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Allogeneic hematopoietic stem cell transplantation is the only curative option for a variety of diseases. Despite advances, it is associated with considerable morbidity and mortality, often involving liver complications. Liver disease can be characterized using ultrasound-based liver stiffness measurement. To assess its prognostic value, consecutive patients undergoing allogeneic hematopoietic stem cell transplantation were prospectively evaluated in a single-center study. Endpoints included liver event-free survival and all-cause mortality at 1 year. Competing risk and Cox-regression were used for analysis. We evaluated 106 patients (42 female, age 57) and observed 33 life-threatening events (14 died) including 16 liver complications at 100 days. At 1 year, 36 patients had died, 20 with disease relapse. The hazard ratios for liver-related complications at 100 days were 3.2 (95% CI: 1.8–14.6, p= 0.0022) and 4.4 (95% CI: 1.6–11.9, p= 0.0042) for elevated transient elastography (n= 11) and shear-wave velocity (n= 31), respectively. Results were analogous for all-cause mortality at 1 year. Prior stem cell therapy and elevated gamma glutamyltransferase were also associated with outcome. This demonstrates that elastography is a promising and viable tool for risk prediction and should be included in upcoming multi-center trials to establish new means of guiding treatment and prophylaxis.

Age-related somatic mutations linked to clonal hematopoiesis have been found in apparently healthy individuals and increase the risk of developing hematologic malignancies. In acute myeloid leukemia (AML) the clinical relevance of clonal hematopoiesis remains controversial and data on patients with detectable clonal hematopoiesis, consolidated with hematopoietic stem cell transplantation are limited. We analyzed samples from 113 AML patients in complete remission prior to hematopoietic stem cell transplantation for the presence of clonal hematopoiesis-associated mutations. The results were correlated with clinical and biological data. In complete remission we found 75 mutations previously linked to clonal hematopoiesis in 47 patients (41.6%). Twenty patients had ≥2 mutations linked to clonal hematopoiesis. DNMT3A, TET2, and ASXL1were most frequently mutated. When compared to pre-treatment samples we found variable patterns of mutation persistence depending on the gene mutated. In AML patients after allogeneic hematopoietic stem cell transplantation the presence of clonal hematopoiesis-associated mutations in complete remission did not associate with inferior clinical outcome. This study demonstrates that clonal hematopoiesis is a frequent phenomenon in AML patients. Presence of clonal hematopoiesis has no negative prognostic impact in the context of an allogeneic hematopoietic stem cell transplantation and might be beneficial if certain genes are affected.

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.

Transcriptional mechanisms responsible for low DAPK2 levels in particular AML subtypes. DAPK2 is a proapoptotic protein that is mostly expressed in the hematopoietic tissue. A detailed DAPK2 expression analysis in two large AML patient cohorts revealed particularly low DAPK2 mRNA levels in APL. DAPK2 levels were restored in APL patients undergoing ATRA therapy. PML‐RARA is the predominant lesion in APL causing transcriptional repression of genes important for neutrophil differentiation. We found binding of PML‐RARA and PU.1, a myeloid master regulator, to RARA and PU.1 binding sites in the DAPK2 promoter. Ectopic expression of PML‐RARA in non‐APL, as well as knocking down PU.1 in APL cells, resulted in a significant reduction of DAPK2 expression. Restoring DAPK2 expression in PU.1 knockdown APL cells partially rescued neutrophil differentiation, thereby identifying DAPK2 as a relevant PU.1 downstream effector. Moreover, low DAPK2 expression is also associated with C/EBPα‐mutated AML patients, and we found C/EBPα‐dependent regulation of DAPK2 during APL differentiation. In conclusion, we identified first inhibitory mechanisms responsible for the low DAPK2 expression in particular AML subtypes, and the regulation of DAPK2 by two myeloid transcription factors underlines its importance in neutrophil development.

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.

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 CEBPAmutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1as 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 CEBPAmutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPAgene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPAmutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPAmutations. These results define miR-34a as a novel therapeutic target in AML with CEBPAmutations.

