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.
•Granulocyte activation and inflammatory cytokine signaling by interferon gamma and IL-6 drive TCL progression and disease symptoms.•Jak1/2 inhibitors simultaneously target malignant T cells and ...inflammatory granulocytes in ITK-SYK mice and human PTCL–derived xenografts.
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Peripheral T-cell lymphomas (PTCLs), especially angioimmunoblastic and follicular TCLs, have a dismal prognosis because of the lack of efficient therapies, and patients’ symptoms are often dominated by an inflammatory phenotype, including fever, night sweats, weight loss, and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on T-cell follicular helper–type PTCL (TFH-PTCL) disease progression and symptoms. We showed that ITK-SYK–driven murine PTCLs and primary human TFH-PTCL xenografts both induced inflammation in mice, including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving interferon gamma (CD4+ malignant T cells) and interleukin 6 (IL-6; activated granulocytes), ultimately inducing broad JAK activation (JAK1/2/3 and TYK2) in both cell types. Inflammatory granulocyte depletion via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox), or IL-6 deletion within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration, and enhanced mouse survival. Furthermore, unselective JAK inhibitors (ruxolitinib) inhibited both TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions, and an intact JAK/STAT signaling pathway for TFH-PTCL development and also support broad JAK inhibition as an effective treatment strategy in early disease stages.
Jaeger et al elucidated the role of neutrophils in peripheral T-cell lymphomas with a strong inflammatory phenotype. The authors focused on follicular T-cell lymphoma (T-follicular helper type). They show that these tumors induce inflammation and neutrophil expansion accompanied by marked cytokine release. Depletion of neutrophils, removal of interleukin-6, and inhibition of JAK-STAT signaling with ruxolitinib all reduced inflammation and tumor progression.
•RUNX1 isoform disequilibrium toward RUNX1A and its interaction with MYC:MAX are key in the pathogenesis of trisomy 21–associated ML.•Restoration of RUNX1A:RUNX1C equilibrium and pharmacological ...interference with MYC:MAX dimerization reverses the oncogenic phenotype.
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Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR–CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.
Trisomy 21 (+21) is a frequent alteration in acute myeloid leukemias (AML) and constitutive in Down syndrome myeloid leukemia (ML-DS). In this Plenary Paper, Gialesaki et al show that ML-DS blasts are dependent on RUNX1 and reveal that disequilibrium among RUNX1 transcripts with disproportionately increased level of RUNX1A is pivotal for DS leukemogenesis and prominent in sporadic AML. This validates a new model for the early stages of ML-DS development and suggests new therapeutic targets for +21 AMLs in general.
Late-onset hypogonadism (LOH), defined as a combination of low serum testosterone (T) levels in combination with clinical signs and symptoms of androgen deficiency in ageing men, is nowadays a ...well-characterized disease. Testosterone therapy in males affected by hypogonadism leads to a significant decrease of fat mass. In humans, the exact molecular mechanism of T effects on inhibition of adipogenesis is still unknown. We hypothesized that specific microRNAs could be regulated by androgens which might cause an inhibition of adipogenic differentiation. To confirm this hypothesis, human mesenchymal stem cells and a preadipocyte cell line were differentiated into mature adipocytes and in parallel treated with testosterone and dihydrotestosterone. The expression level of miR-375 was upregulated during adipogenic differentiation and downregulated after androgen treatment. Furthermore, we could show that after androgen treatment the decreased expression of miR-375 led to increased expression levels of adiponectin receptor 2 (ADIPOR2) compared to untreated adipocytes. Moreover, inhibition of miR-375 also mediated a decreased adipogenic differentiation and increased ADIPOR2 expression levels. In summary, we identified miR-375 as an androgen regulated microRNA, which could play an important role for understanding the mechanism of the increase in visceral fat mass and the associated insulin resistance caused by testosterone deficiency.
•Androgens inhibit adipogenic differentiation of mesenchymal progenitor cells.•In response to androgenic stimulus, alterations in the microRNA expression are induced.•Human adiponectin receptor 2 is a target of androgen-repressed miR-375.•Both are factors in androgen-dependent inhibition of adipogenesis.
In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. microRNAs are known to be critical players in the ...formation of the leukemic phenotype. In this study, we report downregulation of the miR-181a/b gene cluster in APL blasts and NB4 leukemia cells upon ATRA treatment as a key event in the drug response. We found that miR-181a/b expression was activated by the PML/RARα oncogene in cells and transgenic knock-in mice, an observation confirmed and extended by evidence of enhanced expression of miR-181a/b in APL patient specimens. RNA interference (RNAi)-mediated attenuation of miR-181a/b expression in NB4 cells was sufficient to reduce colony-forming capacity, proliferation, and survival. Mechanistic investigations revealed that miR-181a/b targets the ATRA-regulated tumor suppressor gene RASSF1A by direct binding to its 3'-untranslated region. Enforced expression of miR-181a/b or RNAi-mediated attenuation of RASSF1A inhibited ATRA-induced granulocytic differentiation via regulation of the cell-cycle regulator cyclin D1. Conversely, RASSF1A overexpression enhanced apoptosis. Finally, RASSF1A levels were reduced in PML/RARα knock-in mice and APL patient samples. Taken together, our results define miR-181a and miR-181b as oncomiRs in PML/RARα-associated APL, and they reveal RASSF1A as a pivotal element in the granulocytic differentiation program induced by ATRA in APL.
