Iria Vázquez

Abnormal methylation of the common PARK2 and PACRG promoter is associated with downregulation of gene expression in acute lymphoblastic leukemia and chronic myeloid leukemia.

The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm.

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P=0.03) and T-ALL vs B-ALL (50 vs 9%, P=0.001). Relapse rate (62 vs 44%, P=0.05) and mortality (59 vs 43%, P=0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P<0.001 y P<0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

Molecular heterogeneity in AML/MDS patients with 3q21q26 rearrangements.

Patients with 3q21q26 rearrangements seem to share similar clinicopathologic features and a common molecular mechanism, leading to myelodysplasia or acute myeloid leukemia (AML). The ectopic expression of EVI1 (3q26) has been implicated in the dysplasia that characterizes this subset of myeloid neoplasias. However, lack of EVI1 expression has been reported in several cases, and overexpression of EVI1 was detected in 9% of AML cases without 3q26 abnormalities. We report the molecular characterization of seven patients with inv(3)(q21q26), t(3;3)(q21;q26) or related abnormalities. EVI1 expression was detected in only one case, and thus ectopic expression of this gene failed to explain all of these cases. GATA2 (3q21) was found to be overexpressed in 5 of the 7 patients. GATA2 is highly expressed in stem cells, and its expression dramatically decreases when erythroid and megakaryocytic differentiation proceeds. No mutations in GATA1 were found in any patient, excluding loss of function of GATA1 as the cause of GATA2 overexpression. We report finding molecular heterogeneity in patients with 3q21q26 rearrangements in both breakpoints and in the expression pattern of the genes near these breakpoints. Our data suggest that a unique mechanism is not likely to be involved in 3q21q26 rearrangements.

Overexpression of GATA2 predicts an adverse prognosis for patients with acute myeloid leukemia and it is associated with distinct molecular abnormalities

Overexpression of GATA2 predicts an adverse prognosis for patients with acute myeloid leukemia and it is associated with distinct molecular abnormalities

Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia

Background The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia. Design and Methods We analyzed the prevalence and prognostic impact of EVI1 over-expression in a series of 476 patients with acute myeloid leukemia, and investigated the epigenetic modifications of the EVI1 locus which could be involved in the transcriptional regulation of this gene. Results Our data provide further evidence that EVI1 over-expression is a poor prognostic marker in acute myeloid leukemia patients less than 65 years old. Moreover, we found that patients with no basal expression of EVI1 had a better prognosis than patients with expression/over-expression (P=0.036). We also showed that cell lines with over-expression of EVI1 had no DNA methylation in the promoter region of the EVI1 locus, and had marks of active histone modifications: H3 and H4 acetylation, and trimethylation of histone H3 lysine 4. Conversely, cell lines with no expression of EVI1 have DNA hypermethylation and are marked by repressive trimethylation of histone H3 lysine 27 at the EVI1 promoter. Conclusions Our results identify EVI1 over-expression as a poor prognostic marker in a large, independent cohort of acute myeloid leukemia patients less than 65 years old, and show that the total absence of EVI1 expression has a prognostic impact on the outcome of such patients. Furthermore, we demonstrated for the first time that an aberrant epigenetic pattern involving DNA methylation, H3 and H4 acetylation, and trimethylation of histone H3 lysine 4 and histone H3 lysine 27 might play a role in the transcriptional regulation of EVI1 in acute myeloid leukemia. This study opens new avenues for a better understanding of the regulation of EVI1 expression at a transcriptional level.

Downregulation of DBC1 expression in acute lymphoblastic leukaemia is mediated by aberrant methylation of its promoter

The DBC1 gene is a potential tumour suppressor gene that is commonly hypermethylated in epithelial cancers. We studied the role of promoter hypermethylation in the regulation of DBC1 in acute lymphoblastic leukaemia (ALL) cell lines and 170 ALL patients at diagnosis. Abnormal methylation of DBC1 was observed in all ALL cell lines and in 17% of ALL patients. Moreover, DBC1 methylation was associated with decreased DBC1 expression, while treatment of ALL cells with 5‐Aza‐2′‐deoxycytidine resulted in demethylation of the promoter and upregulation of DBC1 expression. Fluorescence in situ hybridisation identified the deletion of one allele of DBC1 in some ALL cell lines, which indicated that the lack of DBC1 expression was due to deletion of one allele and methylation of the other. In conclusion, these results demonstrate, for the first time, that the expression of DBC1 is downregulated in a percentage of patients with ALL due to the hypermethylation of its promoter and/or gene deletion.

Silencing of hsa-miR-124 by EVI1 in cell lines and patients with acute myeloid leukemia

We read with great interest the work published by Dickstein et al. (1) showing that induced EVI1 expression in a murine model silences miR-124 expression by DNA methylation. The EVI1 gene codes for a transcription factor implicated in the development and progression of high-risk acute myeloid leukemia (AML) (2, 3). We quantify the expression of 250 microRNAs (miRNAs; TaqManHuman miRNA assay set) in 15 myeloid cell lines. Statistical analysis comparing cell lines with and without EVI1 protein identified miRNAs differentially expressed (B > 0). Among them, cell lines with EVI1 protein had no expression of hsa-miR-124, whereas most cell lines with no EVI1 had high hsa-miR-124 expression (Table 1). Interestingly, MEG-01 cells, with EVI1 overexpression and no protein, expressed low levels of hsa-miR-124 (Fig. 1 and Table 1). We first considered whether EVI1 was a target of hsa-miR-124. Transiently, transfection of pre–hsa-miR-124 in HEL and KU-812 cell lines, both with EVI1 protein and no hsa-miR-124 expression, showed a dramatic increase of hsa-miR-124; however, no changes in EVI1 expression either at the mRNA or protein level were detected. These results indicate that hsa-miR-124 does not regulate EVI1 expression. The results of Dickstein et al. (1) prompted us to examine whether DNA methylation could be responsible for the low expression of hsa-miR-124 in cell lines with EVI1 protein (1). We analyzed the methylation status of hsa-miR-124-1 by methylation-specific PCR as previously described (4). All of the cell lines analyzed that had low expression of hsa-miR-124 had the promoter methylated: four had EVI1, and three had no protein. Conversely, the two cell lines with high expression of hsa-miR-124 had no methylation and no EVI1 (Table 1). These results strongly support the hypothesis that EVI1 silences hsa-miR-124 expression by DNA methylation (1), although they would also indicate that, in some cases, the expression of hsa-miR-124 might be regulated by other mechanisms. To check the clinical importance of these results, we analyzed 42 AML patients, 19 of which had EVI1 overexpression (Table 1). Consistent with our results in cells lines, expression of EVI1 in patients was associated with decreased expression of hsa-miR-124 (P = 0.036), supporting that EVI1 could play a role in the transcriptional regulation of hsa-miR-124 (Fig. 1). Nevertheless, as in cell lines, some cases with low hsa-miR-124 expression had no EVI1 overexpression.

Molecular characterization of a t(1;3)(p36;q21) in a patient with MDS. MEL1 is widely expressed in normal tissues, including bone marrow, and it is not overexpressed in the t(1;3) cells

Patients with myeloid malignancies and either the 3q21q26 syndrome or t(1;3)(p36;q21) have been reported to share similar clinicopathological features and a common molecular mechanism for leukemogenesis. Overexpression of MDS1/EVI1 (3q26) or MEL1/PRDM16 (1p36), both members of the PR-domain family, has been directly implicated in the malignant transformation of this subset of neoplasias. The breakpoints in both entities are outside the genes, and the 3q21 region, where RPN1 is located, seems to act as an enhancer. MEL1 has been reported to be expressed in leukemia cells with t(1;3) and in the normal uterus and fetal kidney, but neither in bone marrow (BM) nor in other tissues, suggesting that this gene is specific to t(1;3)-positive MDS/AML. We report the molecular characterization of a t(1;3)(p36;q21) in a patient with MDS (RAEB-2). In contrast to previous studies, we demonstrate that MEL1, the PR-containing form, and MEL1S, the PR-lacking form, are widely expressed in normal tissues, including BM. The clinicopathological features and the breakpoint on 1p36 are different from cases previously described, and MEL1 is not overexpressed, suggesting a heterogeneity in myeloid neoplasias with t(1;3).

JAK2-V617F activating mutation in acute myeloid leukemia: prognostic impact and association with other molecular markers

JAK2-V617F activating mutation in acute myeloid leukemia: prognostic impact and association with other molecular markers

Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription

The EVI1 gene (3q26) codes for a transcription factor with important roles in normal hematopoiesis and leukemogenesis. High expression of EVI1 is a negative prognostic indicator of survival in acute myeloid leukemia (AML) irrespective of the presence of 3q26 rearrangements. However, the only known mechanisms that lead to EVI1 overexpression are 3q aberrations, and the MLL-ENL oncoprotein, which activates the transcription of EVI1 in hematopoietic stem cells. Our aim was to characterize the functional promoter region of EVI1, and to identify transcription factors involved in the regulation of this gene. Generation of seven truncated constructs and luciferase reporter assays allowed us to determine a 318-bp region as the minimal promoter region of EVI1. Site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays identified RUNX1 and ELK1 as putative transcription factors of EVI1. Furthermore, knockdown of RUNX1 and ELK1 led to EVI1 downregulation, and their overexpression to upregulation of EVI1. Interestingly, in a series of patient samples with AML at diagnosis, we found a significant positive correlation between EVI1 and RUNX1 at protein level. Moreover, we identified one of the roles of RUNX1 in the activation of EVI1 during megakaryocytic differentiation. EVI1 knockdown significantly inhibited the expression of megakaryocytic markers after treating K562 cells with TPA, as happens when knocking down RUNX1. In conclusion, we define the minimal promoter region of EVI1 and demonstrate that RUNX1 and ELK1, two proteins with essential functions in hematopoiesis, regulate EVI1 in AML. Furthermore, our results show that one of the mechanisms by which RUNX1 regulates the transcription of EVI1 is by acetylation of the histone H3 on its promoter region. This study opens new directions to further understand the mechanisms of EVI1 overexpressing leukemias.