The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPα is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPα, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPα, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPα is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPα, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPα, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

The translocation t(15;17) generates the chimeric PML-RARα transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARα transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)–chip experiments identified 372 direct genomic PML-RARα targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARα targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARα binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARα to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARα regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

The translocation t(15;17) generates the chimeric PML-RARα transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARα transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)–chip experiments identified 372 direct genomic PML-RARα targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARα targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARα binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARα to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARα regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

CCAAT/enhancer-binding protein α (C/EBPα) is a critical regulator for early myeloid differentiation. Mutations in C/EBPα occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominant-negative isoform (C/EBPαp30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPαp30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBPαp30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBPαp30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBPαp42 decreases the transactivation capacity on a minimal C/EBPα promoter. Importantly, overexpression of C/EBPαp30 in granulocyte colony-stimulating factor (G-CSF)–stimulated human CD34+ cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBPαp30 target through which C/EBPαp30 enhances the sumoylation of C/EBPαp42 to inhibit granulocytic differentiation.

CCAAT/enhancer-binding protein α (C/EBPα) is a critical regulator for early myeloid differentiation. Mutations in C/EBPα occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominant-negative isoform (C/EBPαp30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPαp30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBPαp30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBPαp30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBPαp42 decreases the transactivation capacity on a minimal C/EBPα promoter. Importantly, overexpression of C/EBPαp30 in granulocyte colony-stimulating factor (G-CSF)–stimulated human CD34+cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBPαp30 target through which C/EBPαp30 enhances the sumoylation of C/EBPαp42 to inhibit granulocytic differentiation.

The clinical progression of chronic myeloid leukemia (CML) from chronic phase to blast crisis is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes. This study was undertaken to investigate the influence of Bcr-Abl and of the small molecule Abl tyrosine–kinase inhibitor imatinib mesylate on granulocyte colony-stimulating factor (G-CSF)–induced neutrophilic differentiation. We show that differentiation of 32Dcl3 cells into mature granulocytes is accompanied by the increased expression of the antigens macrophage adhesion molecule–1 (Mac-1) and Gr-1, of the G-CSF receptor (G-CSFR), of myeloid transcription factors (CCAAT/enhancer-binding protein–α [C/EBPα], C/EBPε, and PU.1), and of the cyclin-dependent kinase inhibitor p27Kip1. In 32Dcl3 cells transfected with thebcr-abl gene (32DBcr-Abl), G-CSF did not trigger either granulocytic differentiation or the up-regulation of C/EBPα, C/EBPε, and the G-CSFR. This could be correlated to a defect in c-Myc down-regulation. In contrast, the up-regulation of PU.1 and p27Kip1 by G-CSF was not affected by Bcr-Abl. Importantly, incubation of 32DBcr-Ablwtcells with the kinase inhibitor imatinib mesylate prior to G-CSF stimulation completely neutralized the effects of Bcr-Abl on granulocytic differentiation and on C/EBPα and C/EBPε expression. Taken together, the results suggest that the Bcr-Abl kinase induces a reversible block of the granulocytic differentiation program in myeloid cells by disturbing regulation of hematopoietic transcription factors such as C/EBPα and C/EBPε.

The clinical progression of chronic myeloid leukemia (CML) from chronic phase to blast crisis is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes. This study was undertaken to investigate the influence of Bcr-Abl and of the small molecule Abl tyrosine–kinase inhibitor imatinib mesylate on granulocyte colony-stimulating factor (G-CSF)–induced neutrophilic differentiation. We show that differentiation of 32Dcl3 cells into mature granulocytes is accompanied by the increased expression of the antigens macrophage adhesion molecule–1 (Mac-1) and Gr-1, of the G-CSF receptor (G-CSFR), of myeloid transcription factors (CCAAT/enhancer-binding protein–α [C/EBPα], C/EBPε, and PU.1), and of the cyclin-dependent kinase inhibitor p27Kip1. In 32Dcl3 cells transfected with thebcr-ablgene (32DBcr-Abl), G-CSF did not trigger either granulocytic differentiation or the up-regulation of C/EBPα, C/EBPε, and the G-CSFR. This could be correlated to a defect in c-Myc down-regulation. In contrast, the up-regulation of PU.1 and p27Kip1by G-CSF was not affected by Bcr-Abl. Importantly, incubation of 32DBcr-Ablwtcells with the kinase inhibitor imatinib mesylate prior to G-CSF stimulation completely neutralized the effects of Bcr-Abl on granulocytic differentiation and on C/EBPα and C/EBPε expression. Taken together, the results suggest that the Bcr-Abl kinase induces a reversible block of the granulocytic differentiation program in myeloid cells by disturbing regulation of hematopoietic transcription factors such as C/EBPα and C/EBPε.

The transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1−/−mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion geneAML1-ETO. Here we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1. AML1-ETO physically binds to PU.1 in t(8;21)+Kasumi-1 cells. AML1-ETO binds to the β3β4region in the DNA-binding domain of PU.1 and displaces the coactivator c-Jun from PU.1, thus down-regulating the transcriptional activity of PU.1. This physical interaction of AML1-ETO and PU.1 did not abolish the DNA-binding capacity of PU.1. AML1-ETO down-regulates the transactivation capacity of PU.1 in myeloid U937 cells, and the expression levels of PU.1 target genes in AML French-American-British (FAB) subtype M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits antiproliferative function of PU.1. Overexpression of PU.1, however, differentiates AML1-ETO–expressing Kasumi-1 cells to the monocytic lineage. Thus, the function of PU.1 is down-regulated by AML1-ETO in t(8;21) myeloid leukemia, whereas overexpression of PU.1 restores normal differentiation.

The transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1−/− mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion geneAML1-ETO. Here we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1. AML1-ETO physically binds to PU.1 in t(8;21)+ Kasumi-1 cells. AML1-ETO binds to the β3β4 region in the DNA-binding domain of PU.1 and displaces the coactivator c-Jun from PU.1, thus down-regulating the transcriptional activity of PU.1. This physical interaction of AML1-ETO and PU.1 did not abolish the DNA-binding capacity of PU.1. AML1-ETO down-regulates the transactivation capacity of PU.1 in myeloid U937 cells, and the expression levels of PU.1 target genes in AML French-American-British (FAB) subtype M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits antiproliferative function of PU.1. Overexpression of PU.1, however, differentiates AML1-ETO–expressing Kasumi-1 cells to the monocytic lineage. Thus, the function of PU.1 is down-regulated by AML1-ETO in t(8;21) myeloid leukemia, whereas overexpression of PU.1 restores normal differentiation.

ABSTRACTThe transcription factor C/EBPα is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and C/EBPα can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPα knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPα knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPα downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPα expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPα. C/EBPα and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPα-induced granulocytic differentiation. Thus, we propose a model in which C/EBPα needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.

The transcription factor C/EBPα is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and C/EBPα can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPα knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPα knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPα downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPα expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPα. C/EBPα and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPα-induced granulocytic differentiation. Thus, we propose a model in which C/EBPα needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.

Recent results indicate that interactions of transcription factors with other nuclear proteins play an important role in stem cell development, lineage commitment and differentiation in the haematopoietic system, and the pathogenesis of myeloid leukaemias. High-throughput proteomics by mass spectrometric analysis of gel-separated proteins can identify multi-protein complexes and changes in the expression of multiple proteins simultaneously. This review describes an application of proteomic methods (2D gel electrophoresis (GE) and mass spectrometry (MS)), which can be used to identify regulated protein targets of transcription factors important in myeloid differentiation and leukaemia. This global high-throughput functional proteomics approach could lead to new insights into the network of protein–protein interactions and target proteins involved in myeloid stem cell development and leukaemia as well as provide new targets for rational pathogenesis-based therapies of leukaemia and cancer.

The transcription factor PU.1 is required for normal blood cell development. PU.1 regulates the expression of a number of crucial myeloid genes, such as the macrophage colony-stimulating factor (M-CSF) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. Myeloid cells derived from PU.1−/−mice are blocked at the earliest stage of myeloid differentiation, similar to the blast cells that are the hallmark of human acute myeloid leukemia (AML). These facts led us to hypothesize that molecular abnormalities involving the PU.1 gene could contribute to the development of AML. We identified 10 mutant alleles of the PU.1 gene in 9 of 126 AML patients. The PU.1 mutations comprised 5 deletions affecting the DNA-binding domain, and 5 point mutations in 1) the DNA-binding domain (2 patients), 2) the PEST domain (2 patients), and 3) the transactivation domain (one patient). DNA binding to and transactivation of the M-CSF receptor promoter, a direct PU.1 target gene, were deficient in the 7 PU.1 mutants that affected the DNA-binding domain. In addition, these mutations decreased the ability of PU.1 to synergize with PU.1-interacting proteins such as AML1 or c-Jun in the activation of PU.1 target genes. This is the first report of mutations in the PU.1 gene in human neoplasia and suggests that disruption of PU.1 function contributes to the block in differentiation found in AML patients.