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.
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.
•miR-30c is a direct target of C/EBPα and upregulated by C/EBPα-p42.•NOTCH1 is a direct target of miR-30c and regulated by C/EBPα and miR-30c.
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.
Acute myeloid leukemia (AML) is a fatal malignant hematologic disorder. Although many patients with AML initially respond to standard chemotherapy and achieve complete remission (CR), the majority of ...patients relapse due to the development and clonal expansion of chemoresistant subclones. Albeit being a heterogenous disease, individual mutations are still scarcely considered in the current treatment regimen.
Approximately 30% of AML patients exhibit gain-of-function mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-receptor) gene. Here, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor is among the most frequently observed mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways, leading to differential gene expression patterns and aberrant cell growth. Clinically, FLT3-ITD mutations confer adverse prognosis. Thus, many promising FLT3 signaling inhibitor-based therapies are currently utilized to improve patient survival. However, the phenomenon of treatment resistant subclones persists and constantly calls for new angles of treatment.
MicroRNAs, a group of small non-coding RNAs, regulate gene expression on a posttranscriptional level by a complementary binding to 3'UTRs of protein-coding mRNAs. They have been shown to influence healthy and malignant hematopoiesis and to predict treatment response in cancer. In addition, several candidates have been connected to treatment resistance or sensitivity in AML.
Within this study, we hypothesized that microRNAs are biological important players in the adverse treatment response and the development of chemoresistance in FLT3-ITD associated AML.
Initially, we were interested in the microRNA expression pattern in FLT3-ITD associated AML. Therefore, we performed a next generation sequencing screen in bone marrow mononuclear cells from healthy donors, AML patients with FLT3-wildtype and AML patients with FLT3-ITD mutation. Here, we identified miR-451 to be significantly lower expressed in AML compared to healthy individuals. Furthermore, it was nearly absent in FLT3-ITD associated AML. In addition, we compared our results to the AML patient cohort of “The Cancer Genome Atlas” (TCGA) and found a similar miR-451 expression distribution. In agreement with this, miR-451 expression increased during differentiation of primary human and mouse hematopoietic cells. Moreover, transduction of U937 as well as 32D cells with FLT3-ITD led to a decrease in miR-451 expression.
Downregulation of miR-451 has been shown for various cancerous diseases to correlate with an increased cancer persistence as well as recurrence, as one established target of miR-451 is multi drug resistance protein 1 (MDR1). MDR1 is an ATP dependent efflux pump that mediates chemoresistance by transporting drugs out of the cell. Today, only limited data are available on the miR-451-MDR1 axis in FLT3-ITD associated AML. Thus, we hypothesized that low miR-451 expression and high MDR1 activity is associated with treatment resistance.
Therefore, we showed an inverse correlation of miR-451 expression and MDR1 protein levels in various leukemic cells lines. Furthermore, we observed that a repression of FLT3-ITD signaling by both Quizartinib and Gilteritinib causes a miR-451 expression increment and a subsequent reduction of MDR1 protein levels. By luciferase activity assay, we could demonstrate a direct binding of miR-451 to the 3'UTR of MDR1. In order to investigate whether miR-451 alone is able to abrogate treatment resistance, we overexpressed miR-451 in both normal MV4;11 and treatment resistant MV4;11 cells (MV4;11-R). Subsequently, MDR1 protein levels were decreased and cells exhibited an enhanced sensitivity towards both FLT3-ITD inhibitor and chemotherapeutic agent-based treatment. Moreover, exogenous miR-451 was able to restore drug sensitivity in MV4;11-R cells comparable to that of normal MV4;11 cells. As proof of principle, MDR1 inhibitor Tariquidar mimicked miR-451-mediated effects and sensitized MV4;11-R cells to FLT3-ITD inhibitor Quizartinib.
In conclusion, we identified a novel mechanism by which FLT3-ITD is able to alter treatment response and confer a poor prognosis. Hence, not only inhibiting FLT3-ITD but mimicking the effects of negatively regulated downstream targets such as miR-451 presents as promising new avenue for FLT3-ITD AML treatment.
Thiede:Bayer: Consultancy, Speakers Bureau; Roche: Consultancy; Novartis: Consultancy, Speakers Bureau; Agendix: Employment.
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