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Blood, 15 May 2007, Vol. 109, No. 10, pp. 4432-4440. Prepublished online as a Blood First Edition Paper on January 23, 2007; DOI 10.1182/blood-2006-09-045781.
NEOPLASIA Heterochromatic gene repression of the retinoic acid pathway in acute myeloid leukemia1 San Raffaele Bio-medical Park Foundation, Rome, Italy; 2 Departments of Histology and Medical Embryology and 3 Cellular Biotechnologies and Hematology, University La Sapienza, Rome, Italy; 4 Department of Experimental Oncology, European Institute of Oncology, Milan Italy, 5 Institute Pasteur Cenci-Bolognetti and Department of Genetics and Molecular Biology, University La Sapienza, Rome, Italy; 6 Department of Clinical and Experimental Medicine, University of Perugia, Italy; 7 Department of Biopathology, University Tor Vergata, Rome, Italy
Alteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RAR  ) at RA regulatory regions on RARß2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARß2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARß2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.
The postgenomic era has shown that correct gene expression is regulated by epigenetic mechanisms, such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and expression of small regulatory RNAs. These events are essential during development and for the maintenance of tissue- and cell-type–specific functions.1–3 The contribution of epigenetic mechanisms for a correct cell function is highlighted by the effects of their deregulation that in cooperation with genetic alterations lead to the establishment and progression of tumors.4–9
In vivo and in vitro models of hematopoiesis indicate a physiological role for retinoic acid (RA), a natural derivative of vitamin A, in regulating myelopoiesis through its binding to the RA receptor alpha (RAR
In APL, the aberrant DNA methylation of RARß2 and consequent gene silencing is caused by the leukemogenetic PML/RAR
The t(8;21) generating the AML1/ETO fusion product is one of the most common genetic events associated with AML. This chromosomal translocation is present in up to 40% in AML FAB M2 cases and, at a lower frequency, in AML-M4 and other subsets.26–28 The AML1/ETO product shares several features with PML/RAR Non-APL AMLs are resistant to the differentiating action of RA.36 A dysfunctional control of RA activity in myelopoiesis might represent per se an oncogenic hit relevant to the development and progression of leukemia. In agreement with this hypothesis, we have shown that the RA signaling pathway is silenced in AMLs regardless of their underlying genetic lesion.37,38
In this study, we identify the epigenetic silencing of the RA signaling pathway as an additional genomic alteration caused by the AML1/ETO fusion product in myeloid cells. Here, we show AML1/ETO and RAR
Reagents All-trans-retinoic acid (RA) and 5-azacytidine were from Sigma-Aldrich (Milan, Italy) and both used at a concentration of 1µM. Clinical samples Bone marrow (BM) and/or peripheral blood (PB) samples were obtained from 20 informed, newly diagnosed AML patients showing a percentage of BM blasts of at least 80%. AML cases were classified as AML-M2 (9 cases), AML-M3 (1 case), and AML-M4 (10 cases) according to the FAB classification.16 Blasts isolation and molecular analysis to evaluate the presence of the AML-associated fusion genes were performed as described.39 CD34+ cells were isolated from the BM of informed healthy donors as reported.40 Cell lines and cell cultures
