia, characterized by proliferation of undifferentiated cells, or bone marrow failure (BMF), characterized by impaired hematopoiesis involving one or multiple hematopoietic lineages 5,6. Proliferation and differentiation of HSPCs are coordinated by gene expression programs driven by endogenous and exogenous factors. MicroRNAs (miRNAs) are a class of non-coding RNAs which function as regulators of gene expression. In the studies described in this thesis the role of miRNAs in normal myelopoiesis and their involvement in acute myeloid leukemia (AML) and Fanconi anemia (FA), the most frequent inherited form of BMF syndromes are investigated. AML, FA and miRNAs will be further introduced in the following sections

Stop the dicing in hematopoiesis: What have we learned?

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis

Dicer1 deletion in myeloid-committed progenitors causes neutrophil dysplasia and blocks macrophage/dendritic cell development in mice

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered geneexpression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huë t anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs

ICL-induced miR139-3p and miR199a-3p have opposite roles in hematopoietic cell expansion and leukemic transformation

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1- deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors