Nerea Marcotegui

SETBP1 overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia

Acute myeloid leukemias (AMLs) result from multiple genetic alterations in hematopoietic stem cells. We describe a novel t(12;18)(p13;q12) involving ETV6 in a patient with AML. The translocation resulted in overexpression of SETBP1 (18q12), located close to the breakpoint. Overexpression of SETBP1 through retroviral insertion has been reported to confer growth advantage in hematopoietic progenitor cells. We show that SETBP1 overexpression protects SET from protease cleavage, increasing the amount of full-length SET protein and leading to the formation of a SETBP1-SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. The prevalence of SETBP1 overexpression in AML at diagnosis (n = 192) was 27.6% and was associated with unfavorable cytogenetic prognostic group, monosomy 7, and EVI1 overexpression (P < .01). Patients with SETBP1 overexpression had a significantly shorter overall survival, and the prognosis impact was remarkably poor in patients older than 60 years in both overall survival (P = .015) and event-free survival (P = .015). In summary, our data show a novel leukemogenic mechanism through SETBP1 overexpression; moreover, multivariate analysis confirms the negative prognostic impact of SETBP1 overexpression in AML, especially in elderly patients, where it could be used as a predictive factor in any future clinical trials with PP2A activators.

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.

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.

EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2.

Bakground:The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.Methods:We performed QT-RT–PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT.Results:Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines.Conclusions:Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

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

Integration of SNP and mRNA arrays with microRNA profiling reveals that MiR-370 is upregulated and targets NF1 in acute myeloid leukemia

Background Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. Results Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. Conclusions NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.

MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia

The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.

CIP2A high expression is a poor prognostic factor in normal karyotype acute myeloid leukemia

Acute myeloid leukemia (AML) comprises a biologically and clinically heterogeneous group of aggressive disorders that occur as a consequence of a wide variety of genetic and epigenetic abnormalities in hematopoietic progenitors. Despite significant advances in the understanding of AML biology,

The MDS and EVI1 complex locus (MECOM) isoforms regulate their own transcription and have different roles in the transformation of hematopoietic stem and progenitor cells

Transcriptional activation of the EVI1 oncogene (3q26) leads to aggressive forms of human acute myeloid leukemia (AML). However, the mechanism of EVI1-mediated leukemogenesis has not been fully elucidated. Previously, by characterizing the EVI1 promoter, we have shown that RUNX1 and ELK1 directly regulate EVI1 transcription. Intriguingly, bioinformatic analysis of the EVI1 promoter region identified the presence of several EVI1 potential binding sites. Thus, we hypothesized that EVI1 could bind to these sites regulating its own transcription. In this study, we show that there is a functional interaction between EVI1 and its promoter, and that the different EVI1 isoforms (EVI1-145kDa, EVI1-Δ324 and MDS1-EVI1) regulate the transcription of EVI1 transcripts through distinct promoter regions. Moreover, we determine that the EVI1-145kDa isoform activates EVI1 transcription, whereas EVI1-Δ324 and MDS1-EVI1 act as repressors. Finally, we demonstrate that these EVI1 isoforms are involved in cell transformation; functional experiments show that EVI1-145kDa prolongs the maintenance of hematopoietic stem and progenitor cells; conversely, MDS1-EVI1 repressed hematopoietic stem and progenitor colony replating capacity. We demonstrate for the first time that EVI1 acts as a regulator of its own expression, highlighting the complex regulation of EVI1, and open new directions to better understand the mechanisms of EVI1 overexpressing leukemias.