Rewiring of the Transcription Factor Network in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous cancer associated with different patterns of gene expression determined by the nature of their DNA mutations. These mutations mostly act to deregulate gene expression by various mechanisms at the level of the nucleus. By performing genome-wide epigenetic profiling of cis-regulatory elements, we found that AML encompasses different mutation-specific subclasses associated with the rewiring of the gene regulatory networks that drive differentiation into different directions away from normal myeloid development. By integrating epigenetic profiles with gene expression and chromatin conformation data, we defined pathways within gene regulation networks that were differentially rewired within each mutation-specific subclass of AML. This analysis revealed 2 major classes of AML: one class defined by mutations in signaling molecules that activate AP-1 via the mitogen-activated protein (MAP) kinase pathway and a second class defined by mutations within genes encoding transcription factors such as RUNX1/CBFβ and C/EBPα. By identifying specific DNA motifs protected from DNase I digestion at cis-regulatory elements, we were able to infer candidate transcription factors bound to these motifs. These integrated analyses allowed the identification of AML subtype-specific core regulatory networks that are required for AML development and maintenance, which could now be targeted in personalized therapies.

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MiR-132 and MiR-212 as Regulators of WT1 and GATA2 in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.1283.1283 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1283-1283 ◽

Cited By ~ 1

Author(s):

Maaike Luesink ◽

Jeannet Nigten ◽

Ruth H.J.N. Knops ◽

Theo J.M. de Witte ◽

Bert A. van der Reijden ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Transcription Factors ◽

Quantitative Pcr ◽

Blood Cells ◽

Myeloid Leukemia ◽

Granulocytic Differentiation ◽

Aberrant Expression ◽

Differentiation Induction ◽

Acute Myeloid

Abstract Abstract 1283 Poster Board I-305 Wilms' tumor 1 (WT1) and GATA binding protein 2 (GATA2) transcription factors are highly expressed in hematopoietic stem cells and progenitors. Differentiation of precursor blood cells towards mature blood cells is accompanied by rapid downregulation of both transcription factors. Overexpression of WT1 has been observed in the majority of acute myeloid leukemia (AML) cases. Furthermore, in 10-15% of the AML cases mutations in the WT1 gene occur, which have been correlated with poor prognosis. Aberrant expression of GATA2 in AML has been described as well, but no mutations in this gene have been reported in AML so far. How the (aberrant) expression of WT1 and GATA2 is controlled is not completely clear. A regulatory role for microRNAs (miRNAs) has been described for several transcription factors which regulate hematopoiesis. MiRNAs negatively regulate gene expression by translational repression or degradation of target messenger RNAs (mRNAs). In the present study we investigated the interplay between miRNAs and transcription factors that are involved in myeloid development and malignant transformation towards AML. We studied the expression of 158 miRNAs in the APL cell line NB4 during induction of granulocytic differentiation with all-trans retinoic acid (ATRA). Quantitative PCR specific for mature miRNAs was performed (Applied Biosystems). Twenty out of 158 miRNAs were more than 10-fold upregulated upon differentiation induction with ATRA. MiR-132 and miR212, which are derived from the same pri-miRNA transcript, were most strongly upregulated during ATRA-induced granulocytic differentiation (1200- and 350-fold respectively at 96 hours after ATRA-stimulation). In vitro ATRA-induction of primary APL cells also resulted in upregulation of miR-132 and miR-212. Computational target prediction algorithms were used to identify transcription factors which may be targeted by miR-132 and miR-212. Subsequently, the expression pattern of the predicted targets was determined experimentally in NB4 cells before and after differentiation induction with ATRA using microarray-based mRNA profiling (Affymetrix). In addition, further verification of target gene expression during ATRA-induced differentiation was performed using quantitative PCR. The transcription factors WT1 and GATA2 were predicted as targets of miR-132 and miR-212 by two out of four different prediction programs that were used. Both transcription factors contained putative binding sites for miR-132 and miR-212 in their 3'UTR. When tested on microarray and by quantitative PCR, the expression of WT1 and GATA2 was indeed strongly downregulated during ATRA-induced granulocytic differentiation of NB4 cells (65- and 165-fold respectively at 96 hours after ATRA stimulation) as well as in primary leukemia cells derived from APL patients (30- and 10-fold respectively at 48 hours after ATRA-stimulation). During ATRA-induced differentiation the expression levels of WT1 were positively correlated with the expression levels of GATA2. In addition, WT1 expression was also strongly correlated with GATA2 expression in a cohort of 27 pre-treatment AML cases as well as in 7 healthy controls, suggesting that these genes might be co-regulated to a large extent. To directly prove that WT1 and GATA2 are indeed targeted by miR-132 and miR-212, we are currently performing lentiviral-based overexpression studies of both miRNAs to determine the effect on endogenous WT1 and GATA2 mRNA expression. MicroRNAs which target WT1 and GATA2 may be valuable tools in controlling the aberrant expression of WT1 and GATA2 observed in AML. Disclosures No relevant conflicts of interest to declare.

