Alexander Arthur Wurm

Transcription factor C/EBPα-induced microRNA-30c inactivates Notch1 during granulopoiesis and is downregulated in acute myeloid leukemia

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBPα exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBPα during granulopoiesis. Thus, wild-type C/EBPα-p42 directly upregulates miR-30c expression, whereas C/EBPα-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBPα-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBPα, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.

The Janus-faced Nature of miR-22 in Hematopoiesis: Is It an Oncogenic Tumor Suppressor or Rather a Tumor-Suppressive Oncogene?

Hematopoiesis is a complex, multistep process originating from hematopoietic stem cells and structured into different lineages. Cell fate decision is well organized and strictly regulated by extrinsic and intrinsic molecules (such as cytokines and transcription factors) whose deregulation is connected to leukemic transformation. In recent years, the importance of microRNAs in controlling hematopoiesis has been discussed [1]. Since their discovery in 1993 [2], microRNAs have been intensively studied in multidisciplinary research fields and have even attracted the attention of the pharmaceutical industry as potential therapeutic targets in various diseases. The first drugs based on microRNA inhibition have already been approved [3]. Of note, increasing numbers of scientific publications focus on their role in oncology. It has been shown that microRNAs have great impact on cancer initiation or prevention, and several candidates were described to act as either tumor suppressors or oncogenes. While certain microRNAs can act as either tumor suppressors or oncogenes in different tissues, the observation of contradictory functions of a single microRNA in the same tissue and even the same cell type is rare and unusual. Looking at the myeloid lineage in the hematopoietic system, miR-181a is such a candidate: while Hickey et al. postulated its tumor-suppressive function in acute myeloid leukemia (AML) [4], several other groups revealed the oncogenic potential of miR-181a in the myeloid background [5, 6].

EVI2B is a C|[sol]|EBP|[alpha]| target gene required for granulocytic differentiation and functionality of hematopoietic progenitors

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

miR-451a abrogates treatment resistance in FLT3-ITD-positive acute myeloid leukemia

Acute myeloid leukemia (AML) is a fatal disease with up to 95% of patients remaining incurable. While supportive care has been increased in quantity and quality; substantial progress of AML therapy itself is still lacking. Primarily two predicaments challenge current therapy: first, the heterogeneity of the disease; second, the phenomenon of treatment resistance. Although many AML patients initially respond to therapy, the majority of patients relapse caused by chemoresistant clones, ultimately leading to the incurability of the disease. Approximately a quarter of AML patients present with FLT3-gain-of-function mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways and changes gene expression patterns from healthy to malignant. On account of the poor prognosis of FLT3-ITD AML patients, FLT3-inhibitors are under development and in clinical testing. Nevertheless, acquired treatment resistance persists. This underlines the necessity of a greater knowledge on the origins of resistance as well as new approaches abrogating treatment resistance. Therefore, we propose to deviate from the common strategy of target inhibition and focus on the reinforcement of negatively regulated downstream targets of wellknown oncogenes such as FLT3-ITD. Because microRNA (miR) expression was shown to be altered by FLT3-ITD, this group of small non-coding RNAs is of interest in this work. By binding to the 3’UTRs of mRs, miRs post-transcriptionally regulate gene expression. Thus, miRs hold key regulatory functions in processes such as hematopoiesis and leukemogenesis. In accordance with this, various miRs have been shown to impact treatment resistance as well as sensitivity toward AML therapy. First clinical trials disclosed the potential of miR-inhibitors to be a promising therapeutic option. Nevertheless, no miR-based experimental drugs are currently available in AML. Therefore, we screened for differentially expressed miRs in bone marrow mononuclear cells from healthy donors and AML patients with either FLT3-wildtype, mutations in the FLT3-tyrosine kinase domain (TKD) or FLT3-ITD mutations (Fig. 1a, Supplementary Table 1). Here, we found miR-451a to be significantly (p= 0.002) lower expressed in FLT3-ITD AML patients compared to healthy individuals; while among the remaining AML samples screened, miR-451a levels were lowest in FLT3ITD patient samples (Fig. 1b). In agreement with this, we observed increasing miR-451a levels upon differentiation of human and murine hematopoietic cells (Supplementary Figure 1a-c). In addition, we analyzed the AML patient cohort of “The Cancer Genome Atlas” (TCGA) and discovered a similar miR-451a expression distribution (Fig. 1c). In order to confirm our observations in vitro, we assessed miR-451a levels by qPCR in FLT3-ITDtransduced U937 and 32D cells (Fig. 1d,e). Indeed, a stable overexpression of FLT3-ITD led to a decrease in miR-451a levels. In contrast, interruption of FLT3-ITD signaling by treatment of MV4;11 cells with FLT3 inhibitors quizartinib and gilteritinib (currently evaluated in clinical trials) caused an increase in miR-451a expression (Fig. 1f). As a healthy control, we treated mononuclear cells from umbilical cord blood (UCB) with quizartinib as well as gilteritinib and observed neither a significant change in