The transcription factor PU.1 is required for normal blood cell development. PU.1 regulates the expression of a number of crucial myeloid genes, such as the macrophage colony-stimulating factor (M-CSF) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. Myeloid cells derived from PU.1−/− mice are blocked at the earliest stage of myeloid differentiation, similar to the blast cells that are the hallmark of human acute myeloid leukemia (AML). These facts led us to hypothesize that molecular abnormalities involving the PU.1 gene could contribute to the development of AML. We identified 10 mutant alleles of the PU.1 gene in 9 of 126 AML patients. The PU.1 mutations comprised 5 deletions affecting the DNA-binding domain, and 5 point mutations in 1) the DNA-binding domain (2 patients), 2) the PEST domain (2 patients), and 3) the transactivation domain (one patient). DNA binding to and transactivation of the M-CSF receptor promoter, a direct PU.1 target gene, were deficient in the 7 PU.1 mutants that affected the DNA-binding domain. In addition, these mutations decreased the ability of PU.1 to synergize with PU.1-interacting proteins such as AML1 or c-Jun in the activation of PU.1 target genes. This is the first report of mutations in the PU.1 gene in human neoplasia and suggests that disruption of PU.1 function contributes to the block in differentiation found in AML patients.

The transcription factor C/EBPα regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBPα gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBPα function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBPα is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBPα to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBPα via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBPα transactivation domain and does not change C/EBPα DNA binding. Ras acts on serine 248 of the C/EBPα transactivation domain, because it does not enhance the transactivation function of a C/EBPα serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBPα is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EBPα by Ras. Ras signaling leads to phosphorylation of C/EBPαin vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBPα to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBPα to induce granulocytic differentiation by phosphorylation of serine 248.

Several transcription factors have been implicated as playing a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of myeloid and lymphoid lineages, whereas the transcription factor CCAAT–enhancer binding protein family member C/EBPα is essential for granulocyte development. We present here the first evidence that C/EBPα blocks the function of PU.1. PU.1 and C/EBPα interact physically and colocalize in myeloid cells. As a consequence of this interaction, C/EBPα can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA-binding sites. We further demonstrate that the leucine zipper in the DNA-binding domain of C/EBPα interacts with the β3/β4 region in the DNA-binding domain of PU.1 and as a result displaces the PU.1 coactivator c-Jun. Finally, C/EBPα blocks PU.1-induced dendritic cell development from CD34+ human cord blood cells. The functional blocking of PU.1 by C/EBPα could be the mechanism by which C/EBPα inhibits cell fates specified by PU.1 and directs cell development to the granulocyte lineage.

Several transcription factors have been implicated as playing a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of myeloid and lymphoid lineages, whereas the transcription factor CCAAT–enhancer binding protein family member C/EBPα is essential for granulocyte development. We present here the first evidence that C/EBPα blocks the function of PU.1. PU.1 and C/EBPα interact physically and colocalize in myeloid cells. As a consequence of this interaction, C/EBPα can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA-binding sites. We further demonstrate that the leucine zipper in the DNA-binding domain of C/EBPα interacts with the β3/β4 region in the DNA-binding domain of PU.1 and as a result displaces the PU.1 coactivator c-Jun. Finally, C/EBPα blocks PU.1-induced dendritic cell development from CD34+human cord blood cells. The functional blocking of PU.1 by C/EBPα could be the mechanism by which C/EBPα inhibits cell fates specified by PU.1 and directs cell development to the granulocyte lineage.

