Hematopoiesis, the formation of blood cells from hematopoietic stem cells (HSC), is a highly regulated process. Since the discovery of microRNAs (miRNAs), several studies have shown their significant ...role in the regulation of the hematopoietic system. Impaired expression of miRNAs leads to disrupted cellular pathways and in particular causes loss of hematopoietic ability. Here, we report a previously unrecognized function of miR-143 in granulopoiesis. Hematopoietic cells undergoing granulocytic differentiation exhibited increased miR-143 expression. Overexpression or ablation of miR-143 expression resulted in accelerated granulocytic differentiation or block of differentiation, respectively. The absence of miR-143 in mice resulted in a reduced number of mature granulocytes in blood and bone marrow. Additionally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia (AML). Overexpression of miR-143 in AML cells impaired cell growth, partially induced differentiation, and caused apoptosis. Argonaute2-RNA-Immunoprecipitation assay revealed ERK5, a member of the MAPK-family, as a target of miR-143 in myeloid cells. Further, we observed an inverse correlation of miR-143 and ERK5 in primary AML patient samples, and in CD34
HSPCs undergoing granulocytic differentiation and we confirmed functional relevance of ERK5 in myeloid cells. In conclusion, our data describe miR-143 as a relevant factor in granulocyte differentiation, whose expression may be useful as a prognostic and therapeutic factor in AML therapy.
Acute myeloid leukemia (AML) is a very heterogeneous disease of the hematopoietic system which is characterized by a fast progress with poor survival options. Despite intensive research standard ...treatment strategies rely since decades on the same chemotherapeutics, and the overall survival options still remain poor.
In recent years, there is an emerging role of microRNAs (miRNAs) in the regulation of cancer development and resistance to cancer therapies. Mechanistically, miRNAs regulate gene expression through modulation of multiple target mRNAs by mRNA degradation or block of translation. It is shown that miRNAs can act as tumor suppressors and oncogenes dependent on the type of cancer. The miRNA-143/145 cluster is shown to be down-regulated in most cancers and is able to modulate tumorigenesis by targeting tumor associated genes. Several miR-143/145 targets are well described oncogenes, such as ERK5, DNMT3A or Bcl2. Interestingly, recent studies showed that the miR-143/145 cluster can regulate multidrug resistance proteins (MDR) in various cancer types and that enforced expression of miR-143/145 leads to increased sensitivity to various drugs. Besides its well-studied function mainly in solid tumors the role of the miR-143/145 cluster or miR-143 alone especially in the hematopoietic system remains largely unknown.
In this study, we report a previously unrecognized function of miR-143 in granulopoiesis. Based on our observation that hematopoietic cells undergoing granulocytic differentiation exhibit increased miR-143 expression, we show that the absence of miR-143 in mice results in a reduced number of mature granulocytes but not monocytes in blood and bone marrow. Additionally, we show that overexpression or ablation of miR-143 resulted in accelerated granulocytic differentiation or block of differentiation in several in vitro systems, including leukemic cell lines and G-CSF treated CD34+ HSPCs, respectively.
To identify the mRNA targets affected by miR-143 in hematopoietic cells we performed Argonaut2-RNA-Immunoprecipitation coupled with next generation sequencing. Here we could demonstrate a strong enrichment of members of the MAPK- and mTOR-pathways in cells overexpressing miR-143. Besides several potential targets of miR-143 we found ERK5 as particularly interesting since ERK5 was found to be a central mediator of cell survival, proliferation, motility, differentiation and apoptotic regulation of normal cells. To test whether miR-143 overexpression would also affect ERK5 protein abundance we performed western blot analysis of ERK5 and activated ERK5 in hematopoietic cells infected with miR-143 O/E constructs. Here we could show that miR-143 leads to reduced ERK5 protein levels in these cells. Since deregulation of both factors miR-143 as well as ERK5 have been implicated in the response to chemotherapeutic agents we analyzed the impact of miR-143 expression on treatment response. Here we show that AML patients receiving 5-azacytidine (Vidaza) and respond to the therapy had significant higher miR-143 levels than patients which did not respond. To test whether miR-143 expression would affect the response to chemotherapeutics, we overexpressed miR-143 in leukemic cells followed by Vidaza and AC220 treatment. Our results show a significant increase of apoptosis in cells overexpressing miR-143. Finally, we observed an association of high miR-143 expression levels with a higher probability of survival in two different cohorts of patients with acute myeloid leukemia. Taken together, we identified miR-143 as a relevant factor in granulocytic differentiation, which expression may be useful as prognostic factor and as new target for therapeutic strategies in AML.