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Pathogenesis of Acute Myeloid Leukemia: In Vitro Studies Using Primary Human Cells.

Blood ◽

10.1182/blood.v112.11.2264.2264 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2264-2264

Author(s):

Anmaar Abdul-Nabi ◽

Enas R Yassin ◽

Akiko Takeda ◽

Nabeel R. Yaseen

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Transcription Factors ◽

Myeloid Leukemia ◽

Human Peripheral Blood ◽

Gene Profiling ◽

Number Of Genes ◽

Acute Myeloid ◽

Time Point

Abstract Many cases of acute myeloid leukemia (AML) are associated with non-random chromosomal rearrangements and most of these result in fusions involving retinoic acid receptor α (RARα), CBF transcription factors, MLL, and nucleoporins. Here, we report the effects of key members of these four major groups of AML-associated chimeric fusion proteins on differentiation, proliferation, self-renewal and gene expression in primary human CD34+ hematopoietic cells. We expressed the PML-RARα, AML1-ETO, MLL-AF9 and NUP98-HOXA9 fusion genes in human peripheral blood CD34+ cells by retroviral transduction and compared them to cells transduced with empty vector. By colony forming assays, morphological examination and flow cytometric immunophenotyping we found that PML-RARα causes some degree of myeloid differentiation block. AML1-ETO had no obvious effect on differentiation, while MLL-AF9 and NUP98-HOXA9 caused a block in both myeloid and erythroid differentiation. All fusion oncoproteins, except PML-RARα, induced dramatic long-term proliferation in liquid culture and a marked increase in the numbers of primitive long-term culture-initiating cells (LTC-ICs). In order to understand the molecular basis of these effects, gene expression profiling was performed for each fusion gene by oligonucleotide microarray analysis at 3 different time points (6 h, 3 days, and 8 days post transduction). At the 6 h time point both AML1-ETO and PML-RARα caused extensive changes in gene expression with a predominance of downregulated genes. The number of genes dysregulated by AML1-ETO and PML-RARα decreased dramatically at the 3-day time point. These data are consistent with the fact that these two oncoproteins are DNA-binding transcription factors that are known to repress transcription. In contrast, MLL-AF9 and NUP98-HOXA9 altered the expression of fewer genes at the 6 h time point with a preponderance of upregulated genes; at the 3 day time point, the number of genes dysregulated by NUP98-HOXA9 and MLL-AF9 increased. These findings suggest that NUP98-HOXA9 and MLL-AF9 may have delayed effects that are not due to direct transcriptional regulation. Homeobox transcription factors were upregulated by both MLL-AF9 and NUP98-HOXA9, but not by either AML1-ETO or PML-RARα. The results of the biological assays and gene profiling show marked similarities between NUP98-HOXA9 and MLL-AF9, and suggest that they transform cells by similar pathways that are different from those used by AML1-ETO and PML-RARα.