Bei der Gewinnung von Bodenlösung mittels Saugkerzen können die Konzentrationen gelöster Stoffe in Abhängigkeit vom Kerzenmaterial durch Ad- und Desorptionsprozesse stark verändert werden. Zum Vergleich der bislang überwiegend üblichen Keramik-Saugkerzen mit neuartigen Saugkerzen aus Borosilikatglas wurden wässrige Lösungen von Pflanzenschutzmitteln (PSM) und DOC durch diese Kerzentypen geleitet. Dabei zeigte sich, dass die Adsorption der PSM Pendimethalin, Terbuthylazin, Metolachlor und Chlortoluron (Konzentrationen: 2, 20 und 200 μg 1⁻¹) an Saugkerzen aus Borosilikatglas z. T. erheblich geringer war als an Saugkerzen aus Keramik. Während bei der Perkolation durch die Keramik-Saugkerzen im Mittel 10% (1,1—31%) der Wirkstoffe adsorbiert wurden, betrugen die Verluste bei den Glas-Saugkerzen im Mittel nur 3,1% (0—11%). Die Unterschiede zwischen den beiden Saugkerzentypen nahmen mit steigender Hydrophobie der PSM-Wirkstoffe und sinkender Lösungskonzentration deutlich zu. So wurden bei einer Lösungskonzentration von 2 μg 1⁻¹ in den Keramik-Saugkerzen 31% des gelösten Pendimethalins adsorbiert, während die Verluste in den Glas-Saugkerzen mit 7,7% deutlich geringer waren. Entsprechende Adsorptionstests mit bodeneigenem gelöstem organischem Kohlenstoff (DOC) führten zu ähnlichen Ergebnissen. Vom DOC verschiedener Bodenextrakte wurden in den Glas-Saugkerzen durchschnittlich 2,4% adsorbiert, während die Keramik-Saugkerzen bis zu 50% der gelösten organischen Substanz retardierten. Da bodeneigene gelöste organische Substanzen bedeutende Sorbenten und Carrier für Pflanzenschutzmittel darstellen, wird durch die weitgehende Neutralität des Glas-Materials gegenüber PSM und DOC die Eignung dieses Werkstoffs für die Gewinnung von Saugkerzen-Lösungen in Pflanzenschutzmittel-Studien nachdrücklich unterstrichen.

Acute myeloid leukemia (AML) is still associated with a mortality of 60 to 80%. AML is characterized by a block in myeloid differentiation. The transcription factors PU.1 and C/EBPα are responsible for normal myeloid differentiation from stem cells to monocytes or granulocytes. In particular, PU.1 induces expression of the macrophage colony-stimulating factor (M-CSF) receptor and the development of monocytes, whereas C/EBPα increases the expression of the granulocyte colony-stimulating factor (G-CSF) receptor and leads to mature granulocytes. In AML, chromosomal aberrations result in oncoproteins such as AML1/ETO, PML/RARα, or activated Ras, which can deregulate genes important for normal myelopoiesis. Thus, AML1/ETO can bind to the transcription factor C/EBPα, inhibit C/EBPα-dependent transcription, and block granulocytic differentiation. However, AML1/ETO can also synergize with the transcription factor AML1 to enhance the activity of the M-CSF receptor promoter. On the other hand, the PML/RARα fusion protein causes transcriptional repression by recruiting the nuclear corepressor (N-CoR) histone deacetylase complex to the DNA, which results in decreased histone acetylation and a repressive chromatin organization. Here we describe methods to investigate whether and how signaling agonists induce myeloid differentiation and how oncoproteins might cause AML by modulating the activity of transcription factors that are pivotal for normal myeloid development.

Invasive fungal infections have increasingly become a matter of concern with regard to patients receiving intensive myelosuppressive therapy for hematologic malignancies. Such infections, especially prolonged neutropenia systemic fungal infections, may contribute substantially to infectious complications and early death. Measures for early detection and effective prophylactic strategies using active and nontoxic antifungal agents are therefore urgently needed. The current randomized study was initiated to assess the efficacy of oral fluconazole as systemic antifungal prophylaxis for high risk patients with recurrent acute myeloid leukemia undergoing intensive salvage therapy. Of 68 fully evaluable patients, 36 were randomized to fluconazole in addition to standard prophylaxis with oral co-trimoxazol, colistin sulphate, and amphotericin B suspension, and 32 were randomized to standard prophylaxis only. No major differences between the two groups were observed in the number of episodes of fever of unknown origin (61% vs. 50%) or clinically defined infections (56% vs. 50%). Microbiologically defined infections were more frequent in the fluconazole group (50% vs. 31%), mainly due to a higher incidence of bacteremias (42% vs. 22%). There were two cases of proven invasive fungal infections in each group. Systemic amphotericin B was administered more frequently to patients receiving fluconazole prophylaxis (56% vs. 28%). Fluconazole prophylaxis had no impact on the rate of early death or overall survival. For patients with high risk recurrent acute myeloid leukemia undergoing intensive salvage therapy, antifungal prophylaxis with fluconazole was not superior to standard prophylaxis only. Cancer 1998;83:291-301. © 1998 American Cancer Society.

In the original article, the affiliations were presented incorrectly. David Petroff is in fact the only author at affiliation 2. All other authors listed as being at affiliation 2 (Tina Weiße, Sebastian Beer, Franziska Gnatzy, Joachim Mössner, Michael Tröltzsch, Johannes Wiegand and Volker Keim) are in fact just at affiliation 1. These have now been corrected in the original article.