The U937–A/E-HA clone was obtained by electroporation into U937 wild-type (WT) cells of a hemagglutinin (HA)–tagged AML1/ETO cDNA subcloned into a vector carrying the Zn2+-inducible mouse MT1 promoter as described.29,37 Mock cells were U937 WT cells transfected with an empty MT1 promoter vector. The leakiness of the MT1 promoter was used to select the U937 MT-MHA-AE clone 9, which in the absence of Zn2+ expressed an amount of AML1/ETO fusion product at levels comparable to those expressed by the t(8;21) SKNO-1 cell line,41 as detected by Western blot analysis using an Immunophenotypic analysis Cell differentiation was evaluated by direct immunofluorescence staining of cells using an allophycocyanin (APC)–conjugated mouse anti–human CD11b antibody and a peridinin chlorophyll protein (PerCP)– conjugated mouse anti–human CD14 antibody (Becton Dickinson, San Jose, CA). A minimum of 50 000 events was recorded for each sample by a FACScan flow cytometer (Becton Dickinson) using CellFit software (Becton Dickinson) for data acquisition and analysis. Assay for RA binding activity Nuclear extracts were prepared from 2 x 106 cells, incubated for 18 hours at 4°C with 10 nM [3H]-RA, and fractionated at 4°C by high-performance liquid chromatography (HPLC) using a Superpose 6 HR 10/30 column (Pharmacia, Milan, Italy) at a flow rate of 0.4 mL/min as described.43 Plasmid constructs and siRNA assay The sequence of the siRNA oligonucleotide siAGF144 was used to derive a short hairpin that, upon cloning into the plasmid psiUx,45 allowed the stable expression of siRNAs against the AML1/ETO mRNA fusion (siA/E-RNAs). This psiU-derived expression cassette was subcloned into the long terminal repeat (LTR) of the lentiviral vector pRRLcPPT.hPGK.EGFP.WPRE. Cells were infected with empty control vector (mock) or vector encoding the siAGF1 oligonucleotide (siA/E) and purified by fluorescence-activated cell sorting (FACS) as reported.46 Transient cotransfection and transactivation assays Human embryonic kidney 293T cells were transiently cotransfected by the Ca3(PO4)2 method with 20 ng or 40 ng of the pcDNA3 vector containing the HA-tagged AML1/ETO cDNA29 and 200 ng of the –5kb+155bp RARß2pr-LUC reporter15,37 carrying (RAREmut) or not (RAREwt) the 5'-GGTTCAC-3' direct motif of the ßRARE site substituted with the 5'-aacTCAC-3' sequence. A cotransfected plasmid encoding ß-galactosidase (pCMV-ßgal) was used as a control for normalization of the reactions. Twelve hours after transfection, cells were treated with 1 µM RA for 24 hours, lysed, and assayed using the Luciferase Assay Kit (Promega, Milan, Italy). RNA extraction and analysis Total RNA was extracted from cells using the TRIzol RNA isolation system (Invitrogen, Milan, Italy). Northern blot analysis to evaluate the siRNA oligo (siAGF1) expression was performed using 5 µg total RNA electrophoresed in a 10% polyacrylamide-7M urea gel and transferred by electroblotting onto a HybondN+ membrane (Amersham Biosciences, Milan, Italy). Hybridization was performed with terminally 32P-labeled DNA oligonucleotides as described.44,46 U2 snRNA expression levels were measured to normalize for RNA content among samples.46 The relative quantity of RARß2 mRNA was measured on 1 µg total RNA by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) in the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Monza, Italy), and it was determined by the comparative CT method using GAPDH mRNA levels for normalization as reported.19 AML1/ETO mRNA levels were normalized using c-abl mRNA quantity values and quantified by the absolute standard curve method.39 Semiquantitative RT-PCR was performed to evaluate the RARß2 gene expression in AML blasts.19,37 Western blot and coimmunoprecipitation assays
Western blot analyses were performed on total cell lysates (50 µg) using the following rabbit polyclonal IgG antibodies: anti-RAR Chromatin immunoprecipitation assay
Crosslinking of proteins to DNA was obtained by the addition to cultured cells (2 x 106) of formaldehyde at 1% final concentration for 10 minutes at 37°C. After sonication, the chromatin was immunoprecipitated overnight with 5 µL of the following antibodies recognizing RAR Bisulfite sequencing and methylation-specific PCR assays We digested 5 µg of DNA extracted from SKNO-1, si-A/E, U937, A/E-HA cells, and human BM samples with 5 units of EcoRV. Sodium bisulfite treatment was performed as described.48 For bisulfite sequencing assay, a fragment of 608 bp (nucleotides [nt] –370 to +238) of the RARß2 promoter/exon 1 region was amplified using specific primer pairs as described.18,19,49 Single-band PCR products were gel purified and cloned into the TOPO TA Cloning/pCR2.1 TOPO kit (Invitrogen). We subjected individual bacterial colonies to PCR using vector-specific primers (sequences available upon request), and the products were sequenced for the analyses of DNA methylation. Methylation-specific PCR (MSP) assay was performed on bisulfite-treated genomic DNA as described.18,19
AML1/ETO knockdown restores the transcriptional and differentiation response of t(8;21) AMLs to RA We investigated the consequences of the AML1/ETO knockdown on the response of the SKNO-1 cell line to RA. SKNO-1 is a human AML cell line that is resistant to the differentiation effect of RA and harbors the chromosomal translocation t(8;21) and the AML1/ETO fusion product.41,47 AML1/ETO mRNA interference was obtained by the infection of SKNO-1 cells with a GFP lentiviral vector expressing siRNA against the AML1/ETO mRNA (siA/E-RNA).44,46,50 Northern blot analysis revealed that the siA/E-RNA transcripts were specifically expressed in SKNO-1 cells (si-A/E) (Figure 1A), while qRT-PCR and Western blot analyses assessed a diminution of about 50% in the AML1/ETO mRNA and fusion protein levels in si-A/E cells as compared with mock-infected cells (Figure 1B-C). Notably, this reduction of the AML1/ETO product was sufficient to restore the transcriptional action of RA as indicated by the potent induction of the endogenous RARß2 mRNA transcripts measurable by qRT-PCR in RA-treated si-A/E cells (Figure 1D). The AML1/ETO knockdown also restored the differentiation response of SKNO-1 cells to RA as shown by the specific induction of the expression levels of CD11b in RA-treated si-A/E cells, while the levels of CD14 remained unchanged (Figure 1E). These data suggested that threshold levels of AML1/ETO are required for the silencing of the RA signaling pathway and for inducing the differentiation block, which occurs toward the granulocytic lineage. In agreement with evidence showing the epigenetic silencing of RARß2 as the basis of RA resistance in cells,51 our findings pointed at the RA-induced maturation of SKNO-1 cells as the consequence of a combined effect on RARß2 gene promoter: transcriptional derepression caused by the reduced AML1/ETO levels and transcriptional activation induced by RA treatment.
AML1/ETO expression modulates the RARß2 gene promoter activity through the ßRARE site The transcriptional regulatory functions of AML1/ETO on RARß2 gene were investigated by cotransfecting the human 293T cell line with an AML1/ETO expression vector along with the RARß2Pr-LUC, a reporter vector containing the entire 5 Kb promoter region of RARß2 gene cloned upstream of the luciferase coding region.15 Increasing concentrations of ectopically expressed AML1/ETO down-regulated the RARß2 promoter activity in a dose-dependent manner in untreated and RA-treated 293T cells. Both the transcriptional repression exerted by AML1/ETO product on RARß2pr-LUC basal activity and the RA-dependent activation of RARß2 promoter were abrogated by mutating 3 bases of the 5'-GGTTCAC direct motif of the ßRARE binding site of the RARß2Pr-LUC vector (RAREmut) (Figure 2A-B). These results pointed at the ßRARE site on the RARß2 promoter as a target of the repressive effect exerted by AML1/ETO expression on the RA signaling pathway.