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The expression pattern of erythrocyte/megakaryocyte-related transcription factors GATA-1 and the stem cell leukemia gene correlates with hematopoietic differentiation and is associated with outcome of acute myeloid leukemia

Blood ◽

10.1182/blood.v86.8.3173.bloodjournal8683173 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3173-3180 ◽

Cited By ~ 1

Author(s):

T Shimamoto ◽

K Ohyashiki ◽

JH Ohyashiki ◽

K Kawakubo ◽

T Fujimura ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Transcription Factors ◽

Expression Pattern ◽

Cell Lines ◽

Myeloid Leukemia ◽

Cell Leukemia ◽

Stem Cell Leukemia ◽

Acute Myeloid

To understand the clinical implications of transcription factors and their biologic roles during cellular differentiation in the hematopoietic system, we examined the expression of GATA-1, GATA-2, and stem cell leukemia (SCL) gene in human leukemia cell lines and various leukemia patients using the reverse transcriptase-polymerase chain reaction. Cell lines exhibiting megakaryocytic or erythrocytic phenotypes had GATA-1, GATA-2, and SCL gene transcripts, while monocytic cell lines had no detectable GATA-1, GATA-2, or SCL gene mRNA. In some myeloid cell lines, GATA-1 expression, but not SCL gene expression, was detected; GATA-1 expression in HL-60 cells was downregulated during the process of monocytic differentiation. We next examined GATA-1, GATA-2, and SCL gene expression in 110 leukemia samples obtained from 76 patients with acute myeloid leukemia (AML), 19 with acute lymphoblastic leukemia (ALL), and 15 with chronic myeloid leukemia in blast crisis (CML-BC). SCL gene expression was usually accompanied by GATA-1 expression and was preferentially detected in patients with leukemia exhibiting megakaryocytic or erythrocytic phenotypes, while patients with monocytic leukemia were clustered in the group with no detectable GATA-1 expression. None of the patients with ALL or CML-lymphoid-BC expressed SCL. De novo AML patients with SCL gene expression had a lower complete remission (CR) rate and had a significantly poorer prognosis. Among the patients with AML not expressing SCL, a high percentage of patients with CD7+ AML and CD19+ AML had detectable GATA-1, while patients with GATA-1-negative AML had the best CR rate (87.5%). Our results suggest that the expression pattern of transcription factors reflects the lineage potential of leukemia cells, and GATA-1 and SCL gene expression may have prognostic value for the outcome of patients with AML.

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MicroRNA-155 Promotes Myeloid Proliferation and Is Overexpressed in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.715.715 ◽

2007 ◽

Vol 110 (11) ◽

pp. 715-715

Author(s):

Dinesh S. Rao ◽

Ryan M. O’Connell ◽

Aadel A. Chaudhuri ◽

Mark Boldin ◽

Konstantin Taganov ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Regulatory Networks ◽

Quantitative Polymerase Chain Reaction ◽

Retroviral Infection ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Myeloid Development ◽

Acute Myeloid

Abstract Recent discoveries have implicated microRNAs, which are small 22–24 nucleotide long RNA molecules, as important regulators of many cellular processes, including differentiation and development. The microRNA-155 (miR-155) is known to be overexpressed in subsets of B-cell neoplasms and is thought to be important in the activation and function of B-lymphocytes. Here, we show that miR-155 is signficantly overexpressed in human acute myeloid leukemia and that its overexpression is most consistently seen in acute myelomonocytic leukemia. These findings led us to investigate the effects of overexpression of miR-155 in hematopoietic cells. By utilizing retroviral infection and transfer of murine bone marrow, we introduced miR-155 overexpressing hematopoietic stem cells into lethally irradiated recipient mice. Following reconstitution of hematopoietic organs, the mice demonstrated a profound myeloproliferative condition in the bone marrow characterized by replacement of the marrow by proliferating and dysplastic myelomonocytic cells. In addition, extramedullary hematopoiesis was observed in the spleen and examination of the peripheral blood revealed anemia and thrombocytopenia. To begin to explore the mechanisms whereby miR-155 overexpression results in this myeloproliferative phenotype, we utilized computational methods to identify targets predicted to be regulated by miR-155. This revealed several genes that have previously been implicated in myeloid development and neoplasia, which were confirmed to be targets of miR-155 by reverse-transcription/quantitative polymerase chain reaction (RT/QPCR) and by downregulation of luciferase protein upon fusion of the luc gene with the respective 3′ untranslated regions. These studies show a hitherto uncharacterized role of miR-155 in myeloid development and proliferation. Importantly, these findings lay the groundwork for understanding the complex regulatory networks underlying myeloid development in the context of microRNAs, and may point to new therapeutic opportunities in the treatment of myeloid malignancies.

