5q- syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q- syndrome.

The G protein-coupled receptor 56 (GPR56) was identified as part of the molecular signature of functionally validated leukemic stem cells isolated from patients with acute myeloid leukemia (AML). This report now demonstrates particularly high expression of GPR56 in patients with mutant NPM1 and FLT3-length mutation and association of high GPR56 expression with inferior prognosis in a large patient cohort treated in two independent multicenter phase III trials. Functional relevance of GPR56 expression was validated in mice, in which co-expression of Gpr56 significantly accelerated HOXA9-induced leukemogenesis and vice versa knockdown of Gpr56 delayed onset of HOXA9/MEIS1-induced AML. Overexpression of Gpr56 grossly changed the molecular phenotype of Hoxa9-transduced cells affecting pathways involved in G protein-coupled receptors (GPRCs) and associated intracellular signaling. Blockage of surface GPR56 by an anti-GPR56 antibody successfully impaired engraftment of primary human AML cells. In summary, these data demonstrate that high expression of GPR56 is able to contribute to AML development and characterize the GPR56 as a potential novel target for antibody-mediated antileukemic strategies.

A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell–containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34+ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.

MEIS1 is a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is ratelimiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by IntroductionMultiple lines of evidence now point to the key roles of MEIS1, a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor, in both normal hematopoiesis and leukemogenesis. In the context of normal hematopoiesis, Meis1 is preferentially expressed in primitive bone marrow (BM) cells enriched for hematopoietic stem cells (HSCs), 1 and Meis1 knockout mice are embryonic lethal presenting with severely impaired hematopoiesis. 2,3 Deregulated expression of Meis1 accelerates the onset of disease induced by various Hox and NUP98-HOX fusion genes, including genes without leukemogenic potential on their own (reviewed in Argiropoulos and Humphries 4 ). Meis1 expression is also essential and rate limiting for the leukemogenicity of multiple MLL fusions, 5,6 and HOX and MEIS1 gene expression is frequently dysregulated in leukemia patient samples (reviewed in Argiropoulos and Humphries 4 ).Despite the well-documented role of Meis1 in the promotion of acute myelogenous leukemia (AML), the regulatory networks controlled by Meis1 are not fully known. The identification of Flt3, Cd34, Erg1,7,9 and Trib2 10 as Meis1 target genes have provided valuable insight into the function of Meis1. A group of these genes can replace Meis1 and serve as collaborating genes in leukemic transformation, albeit, with decreased potencies compared with Meis1. [10][11][12] However, as in the case with Flt3, the target may be dispensable. 13 Thus, the potency of Meis1 to induce leukemia appears to be related to its ability to modulate multiple pathways. Interesting in this regard is the recently reported link between Meis1 and cell-cycle control. In the context of mixed lineage leukemia (MLL)-fusion leukemias, Meis1 overexpression was observed to correlate with increased cell-cycle entry and modest up-regulation of Bmi1. 5 Moreover, gene expression profiling of murine Mll-AF9 leukemic BM cells transduced with Meis1 short-hairpin RNA showed reduced expression of genes associated with cell-cycle entry, consistent with the impaired cell growth of these cells. 6 The linkage to cell cycle gains currency in light of 2 recent reports that showed a correlation between Meis1 activity, retinal progenitor cell proliferation, and cyclin D1 expression in the developing chick and zebrafish. 14,15 Together, these data provide the basis for further investigation into the role of Meis1 in cell-cycle regulation.Experiments with the Drosophila melanogaster ortholog of Meis1, homothorax (hth), have shown that HTH fused to the potent ...

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