ABR, a novel inducer of transcription factor C/EBPα, contributes to myeloid differentiation and is a favorable prognostic factor in acute myeloid leukemia

Active BCR related (ABR) gene deactivates ras-related C3 botulinum toxin substrate 1 (RAC1), which plays an essential role in regulating normal hematopoiesis and in leukemia. BCR gene, closely related to ABR, acts as a tumor suppressor in chronic myeloid leukemia and has overlapping functions with ABR. Evidence for a putative tumor suppressor role of ABR has been shown in several solid tumors, in which deletion of ABR is present. Our results show downregulation of ABR 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 ABR 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 ABR expression plays a critical role in both myelopoiesis and AML. Our data indicate the tumor suppressor potential of ABR and underline its potential role in leukemia therapeutic strategies.

Incubation of Immune Cell Grafts With MAX.16H5 IgG1 Anti-Human CD4 Antibody Prolonged Survival After Hematopoietic Stem Cell Transplantation in a Mouse Model for Fms Like Tyrosine Kinase 3 Positive Acute Myeloid Leukemia

Despite the constant development of innovative therapeutic options for hematological malignancies, the gold-standard therapy regimen for curative treatment often includes allogeneic hematopoietic stem cell transplantation (HSCT). The graft-vs.-leukemia effect (GVL) is one of the main therapeutic goals that arises from HSCT. On the other hand, graft-vs.-host disease (GVHD) is still one of the main and most serious complications following allogeneic HSCT. In acute myeloid leukemia (AML), HSCT together with high-dose chemotherapy is used as a treatment option. An aggressive progression of the disease, a decreased response to treatment, and a poor prognosis are connected to internal tandem duplication (ITD) mutations in the Fms like tyrosine kinase 3 (FLT3) gene, which affects around 30% of AML patients. In this study, C3H/HeN mice received an allogeneic graft together with 32D-FLT3ITD AML cells to induce acute GVHD and GVL. It was examined if pre-incubation of the graft with the anti-human cluster of differentiation (CD) 4 antibody MAX.16H5 IgG1 prevented the development of GVHD and whether the graft function was impaired. Animals receiving grafts pre-incubated with the antibody together with FLT3ITD AML cells survived significantly longer than mice receiving untreated grafts. The observed prolonged survival due to MAX.16H5 incubation of immune cell grafts prior to transplantation may allow an extended application of additional targeted strategies in the treatment of AML.

MicroRNA-143 targets ERK5 in granulopoiesis and predicts outcome of patients with acute myeloid leukemia

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.

Correction to: EVI2B is a C/EBP α target gene required for granulocytic differentiation and functionality of hematopoietic progenitors

Since publication of this article, the authors identified an error in the acknowledgments section. The original sentence in acknowledgments reads as follows: