Podcasts about aml1 eto

Redox systems are a major tool for the organism to deal with oxidative stress and the balance of these systems is crucial for life. The glutathione (GSH) dependent system consists of glutathione, glutathione peroxidases and glutathione reductases and is critically involved in the maintenance of intracellular redox balance. In addition, the glutathione dependent system is a promising target for anti-cancer therapy. In particular, glutathione peroxidase 4, an enzyme that protects cell membranes from lipidperoxidation, plays a major role in proliferation, growth and development as Gpx4 disruption leads to growth arrest and embryonic death in a mouse model. GPX4 function plays a regulative role in several kinds of cancer and is highly expressed in leukemias, especially in patients carrying the AML1-ETO fusion gene. In this work, we delineated the role and expression of GPX4 in hematopoiesis and leukemia and investigated its role as potential target for anti-cancer therapy. In a state of non-disease glutathionperoxidase 4 is highly expressed in all hematopoietic lineages with the highest expression in the mouse erythroid lineage and is upregulated in the stem cell compartment in human bone marrow. This suggests that GPX4 is involved as one of the major antioxidative mechanisms in hematopoiesis. In the state of leukemia we could prove an upregulation of the gene in AML1-ETO patients compared to healthy individuals, which was shown by microarray data before. The present study thus could show that a knock-down of GPX4 in leukemic cell lines carrying the AML1-ETO fusion gene led to a strong anti-proliferative effect but no effect on the clonogenicity of the cells was observed. In addition, the knockdown of only this one antioxidative mechanism was sufficient to increase ROS levels in those cell lines. Apoptosis signaling was not involved in these processes. These data shed new light on the role of GPX4 in normal and malignant hematopoiesis and encourage further studies testing the therapeutic efficacy of GPX4 blockage in human AML.

Fri, 13 Feb 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10769/ https://edoc.ub.uni-muenchen.de/10769/1/Mahalakshmi_Naidu_Vegi.pdf Mahalakshmi Naidu, Vegi

Thu, 30 Oct 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9306/ https://edoc.ub.uni-muenchen.de/9306/1/Franzen_Michael.pdf Franzen, Michael

Thu, 9 Oct 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9180/ https://edoc.ub.uni-muenchen.de/9180/1/Heiss-Neumann_Marion_S.pdf Heiß-Neumann, Marion Susanne

Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations that are strong determinants of prognostic outcome and therapeutic response. Because the pathological outcome of AML patients with cytogentic abnormalities differs considerably we hypothesized that their proteome may also differ specifically in their expression pattern, protein interaction pathways and posttranslational modifications. We performed this study using 42 AML patients diagnosed for various cytogenetic abnormalities based on two-dimensional gel electrophoresis and MALDI TOF Tandem MS (MS/MS) analysis. We could identify significant differences in the proteome and posttranslational modifications of peptides, later confirmed by other methods, between cytogenetic groups. The interactome analysis based on computational bioinformatics reveals a major regulating networks, MAPK8 and MYC for complex aberrant karyotype, TP53 for t(8;21), TP53- MYC- PRKAC for 11q23, JUN and MYC for Inv(16). We could show in our validation and characterisation experiments that survivin is a novel target of t(8;21) leukemia and AML1-ETO directly regulates its expression to induce the differentiation block that could be overcome by silencing its expression. Further, we analysed 42 MS spectra representative of hnRNPH1, Calreticulin and hnRNPA2/B1 in a peak explorer which reveals a cytogenetic specific posttranslational modification of β-O-linked N-acetyl glucosamine (O-GlcNAc) of hnRNPH1 in AML patients with 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation and methylation of hnRNPA2/B1 in patients with translocations of t(8;21) and inv(16). This report may lead to a new thinking about the AML pathogenesis as differences at PTM level could be used to distinguish different subtypes of AML besides for testing the therapeutic significance. Further, we characterised the biological role of survivin identified specifically from t(8;21) patients. We could show that AML1-ETO induces the expression of survivin both in a cell line model and in primary human hematopoietic precursors. AML1-ETO activates the basal transcription of the survivin promoter and binds to the only AML1 core enhancer binding sequence, TGTGGT, in survivin promotor. Repression of AML1-ETO mediated induction of survivin expression by a specific short hairpin RNA restores C/EBPα protein and its basal transcriptional activity on its own promotor. This restoration differentiates AML1-ETO positive leukemic cells to terminal granulocytic differentiation and growth arrest. These observations indicate that the antiapoptotic survivin protein, which holds a great therapeutic promise, is a critical mediator of AML1-ETO induced defective granulopoiesis. Thus, proving that AML1-ETO induces inhibition of granulocytic differentiation by activating survivin expression