AML1/ETO interacts with RAR -RXR at specific sites on AML1 and RA target gene promoters
To clarify the molecular events by which AML1/ETO silences the RA signaling pathway, initially we investigated the possibility of an in vivo interaction of AML1/ETO fusion protein with the RAR
We tested whether the in vivo interaction with the AML1/ETO fusion complex affects the RA binding properties of RAR
Thus, we investigated whether in vivo the AML1/ETO fusion protein is aberrantly present on ßRARE binding sites. ChIP experiments were performed in mock, A/E-HA, and SKNO-1 cells using anti-RAR
AML1/ETO recruits chromatin remodeling enzymes on target promoters
We investigated whether specific active multifactor complexes, including chromatin remodeling activities such as DNMTs, HDAC1, and the methyl binding protein MeCP2, are aberrantly recruited by AML1/ETO at promoter sites on RARß2 as demonstrated for the oncoprotein PML/RAR Epigenetic modifications induced by AML1/ETO at RARß2 promoter and exon 1 regions
We addressed whether the aberrant recruitment of chromatin-modifying activities by AML1/ETO on the RARß2 promoter is linked to its transcriptional silencing by investigating in mock, A/E-HA, and SKNO-1 cell lines the modifications of DNA and chromatin structure at the promoter regions including the ßRARE site and the 5'–untranslated region (5'-UTR) exon 1 region of this gene (Figure 4A). ChIP experiments performed with an
Finally, we investigated whether posttranslational modifications of histone tails in AML1/ETO-expressing cells correlated with DNA hypermethylation and with the aberrant recruitment of epigenetic modifiers at these sites on the RARß2 gene. Deacetylated histones within regions of DNA methylation and/or methylation of specific histone residues (ie, lysine 9 of histone 3) are a hallmark of repressive chromatin status.3 ChIP analysis was therefore performed on SKNO-1 cells using antibodies against the acetylated forms of histone H3 and H4 and the acetylated (H3-K9Ac) or methylated (H3-K9Met) lysine 9 on histone H3. By PCR we amplified the RARß2 region encompassing the ßRARE, the TATA box, the transcription start site, and the CpGs present in exon 1 (Figure 4A, arrows and "ChIP"). Figure 4E shows that in SKNO-1 cells histones H3 and H4 were fully deacetylated and low levels of acetylation were measurable on histone H3 at lysine 9 (H3-K9), which indeed was highly methylated at these regulatory regions on the RARß2 gene. Thus, the aberrant presence of chromatin remodeling activities causing a repressive chromatin status of the RARß2 gene appears functionally related to the RA resistance of AML1/ETO-positive blasts. 5-azacytidine relieves AML1/ETO transcriptional silencing of the RA signaling pathway and supports RA-induced differentiation Next we investigated the respective roles of DNA hypermethylation of the RARß2 gene and of AML1/ETO expression in the silencing of the RA signaling pathway and in the differentiation block of SKNO-1 cells. Initially, we tested the ability of the DNA methylation inhibitor 5-azacytidine to restore cell sensitivity to RA in terms of reactivation of the endogenous RARß2 gene through demethylation of its promoter region and of restoration of RA differentiation activity in this RA-resistant AML cell line.47 In untreated SKNO-1 cells the basal percentage of 5-methylcytosine residues on the RARß2 gene in the promoter/ßRARE and in the exon 1 regions was 67% and 99%, respectively (Figures 4C and 5A). Following treatment with 5-azacytidine, the percentage of 5-methylcytosine residues diminished to 26% in the promoter/ßRARE region of RARß2 gene, and a similar percentage was reached when given simultaneously with RA, while RA alone decreased the methylation of this region to 38%. Notably, the knockdown of AML1/ETO in SKNO-1-siA/E–infected cells also resulted in a reduction of the percentage of 5-methylcytosine residues at that promoter/ßRARE site to 32%. Although we did not observe demethylation of the exon 1 region in SKNO-1–treated or siA/E-infected cells, the demethylation of the promoter/ßRARE region following the 5-azacytidine treatment or AML1/ETO knockdown was sufficient to restore RA-dependent induction of RARß2 expression in these cells (Figures 1D and 5B). These results are in agreement with the regulatory function of the ßRARE region and with its RA inducibility that can be accomplished in the presence of a reduced CpGs methylation status at this site, as shown by methylation data. Remarkably, in SKNO-1 cells the expression level of the myeloid-specific cell-surface antigen CD11b is poorly modified by treatment with RA or 5-azacytidine when used as single agents, while it is strongly induced by the combined treatment of RA/5-azacytidine, thus suggesting the ability of 5-azacytidine to restore the differentiation responsiveness of SKNO-1 cells to RA (Figure 5B). These findings also established a correlation between AML1/ETO expression levels, RARß2 demethylation, RARß2 reexpression, and induction of differentiation of RA-treated SKNO-1 cells.
The RARß2 promoter/exon 1 region is methylated in non-APL AML blasts To determine the significance of our findings in human leukemia, we investigated the methylation status of RARß2 promoter/exon 1 and RARß2 mRNA expression levels in primary blasts from AML patients containing or not specific genetic lesions (Figure 6). We used MSP as a sensitive and reliable approach for the identification of CpG sites that differ in their methylation status in DNA samples from a series of AML patients (Figure 6A). Remarkably, by using primers for the promoter/exon 1 regions of RARß2 (P3 primers), we found that the region containing the ßRARE binding site is methylated in 6 of 8 AML1/ETO-positive samples, and all of the samples (8 of 8) presented hypermethylation of a region located in the 5'-UTR exon 1 of RARß2 as detected by using P4 primers. Neither of these 2 regions on the RARß2 gene was found methylated in CD34+ normal hematopoietic precursors (Figure 6A).
To verify whether RARß2 promoter/exon 1 methylation could represent a common lesion in myeloid leukemia, we next analyzed AML-M2 and AML-M4 blasts with apparently normal karyotype or presenting recurrent AML genetic alterations. Notably, the RARß2 region containing the ßRARE binding site was methylated in 7 of 9 AML-M2 samples (P3 primers) and in 9 of 10 AML-M4 samples, whereas the 5'-UTR exon 1 region of RARß2 (P4 primers) was methylated in samples from 9 of 9 AML-M2 and 8 of 10 AML-M4. In agreement with previous results obtained in BM cells from 15 APL (AML-M3) patients,18,19 RARß mRNA was not expressed in samples isolated from the BM of 1 APL or 12 non-APL AML patients (AML-M2–AML-M4) (Figure 6B). However, RARß gene expression was fully detectable in CD34+ hematopoietic progenitors isolated from the BM of healthy donors and, as expected, its expression was restored by RA treatment in primary APL blasts (Figure 6B). Overall, these results indicate the magnitude of the heterochromatic silencing of the RA signaling pathway in myeloid leukemias and support previous in vitro and in vivo evidence indicating that epigenetic deregulation of the developmental program of normal myelopoiesis such as that of RA is shared by different AML subtypes irrespective of the presence of specific genetic lesions.37,38,42
Aberrant DNA methylation seems to be a dominant factor in epigenetic gene silencing. Indeed, disruption of DNA methylation patterns is frequently present in aberrant development and neoplastic transformation.4,5 Here, we show that the expression of AML1/ETO, the most common AML-associated fusion protein, increases the methylation status at genomic loci physiologically regulated by RA, a regulator of myelopoiesis. Our findings identify RAR -RXR heterodimer as a component of the macromolecular complex formed by AML1/ETO. As a consequence, AML1/ETO recruits HDAC1/DNMT/MeCP2 activities on specific DNA binding domains of RAR and AML1 genes and methylates CpG-rich sequences and deacetylates/methylates specific lysine residues on nucleosomal histones, thereby promoting the formation of nonpermissive chromatin at these sites. Therefore, the association of the "gain of function" of the AML1/ETO oncoprotein and the "loss of function" of RAR-RXR heterodimer results in the heterochromatic silencing of a key transcriptional pathway of myelopoiesis, contributing to the differentiation block of myeloid progenitors. This hypothesis is supported by findings indicating that the differentiation response to RA can be restored in AML1/ETO blasts by (1) impairing the interaction between the AML1/ETO and the transcriptional corepressor complex using protein fragments representative of their interaction surfaces47 and (2) changing the methylation status at regulatory sites on the RA target gene RARß2 by AML1/ETO knockdown by RNAi or pharmacologic treatment with the demethylating agent 5-azacytidine.
While this paper was in preparation, Tabe et al addressed the methylation status of the RARß2 gene and the ability of demethylating agents to reverse RA resistance in AML blasts and in PML/RAR Transgenic mouse models and bone marrow transplantation approaches in mice have shown, however, that AML1/ETO expression predisposes myeloid precursors to transformation but is not sufficient to induce leukemia, which appears to require further molecular damages.27,54 In this context, a crucial question is the role of genetic and epigenetic alterations in the initiation or maintenance of AML1/ETO-positive leukemia. RNAi experiments indicated that a titrated AML1/ETO production is necessary for its activity as a transcriptional repressor of RARß2 and for the block of myeloid differentiation. Interestingly, RARß2 is the RA-regulated tumor suppressor gene silenced in a variety of human malignancies.8,16–19 Of note, hypermethylation of RARß2 is commonly detectable in AML blasts independently from the presence of specific genetic lesions.
The relevance of this finding in AMLs is underlined by the fact that aberrant heterochromatic gene silencing can represent an alternative mechanism to gene mutation or deletion for the transcriptional repression of tumor suppressor genes.4,5,48 In AML subtypes lacking the PML/RAR In conclusion, our study provides new insight into molecular pathways deregulated in myeloid leukemogenesis and further supports the reversion of transformed phenotype by targeting of gene silencing as a promising and powerful therapeutic strategy for AMLs.
Contribution: F.F. and G.Z. designed and performed experiments and cowrote the manuscript; V.G., L.T., and A.C. designed and performed experiments; L.D.C., A.R., I.B., and F.G. generated essential new reagents and contributed to experimental design; F.L.-C. provided primary leukemia samples fully characterized at the molecular level and contributed to experimental design; P.G.P. contributed to experimental design; and C.N. designed the experiments and wrote the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Clara Nervi, University La Sapienza Rome & San Raffaele Bio-medical Park Foundation, Via di Castel Romano 100, Rome, 00128, Italy; e-mail: clara.nervi{at}uniroma1.it.
This work was supported by grants from the Italian Association for Cancer Research (AIRC and AIRC–Rome Oncogenomic Center [ROC]), Ministero dell'Istruzione, dell'Università e della Ricerca, University of Rome "La Sapienza," Ministero della Salute, Sixth Research Framework Programme of the European Union, Project RIGHT (LSHB-CT-2004 005276), and a fellowship from University La Sapienza Rome (L.T.). We are grateful to Silvia Di Cesare and Fabrizio Padula for FACS analysis and to Linda Starnes for critical reading of the manuscript.
Submitted September 7, 2006; accepted January 14, 2007.
Prepublished online as Blood First Edition Paper, January 23, 2007
DOI: 10.1182/blood-2006-09-045781
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
) that acts as a ligand-inducible transcription factor by binding to specific response elements (RAREs) in the regulatory regions of target genes.
) or not (
) with 1 µM RA for 24 hours. The results represent the average of 3 independent evaluations ± SD. (C) Coimmunoprecipitation and Western blot experiments performed in human U937 WT, U937 stably transfected with an HA-tagged AML1/ETO cDNA (A/E-HA), or with an empty vector (mock), and in human SKNO-1 cells lines. Ip- indicates the antibody used for the coimmunoprecipitation; IgG, rabbit serum used as nonspecific antibody. Coimmunoprecipitates were analyzed by Western blot (WB) with antibodies detecting the expression levels of AML1/ETO (A/E), AML1, and RAR
) mock and (
) A/E-HA nuclear extracts labeled with 10 nM [3H]-RA in the absence or in the presence of 200-fold molar excess of unlabeled RA (
, mock; 
, A/E-HA) to determine nonspecific binding by HPLC using a Superose 6 HR 10/30 size exclusion column.
) and empty (