Schwind:Novartis: Consultancy.
Active
related (
) gene deactivates ras-related C3 botulinum toxin substrate 1 (RAC1), which plays an essential role in regulating normal hematopoiesis and in leukemia.
gene, closely related to ABR, ...acts as a tumor suppressor in chronic myeloid leukemia and has overlapping functions with
. Evidence for a putative tumor suppressor role of
has been shown in several solid tumors, in which deletion of ABR is present. Our results show downregulation of
in AML. A block of ABR prevents myeloid differentiation and leads to repression of the myeloid transcription factor C/EBPα, a major regulator of myeloid differentiation and functionally impaired in leukemia. Conversely, stable overexpression of ABR enhances myeloid differentiation. Inactivation of the known ABR target RAC1 by treatment with the RAC1 inhibitor NSC23766 resulted in an increased expression of C/EBPα in primary AML samples and in AML cell lines U937 and MV4;11. Finally, AML patients with high
expression at diagnosis showed a significant longer overall survival and patients who respond to azacitidine therapy showed a significant higher ABR expression. This is the first report showing that
expression plays a critical role in both myelopoiesis and AML. Our data indicate the tumor suppressor potential of
and underline its potential role in leukemia therapeutic strategies.
Up to 30% of all acute myeloid leukemias (AMLs) are associated with an activating mutation in the FMS-like tyrosine kinase 3 receptor (FLT3). Two distinct groups of FLT3 mutations are found: (1) the ...most common are internal tandem duplications (ITDs) of the FLT3 juxtamembrane region, and (2) point mutations within the tyrosine kinase domains (TKDs). While FLT3-TKD mutations seem to have no prognostic relevance in AML, patients bearing an FLT3-ITD mutation have a significantly worse outcome compared with AML patients with wild-type FLT3 (FLT3-WT). MicroRNAs (miRNAs) are small (~22 bp) noncoding RNAs, which regulate protein expression posttranscriptionally by recruitment of the RNA-induced silencing complex (RISC) to the 3′-untranslated region (3′-UTR) of target mRNAs. We and others have shown that miRNAs are crucial regulators in myeloid differentiation and in leukemogenesis. Furthermore, it was shown that several miRNAs have a prognostic impact. Hence, we hypothesized that the different FLT3 mutations lead to altered miRNA expression.
To find different expression patterns of miRNAs, we performed next generation sequencing of normal karyotype bone marrow patient samples with FLT3-WT (n=5), FLT3-TKD (n=3) and FLT3-ITD (n=3). Sequencing was performed with an Illumina HighScan-SQ sequencer using version 3 chemistry and flowcell according to the instructions of the manufacturer. For normalization the method of trimmed mean of M values (TMM) was used. Data analyses were performed using the Qlucore Omics Explorer 3.1. In a multi group analyses of miRNA expression pattern, we found 17 significant differentially expressed miRNAs (p ≤ 0.05). The expression of 6 miRNAs (miR-10a-5p, miR-10a-3p, miR-18a-5p, let-7b-3p, miR-155-5p and miR-576-5p) was increased only in the FLT3-ITD associated patient samples. In the FLT3-WT samples we found 8 miRNAs (miR-141-3p, 342-3p, 181a-2-3p, 374b-5p, 30b-5p, 29c-3p, 23b-3p and 125a-3p) with an increased expression. The miR-92a-3p showed an enhanced expression in FLT3-WT and FLT3-ITD patient samples. The multi group analyses showed only 2 miRNAs (miR-3615 and miR-193b-3p) induced in FLT3-TKD patient samples. The two FLT3-ITD induced miRNAs, miR-10a-5p and miR-155-5p were the most abundant and most differentially expressed miRNAs in the screen. From our data we hypothesize that miR-155 and miR-10a could play an important role in disease progression and clinical outcome of FLT3-ITD induced AMLs
To analyze a block of miR-155 in FLT3-ITD driven AML in vivo, we transfected 32D cells, stably expressing human FLT3-ITD, with unspecific scramble or miR-155 specific locked nucleic acids (LNAs (Exiqon)). 24h after transfection, we injected 1x106 cells into C3H mice (scr. n=5; LNA-155 n=5). All animals rapidly developed a leukemia like disease with hepatosplenomegaly. The animals died 17 - 21 days after 32DFLT3-ITD cell injection. We could not observe a difference in survival. In flow cytometry analysis of the peripheral blood we found a strong increase of human FLT3 (huCD135) expressing cells (32DFLT3-ITD) 1 to 2 days before the mice died. At death of the animals we analyzed the accumulation of leukemic cells (32DFLT3-ITD) in bone marrow, spleen and liver by flow cytometry for the human FLT3 (huCD135). Here we could observe a significantly (p≤ 0.05) reduced number of leukemic cells in the bone marrow of the mice with the LNA-155 transfected 32DFLT3-ITD cells in comparison to the group with the scramble transfected 32D cells. In spleen we could not observe a difference in accumulation of leukemic cells, but in the liver we could show a tendentially reduced accumulation of 32DFLT3-ITD cells transfected with LNA-155.
