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Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia.

Vilas-Zornoza A, Agirre X, Martín-Palanco V, Martín-Subero JI, San José-Eneriz E, Garate L, Álvarez S, Miranda E, Rodríguez-Otero P, Rifón J, Torres A, Calasanz MJ, Cruz Cigudosa J, Román-Gómez J, Prósper F - PLoS ONE (2011)

Bottom Line: We found that 154 genes were methylated in more than 10% of ALL samples.The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients.Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis.

View Article: PubMed Central - PubMed

Affiliation: Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

ABSTRACT
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.

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TP53 pathway genes are methylated in ALL cell lines.A. MSP analysis of the methylated and un-methylated sequences of genes implicated in TP53 pathway in ALL derived cell lines. UC: un-methylated control (DNA of peripheral blood from a healthy donor). MC: universally methylated control, M: 100 bp molecular marker. B. Graphic representation of the results of methylation of genes of TP53 pathway and its involvement in TP53 dependent apoptosis, cell cycle or regulation of TP53. Information regarding CpG composition and whether these genes are target of PCR2 in ESC is shown. Red: methylated, Green: unmethylated.
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pone-0017012-g004: TP53 pathway genes are methylated in ALL cell lines.A. MSP analysis of the methylated and un-methylated sequences of genes implicated in TP53 pathway in ALL derived cell lines. UC: un-methylated control (DNA of peripheral blood from a healthy donor). MC: universally methylated control, M: 100 bp molecular marker. B. Graphic representation of the results of methylation of genes of TP53 pathway and its involvement in TP53 dependent apoptosis, cell cycle or regulation of TP53. Information regarding CpG composition and whether these genes are target of PCR2 in ESC is shown. Red: methylated, Green: unmethylated.

Mentions: To further analyze the hypothesis that the TP53 pathway might be inactivated or blocked in ALL independently of TP53 mutations, we extended the analysis of DNA methylation by MSP to a total of 24 genes implicated in the TP53 pathway using six derived ALL cell lines (Figure 3). These genes are implicated in p53 regulation and p53 dependent cell cycle and apoptosis while all of them have a typical CpG island. Interestingly 13 of the 24 genes were aberrantly hypermethylated in ALL derived cell lines (Figure 4A), of which 84.6% (11/13) were HCP, 7.7% (1/13) ICP and 7.7% (1/13) LCP. Unlike the whole group of genes differentially methylated in ALL, only 3 of the 13 genes (23.07%) of the TP53 pathway were PRC2 targets in ESC (Figure 4B). Nine of the 13 methylated genes were involved in the regulation of p53 dependent apoptosis (DBC1, hsa-miR-34b, hsa-miR-34c, POU4F2, AMID, APAF1, ASPP1, TP73 and NOXA), 2 of them in p53 dependent cell cycle regulation (POU4F1 and CDKN1C) and the other two in p53 regulation (LATS2 and DAPK1) (Figure 4B). While we have previously demonstrated most of these genes to be regulated by hypermethylation in ALL [11], [19], that was not the case for AMID, POU4F1, POU4F2 and hsa-miR-34b/c. Expression of these five genes was decreased in hypermethylated ALL cell lines and treatment in vitro with 4 µM of 5-aza-2-deoxycytidine (added one time only) during 4 days, induced a demethylation and upregulation of gene expression (Figure S5). These results indicate that DNA methylation of p53 dependent genes is an important mechanism associated with abnormal function of the TP53 pathway in ALL.


Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia.

Vilas-Zornoza A, Agirre X, Martín-Palanco V, Martín-Subero JI, San José-Eneriz E, Garate L, Álvarez S, Miranda E, Rodríguez-Otero P, Rifón J, Torres A, Calasanz MJ, Cruz Cigudosa J, Román-Gómez J, Prósper F - PLoS ONE (2011)

TP53 pathway genes are methylated in ALL cell lines.A. MSP analysis of the methylated and un-methylated sequences of genes implicated in TP53 pathway in ALL derived cell lines. UC: un-methylated control (DNA of peripheral blood from a healthy donor). MC: universally methylated control, M: 100 bp molecular marker. B. Graphic representation of the results of methylation of genes of TP53 pathway and its involvement in TP53 dependent apoptosis, cell cycle or regulation of TP53. Information regarding CpG composition and whether these genes are target of PCR2 in ESC is shown. Red: methylated, Green: unmethylated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046174&req=5

pone-0017012-g004: TP53 pathway genes are methylated in ALL cell lines.A. MSP analysis of the methylated and un-methylated sequences of genes implicated in TP53 pathway in ALL derived cell lines. UC: un-methylated control (DNA of peripheral blood from a healthy donor). MC: universally methylated control, M: 100 bp molecular marker. B. Graphic representation of the results of methylation of genes of TP53 pathway and its involvement in TP53 dependent apoptosis, cell cycle or regulation of TP53. Information regarding CpG composition and whether these genes are target of PCR2 in ESC is shown. Red: methylated, Green: unmethylated.
Mentions: To further analyze the hypothesis that the TP53 pathway might be inactivated or blocked in ALL independently of TP53 mutations, we extended the analysis of DNA methylation by MSP to a total of 24 genes implicated in the TP53 pathway using six derived ALL cell lines (Figure 3). These genes are implicated in p53 regulation and p53 dependent cell cycle and apoptosis while all of them have a typical CpG island. Interestingly 13 of the 24 genes were aberrantly hypermethylated in ALL derived cell lines (Figure 4A), of which 84.6% (11/13) were HCP, 7.7% (1/13) ICP and 7.7% (1/13) LCP. Unlike the whole group of genes differentially methylated in ALL, only 3 of the 13 genes (23.07%) of the TP53 pathway were PRC2 targets in ESC (Figure 4B). Nine of the 13 methylated genes were involved in the regulation of p53 dependent apoptosis (DBC1, hsa-miR-34b, hsa-miR-34c, POU4F2, AMID, APAF1, ASPP1, TP73 and NOXA), 2 of them in p53 dependent cell cycle regulation (POU4F1 and CDKN1C) and the other two in p53 regulation (LATS2 and DAPK1) (Figure 4B). While we have previously demonstrated most of these genes to be regulated by hypermethylation in ALL [11], [19], that was not the case for AMID, POU4F1, POU4F2 and hsa-miR-34b/c. Expression of these five genes was decreased in hypermethylated ALL cell lines and treatment in vitro with 4 µM of 5-aza-2-deoxycytidine (added one time only) during 4 days, induced a demethylation and upregulation of gene expression (Figure S5). These results indicate that DNA methylation of p53 dependent genes is an important mechanism associated with abnormal function of the TP53 pathway in ALL.

Bottom Line: We found that 154 genes were methylated in more than 10% of ALL samples.The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients.Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis.

View Article: PubMed Central - PubMed

Affiliation: Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

ABSTRACT
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.

Show MeSH
Related in: MedlinePlus