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

Franke: Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; Jazz: Honoraria. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

Rationale: Allogeneic haematopoietic stem cell transplantation (HCT) is the only curative option for a variety of malignant and non-malignant diseases and is increasingly used, even in elderly and medically unfit patients (pts). Despite advances, it is associated with considerable morbidity and mortality. Major obstacles to a successful outcome are infections and graft-versus-host-disease (GvHD) but also liver-related complications like sinusoidal obstruction syndrome (SOS). An important risk factor is a preexisting chronic liver disease. Screening includes hepatitis serology and PCR, ultrasound of the liver and liver related laboratory evaluations of transaminases, gamma-GT and bilirubin. Liver stiffness measurements (LSM) using point shear-wave elastography (pSWE) and transient elastography (TE) have emerged as non-invasive methods to detect hepatic fibrosis. In addition, controlled attenuation parameter (CAP) has good accuracy for detection and differentiation of histologically defined steatosis. Both methods have been extensively evaluated in patients with various chronic liver diseases and represent non-invasive alternatives to liver biopsy. Preliminary reports suggest a potential prognostic role for LSM in the HCT setting.

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.

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.

Introduction:MicroRNA miR-1301is embedded in intron 1 in the DNMT3Agene, which encodes the DNA (cytosine-5)-methyltransferase 3A, is frequently mutated in older acute myeloid leukemia (AML) patients (pts) & has been associated with clonal hematopoiesis. In glioma cells miR-1301suppresses tumor cell invasion by regulating the activity & function of the p53 pathway & inhibits cell proliferation by targeting NRAS. The role of miR-1301in AML remains to be elucidated.

Introduction: Relapse of the disease remains a major burden after stem cell transplantation (HCT) following myeloablative conditioning and even more after reduced intensity and non-myeloablative conditioning. Efforts to reduce relapse incidence involve early immunosuppression withdrawal and prophylactic buffy coat infusion in high risk patients. In this phase II study we analyzed the role of disease-specific chimerism driven immunosuppression.

Introduction:Prognosis remains poor for most acute myeloid leukemia (AML) patients (pts). Enhanced risk-stratification, novel & individualized therapies to improve outcomes are needed. The RAS-pathway (NRAS, KRAS & HRAS& among others the regulator SHP-2 encoded by PTPN11) controls cell proliferation, cell differentiation & apoptosis & mutations in this pathway are common in many human malignancies, including AML. Previous studies of the prognostic impact of RASmutations (mut) in AML demonstrated variable & context specific results. The prognostic impact in pts receiving allogeneic stem cell transplantation (HSCT) remains to be elucidated.

Introduction:Clonal hematopoiesis of indeterminate potential (CHIP) is defined as presence of hematologic malignancy-associated mutations, e.g.in the genes DNMT3A, TET2, ASXL1, but absence of hematologic neoplasms & is a frequent phenomenon in healthy individuals with increasing age. Some individuals with CHIP eventually develop acute myeloid leukemia (AML) & persistent clonal hematopoiesis-associated mutations (CH-mutations) in complete remission (CR) may give rise to relapse. To date in AML little is known about the biological & clinical implications of the presence of CH-mutations in CR, especially in the context of allogeneic hematopoietic stem cell transplantation (HSCT).

Poenisch: Mundipharma: Research Funding.

Poenisch: Mundipharma: Research Funding.

Poenisch: Mundipharma: Research Funding.

Poenisch: Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

In recent years expression levels of several genes & microRNAs (miR) were identified as strong prognostic markers, capable to refine AML risk stratification. So far technical difficulties, including the limitations of established methods for comparable, absolute quantification & the lack of defined cut points prevented translation of these findings into clinical practice. Innovative digital droplet PCR (ddPCR) is a novel technique with high sensitivity, specificity that allows absolute quantification - without the need for standard curves - & promises better inter-laboratory comparability.

Expression levels of miR-29family members (i.e. miR-29a, miR-29b, &miR-29c) are deregulated in various neoplastic diseases, including acute myeloid leukemia (AML), known to affect DNA-methylation profiles by targeting epigenetic modifiers, & have been shown to be important for normal hematopoietic stem cell function. Mir-29is organized in two distinctively regulated bi-cistronic clusters: the miR-29a/b-1cluster & the miR-29b-2/ccluster.

Introduction:The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was shown that ectopic overexpression of Zbtb7ain immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7Aexpression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1&ZBTB7Amutations in t(8;21)-associated AML was suggested.

Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC(Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALCcopy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR.