Promoter hypermethylation of the retinoic acid receptor β2 gene is frequent in acute myeloid leukaemia and associated with the presence of CBFβ–MYH11 fusion transcripts

RUNX3/AML2 is a Runt domain transcription factor like RUNX1/AML1 and RUNX2/ AML3. Regulated by 2 promoters P1 and P2, RUNX3 is frequently inactivated by P2 methylation in solid tumors. Growing evidence has suggested a role of this transcription factor in hematopoiesis. However, genetic alterations have not been reported in blood cancers. In this study on 73 acute myeloid leukemia (AML) patients (44 children and 29 adults), we first showed that high RUNX3 expression among childhood AML was associated with a shortened event-free survival, and RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations. We further demonstrated that this RUNX3 repression was mediated not by P2 methylation, but RUNX1-ETO and CBF␤-MYH11, the fusion products of t(8; 21) and inv(16), via a novel transcriptional mechanism that acts directly or indirectly in collaboration with RUNX1, on 2 conserved RUNX binding sites in the P1 promoter. In in vitro studies, ectopically expressed RUNX1-ETO and CBF␤-MYH11 also inhibited endogenous RUNX3 expression. Taken together, RUNX3 was the first transcriptional target found to be commonly repressed by the t(8;21) and inv (16) IntroductionThe Runt domain transcription factor family includes RUNX1/ AML1, RUNX2/AML3, and RUNX3/AML2. These transcription factors share a conserved Runt domain for binding to a 6-base pair (bp) DNA sequence (TGT/cGGT) and heterodimerization with core-binding factor ␤ (CBF␤). CBF␤ does not bind DNA directly but increases the ability of RUNX proteins to bind DNA and regulate transcription. 1 RUNX1 and RUNX2 are essential for hematopoiesis and osteogenesis, respectively. 2,3 Moreover, RUNX1 regulates neuron and muscle function. 4,5 On the other hand, RUNX3 is involved in neurogenesis and thymopoiesis and acts as a tumor suppressor in gastric cancer. [6][7][8] In addition, RUNX3 is inactivated frequently by promoter methylation and less frequently by gene deletion, point mutations, and protein mislocalization in various solid tumors. 9 RUNX3 and RUNX1 show prominent expression in hematopoietic cells and different subsets of neurons, 4,10 while RUNX2 is expressed mainly in bone and also other tissues including hematopoietic stem cells. 11 The expression of RUNX3 and RUNX1 can be induced by retinoic acid, suggesting that these transcription factors may jointly regulate retinoic acid-mediated hematopoietic differentiation. 10 The functional overlap was supported by the observations that hematopoietic defects due to RUNX1 deficiency could be rescued by RUNX3. 12,13 Further evidence suggesting a role of RUNX3 in hematopoiesis was reduction of mature blood cell formation in zebrafish by RUNX3 depletion. 14 The role of RUNX3 in tumorigenesis and its potential involvement in hematopoiesis suggest a role of this transcription factor in hematological malignancies. However, genetic alterations of RUNX3 have not been reported in acute myeloid leukemia (AML). 15 t(8;21)(q22;q22) and inv(16)(p13;q22) are the 2 most common t...

Section: Discussionmentioning

confidence: 99%

Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia

Cheng

et al. 2008

“…Many genes that are methylated in tumors are not expressed in relevant normal tissues, but silencing genes that are critically important to controlling cell proliferation contributes to the development of a malignant phenotype in the same manner as inactivating mutations of tumor suppressor genes. 31 In leukemia, epigenetic silencing by DNA methylation of cyclin-dependent kinase inhibitors, 6,9,32,33 DNA repair genes, 15 apoptosis mediators, 12,13 nuclear receptors, 8,10,14 transcription factors, 16 cell adhesion molecules, 34 and many other genes has been reported. 2,11,17 We investigated the DNA methylation status of 15 genes in 21 leukemia cell lines using bisulfite pyrosequencing.…”

Section: Discussionmentioning

confidence: 99%

“…Methylation of the tumor suppressor gene p15 is the most frequently reported epigenetic event in myeloid malignancies and is frequently used to assess treatment success. 6,7 Numerous other genes are methylated in AML, 2,[8][9][10][11][12][13][14][15][16][17] but the effects of these changes on the development, progression, and relapse of disease are not completely understood.…”

Section: Introductionmentioning

confidence: 99%

Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse

Kroeger¹,

Jelı́nek²,

Estécio³

et al. 2008

149

131

DNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance. (Blood. 2008;112:1366-1373)

“…Methylation of the RAR␤2 promoter (a RAR␣2 paralog) in AML has also been studied by a number of different investigators, but the results of these studies have been somewhat contradictory in nature. 21,22,[25][26][27] Moreover, RARB is not readily expressed in normal hematopoietic cells or required for proper hematopoiesis, 9,28 and RAR␤2 promoter methylation has no effect on patient survival. 25 In fact, our data indicate that, in contrast to normal cells, RAR␤2 is expressed in a fraction of AML samples ( Figure S2).…”

Section: Figure 2 Mechanisms Underlying Loss Of Rara Expression Diffmentioning

confidence: 98%

DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

Glasow

Barrett

Petrie

et al. 2008

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.