The next generation sequencing screen gives insight into the altered miRNA expression pattern of FLT3-WT, FLT3-TKD and FLT3-ITD related AMLs. The miR-10a-5p and miR-155-5p are highly expressed in FLT3-ITD associated AMLs. The block of the FLT3-ITD induced miR-155 in vivo significantly reduces the accumulation of leukemic cells in the bone marrow of transplanted mice. The results give the evidence that miR-155 could be a novel therapeutic target in FLT3-ITD associated AML.
No relevant conflicts of interest to declare.
ABR (Active BCR-related) is the only protein in humans and mice closely homologous to BCR. BCR acts as a tumor suppressor in different cancers, such as chronic myeloid leukemia and meningiomas. A ...putative anti-oncogenic role of ABR has been shown in tumors of the central nervous system, such as medulloblastoma and astrocytomas, in which deletion of ABR was found. However, the role of ABR in hematopoiesis or leukemia remains unclear. We hypothesized that ABR might be important for myelopoiesis via increasing the expression of C/EBPα, a transcription factor known to be pivotal for myeloid differentiation and functionally impaired in acute myeloid leukemia (AML). In fact, we found that ABR expression is dramatically down-regulated (Figure 1, p=0.01) in bone marrow from AML patients (pts; n=63) compared to bone marrow (BM) mononuclear cells from healthy donors (n=3). In agreement with this finding, Abr is significantly increased during M-CSF and G-CSF-stimulated differentiation of primary wild type mouse BM cells (p<0.05). Additionally, we observe that ABR is necessary for monopoiesis induced by PMA (phorbol 12-myristate 13-acetate), since ABR knockdown in leukemic U937 cells results in a significant reduction of about 50% in the number of CD11b+ cells 48h after PMA treatment (p<0.05). Enforced ABR expression induces C/EBPα and its targets M-CSFR, G-CSFR and microRNA (miR)-223 in U937 cells (p<0.01). Moreover, we prove that ABR knockdown prevents induction of CEBPA, M-CSFR and G-CSFR during PMA-mediated differentiation (p<0.05). ABR overexpression blocks cell-cycle progression and down-regulates the known C/EBPα inhibitor E2F1 (p<0.01) in U937 cells, indicating the functional role of ABR as tumor suppressor. Those data suggest that ABR might induce CEBPA expression via inhibition of cell cycle activator E2F1. Finally, we are the first to identify ABR as a good prognostic factor in AML: patients with high ABR expression (median cut) survive significantly longer after allogeneic hematopoietic stem cell transplantation (Figure 2, p=0.04, log-rank test). Furthermore, high ABR expression associates with a low percentage of blasts in the peripheral blood (p=0.006) and high levels of antileukemic miR-181a (p<0.001). In conclusion, these data indicate that ABR, a novel inducer of C/EBPα, is necessary for myelopoiesis and a prognostic factor in AML. Raising ABR levels might be a goal for future therapeutics in AML. Display omitted
No relevant conflicts of interest to declare.
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