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t(8;21) Acute Myeloid Leukemia as a Paradigm for the Understanding of Leukemogenesis at the Level of Gene Regulation and Chromatin Programming

Cells ◽

10.3390/cells9122681 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2681

Author(s):

Sophie Kellaway ◽

Paulynn S. Chin ◽

Farnaz Barneh ◽

Constanze Bonifer ◽

Olaf Heidenreich

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factors ◽

Myeloid Leukemia ◽

Genome Stability ◽

Regulatory Networks ◽

Growth Regulation ◽

Current Knowledge ◽

Dna Methyltransferases ◽

Dominant Negative ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous disease with multiple sub-types which are defined by different somatic mutations that cause blood cell differentiation to go astray. Mutations occur in genes encoding members of the cellular machinery controlling transcription and chromatin structure, including transcription factors, chromatin modifiers, DNA-methyltransferases, but also signaling molecules that activate inducible transcription factors controlling gene expression and cell growth. Mutant cells in AML patients are unable to differentiate and adopt new identities that are shaped by the original driver mutation and by rewiring their gene regulatory networks into regulatory phenotypes with enhanced fitness. One of the best-studied AML-subtypes is the t(8;21) AML which carries a translocation fusing sequences encoding the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO gene. The resulting oncoprotein, RUNX1/ETO has been studied for decades, both at the biochemical but also at the systems biology level. It functions as a dominant-negative version of RUNX1 and interferes with multiple cellular processes associated with myeloid differentiation, growth regulation and genome stability. In this review, we summarize our current knowledge of how this protein reprograms normal into malignant cells and how our current knowledge could be harnessed to treat the disease.

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The expression pattern of erythrocyte/megakaryocyte-related transcription factors GATA-1 and the stem cell leukemia gene correlates with hematopoietic differentiation and is associated with outcome of acute myeloid leukemia

Blood ◽

10.1182/blood.v86.8.3173.3173 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3173-3180 ◽

Cited By ~ 53

Author(s):

T Shimamoto ◽

K Ohyashiki ◽

JH Ohyashiki ◽

K Kawakubo ◽

T Fujimura ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Transcription Factors ◽

Expression Pattern ◽

Cell Lines ◽

Myeloid Leukemia ◽

Cell Leukemia ◽

Stem Cell Leukemia ◽

Acute Myeloid

Abstract To understand the clinical implications of transcription factors and their biologic roles during cellular differentiation in the hematopoietic system, we examined the expression of GATA-1, GATA-2, and stem cell leukemia (SCL) gene in human leukemia cell lines and various leukemia patients using the reverse transcriptase-polymerase chain reaction. Cell lines exhibiting megakaryocytic or erythrocytic phenotypes had GATA-1, GATA-2, and SCL gene transcripts, while monocytic cell lines had no detectable GATA-1, GATA-2, or SCL gene mRNA. In some myeloid cell lines, GATA-1 expression, but not SCL gene expression, was detected; GATA-1 expression in HL-60 cells was downregulated during the process of monocytic differentiation. We next examined GATA-1, GATA-2, and SCL gene expression in 110 leukemia samples obtained from 76 patients with acute myeloid leukemia (AML), 19 with acute lymphoblastic leukemia (ALL), and 15 with chronic myeloid leukemia in blast crisis (CML-BC). SCL gene expression was usually accompanied by GATA-1 expression and was preferentially detected in patients with leukemia exhibiting megakaryocytic or erythrocytic phenotypes, while patients with monocytic leukemia were clustered in the group with no detectable GATA-1 expression. None of the patients with ALL or CML-lymphoid-BC expressed SCL. De novo AML patients with SCL gene expression had a lower complete remission (CR) rate and had a significantly poorer prognosis. Among the patients with AML not expressing SCL, a high percentage of patients with CD7+ AML and CD19+ AML had detectable GATA-1, while patients with GATA-1-negative AML had the best CR rate (87.5%). Our results suggest that the expression pattern of transcription factors reflects the lineage potential of leukemia cells, and GATA-1 and SCL gene expression may have prognostic value for the outcome of patients with AML.

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