Die meisten genetischen Abweichungen, die bei humanen akuten Leukämien gefunden werden können, lassen sich in zwei Klassen einteilen: Klasse I Mutationen, wie z.B. aktivierende Mutationen in Rezeptortyrosinkinasen (z.B. FLT3 oder c-KIT), die einen Proliferations- und/oder Überlebensvorteil für hämatopoetische Vorläuferzellen bieten und Klasse II Mutationen (wie z.B. AML1-ETO oder PML/RARα), die hämatopoetische Transkriptionsfaktoren betreffen und primär die Reifung der Zellen und die Apoptose unterbinden. Im Zusammenspiel entstehen hämatopoetische Vorläuferzellen, deren Proliferation und Differenzierung empfindlich gestört ist (Gilliland, 2002), was die Ursache für Leukämien sein kann. In dieser Arbeit wurden zwei genetische Alterationen untersucht, die den zwei verschiedenen Klassen entstammen: eine Längenmutation der Rezeptortyrosinkinase FLT3 und das Fusionsgen aus AML1 und ETO, AML1-ETO. Es wurde die Frage gestellt, ob diese Mutationen, die auch gemeinsam in humanen AML Patienten gefunden werden (Care et al., 2003), im Zusammenspiel Leukämie auslösen können. Ein murines Knochenmarktransplantationsmodell wurde etabliert, bei dem Knochenmarkzellen, die entweder AML1-ETO, FLT3-LM, beide Mutationen zusammen, oder GFP alleine exprimierten, in Mäuse injiziert wurden. Die Kontrollmäuse entwickelten keine Erkrankung, wohingegen die Mäuse, die AML1-ETO und FLT3-LM zusammen exprimierten, an aggressiver Leukämie erkrankten. Interessanterweise gab es unterschiedliche Phänotypen: es entstanden sowohl myeloische als auch lymphatische (B- und T-Zell) Leukämien. Alle Leukämien wurden durch FACS und Zytologie, teilweise auch durch Histopathologie bestätigt. Es kann durch die vorliegenden Daten bestätigt werden, dass weder AML1-ETO noch FLT3-LM alleine in der Lage sind Leukämie auszulösen. FLT3-LM stellt aber einen sehr potenten Kooperationspartner dar, um gemeinsam mit AML1-ETO eine Leukämie zu induzieren. Diese Arbeit kann zum Verständnis der Pathophysiologie von akuten Leukämien beitragen, was die Grundvorausetzung zur Entwicklung von Heilmethoden ist. Der nächste Schritt sollte sein, verschiedene Substanzen, wie zum Beispiel Tyrosinkinaseinhibitoren zu testen, um bei entsprechender Wirkung die Prognose für Patienten mit AML1-ETO positiven Leukämien zu verbessern.

Overexpression of proto-oncogene c-jun and constitutive activation of the Jun NH2-terminal kinase (JNK) signaling pathway have been implicated in the leukemic transformation process. However, c-jun expression has not been investigated in acute myeloid leukemia (AML) cells containing the most common chromosomal translocations. t(8;21) is one of the most common AML-associated translocation and results in the AML1-ETO fusion protein. Overexpression of AML1-ETO in NIH3T3 cells leads to increased phosphorylation of Ser63 in c-Jun, which is generally JNK dependent. The role of the JNK signaling pathway for the functional properties of AML1-ETO is, however, unknown. In the present study we found high expression levels of c-jun mRNA in t(8;21), t(15;17) or inv(16) positive patient cells by microarray analysis. Within t(8;21) positive patient samples, there was a correlation between AML1-ETO and c-jun mRNA expression levels. In myeloid U937 cells, c-jun mRNA and c-Jun protein expression levels increased upon induction of AML1-ETO. AML1-ETO transactivated the human c-jun promoter through the proximal AP-1 site via activating the JNK signaling pathway. JNK targets c-Jun and ATF-2, which also bind to the proximal AP-1 site in U937 cells, were also phosphorylated upon AML1-ETO induction. Furthermore, AML1-ETO induction increased the DNA binding capacity of c-Jun and ATF-2 to the proximal AP-1 site of the c-jun promoter, which might result in their enhanced transactivation capacities. Interference with JNK and c-Jun activation by using JIP-1 or a JNK inhibitor reduced the transactivation capacity of AML1-ETO on the c-jun promoter and the pro-apoptotic function of AML1-ETO in U937 cells. AML1-ETO seems to activate the JNK signaling pathway by inducing the expression of a cytoplasmic factor, possibly G-CSF, because supernatant of AML1-ETO expressing cells was sufficient to induce phosphorylation of JNK and c-Jun in wildtype U937 cells. This data demonstrates a novel mechanism of how AML1-ETO might exert positive effects on target gene expression and identifies the proto-oncogene c-jun as a common target gene in AML patient cells.

AML1-ETO is a fusion protein encoded by the translocation t(8;21) and found in 15% of acute myeloid leukemia patients. Here, we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1, an important transcription factor for normal myeloid differentiation. We have demonstrated that AML1-ETO interacts with PU.1 by immunoprecipitation assay in Kasumi-1 cells having t(8;21). On mapping the region of interaction in PU.1, we found that AML1-ETO binds to the b3b4 region in the DNA binding domain of PU.1 and displaces the co-activator c-Jun from PU.1, thus downregulating PU.1’s transcriptional activity. In doing so AML1-ETO does not change the DNA binding capacity of PU.1. The expression levels of PU.1 target genes in acute myeloid leukemia (AML)-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 PU.1 induced differentiation in HL60 cells. Overexpression of PU.1 differentiates AML1-ETO carrying Kasumi-1 cells to the monocytic lineage. Thus, PU.1’s function is downregulated in presence of AML1-ETO in acute myeloid leukemia, whereas overexpression of PU.1 can restore normal differentiation.