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Overexpression of EZH2 associates with a poor prognosis in chronic lymphocytic leukemia
Blood Cells, Molecules, and Diseases
EZH2, a histone methyltransferase, is overexpressed in several human tumors, but whether it exerts any impact in chronic lymphocytic leukemia (CLL) remains unknown. We used real time PCR to investigate the expression profile of EZH1 and EZH2 in 59 CLL patients, 10 samples of purified B-cells from healthy donors and 12 normal adult tissues. EZH2 was overexpressed in CLL patients and correlates with high white blood cell count, ZAP-70 expression and chromosomal abnormalities. EHZ1 expression does not correlate with CLL progression. EZH2 overexpression is related to a poor prognosis of CLL and could be a useful tool to assess its aggressiveness.
Keywords: Chronic lymphocytic leukemia, EZH2, Histone methyltransferase, Gene expression, Cytogenetic.
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small mature B cells present in the blood, bone marrow, and lymphoid organs. This disease is the most frequent adult leukemia in western countries and affects individuals with a median age of 72 years at diagnosis and median age of 79 years at death  . According to the International Workshop on Chronic Lymphocytic Leukemia, CLL diagnosis is defined by the presence of at least 5 × 109 B cells/L in the peripheral blood that co-express the T-cell antigen dim CD5 and B-cell surface antigens CD19, dim CD20, CD23, CD43, CD79a, low density of surface IgM or IgD, with each clone of leukemia cells expressing one of either kappa or lambda immunoglobulin light chains  . The clinical course of CLL is extremely heterogeneous. Many patients present an indolent disease and will live for decades, while others have an aggressive clinical outcome, requiring immediate treatment. Most patients with aggressive clinical form have high expression of ZAP-70, a tyrosine kinase normally expressed in T and natural killer cells, while those with indolent clinical course show no expression of ZAP-70  and . Besides the expression of ZAP-70 and other markers, cytogenetic abnormalities have important prognostic significance, including the most common 13q, 11q, 17p and 6q deletions, and trisomy of chromosome 12  . Molecular genetics also have been contributing to explain the heterogeneity of CLL. Some of the molecular alterations have a well-known clinical implication (TP53 mutation: indication for allogeneic stem cell transplant; ATM disruption: indication for rituximab-based immunochemotherapy), while others still have to be defined (NOTCH1, SF3B1 and BIRC3 mutations)  .
Post-translational modification of histones by methylation, catalyzed by Histone Methyltransferases (HMTase), influence biological processes including normal development, cellular growth and regulation of gene transcription. Recently, associations between various types of cancer and the altered regulation of histone-modifying enzymes have been described  . In this context, beside genetic mutations, epigenetic modifications seem to play a key role in CLL. There is evidence of altered methylation in CLL, with DNA methylation being one of the most studied changes. It seems that other regulators of genome methylation are involved in CLL, in a complex epigenetic pathway that includes HMTase as well. Indeed, it was found that the eukaryotic histone methyltransferase (Eu-HMTaseI) was upregulated in B-cell leukemia, with increased disease severity  . Another family of HMTases, the mixed lineage leukemia (MLL), is also frequently involved in leukemia  .
The Polycomb Group of proteins (PcG) form complexes that modify the chromatin, maintaining gene repression during development and playing an important role in determining cell fate. There are complexes containing PcG proteins such as Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). The PRC2 chromatin mark H3K27me stimulates PRC1 to enact gene silencing at target genes. PRC2 and PRC1 coordinate their functions through regulation of specific microRNAs. Increased PRC2 activity in cancer leads to repression of numerous microRNAs, involved in the maintenance of stem-cell-like phenotypes in cancer cells, and subsequently increases PRC1 components, such as BMI1 and RING2  . PRC2, which catalyzes methylation of histone H3 lysine 27 (H3K27), contains either the enzymatic subunit EZH2 (enhancer of zeste homologue 2, also known as KMT6 or KMT6A) or EZH1 (enhancer of zeste homologue 1, also known as KMT6B). Although PRC2–EZH1 has lower catalytic activity compared to PRC2–EZH2, both complexes contribute to the maintenance of cellular H3K27 methylation states. It seems that PRC2–EZH2 mediated repression and DNA methylation are coordinated in order to maintain gene silencing  . Indeed, EZH2 directly control DNA methylation, recruiting DNA methyltransferases to target genes  , and genes marked by PRC2–EZH2 are major targets for DNA methyltransferases  .
Numerous studies suggest that EZH2 acts as oncogene and is aberrantly overexpressed in several types of cancer and, recently, studies suggest that EZH2 plays an important role in several hematologic malignancies, promoting the proliferation and aggressiveness of neoplastic cells, although the exact mechanisms and pathways it deregulates seem to vary in different malignancies. EZH2 was found to be aberrantly overexpressed in natural killer/T-cell lymphoma (NKTL), conferring a poor prognosis  . In acute myeloid leukemia, EZH2 inhibited differentiation programs in leukemic stem cells, increasing their leukemogenic activity  . In chronic myelomonocytic leukemia, various mutant genotype combinations were observed, including mutation of EZH2 in 5.5% of patients, suggesting that molecular defects affecting distinct pathways can lead to similar clinical phenotypes  . In acute lymphoblastic leukemia cell line Nalm-6, EZH2 is overexpressed and promotes the progression of cancer by directly mediating the inactivation of tumor suppressor genes p21 and PTEN  . In contrast to these results, it was found that loss of EZH2 function in myeloid malignancies can occur through multiple different mechanisms and contribute to leukemogenesis through inappropriate release of genes like HOXA9 from repression by H3K27 trimethylation  .
Although EZH2 is overexpressed in several solid cancers and altered in hematologic malignancies, there are few data about the involvement of this gene in CLL. In the present study, we analyzed the correlation between EZH genes expression patterns with chromosomal aberrations, ZAP-70 expression and white blood cell (WBC) count in 59 cases of CLL and 10 purified B-cells from healthy donors.
Material and methods
Fifty-nine CLL patients were included in the present study. The diagnosis of CLL was established using Matutes immunophenotypic analysis system  . As normal controls, we used peripheral blood mononuclear cells (PBMC) from 10 age-matched hematological healthy donors (age 50 to 84 years). Informed consent was obtained from all patients, and approval for this study was obtained from the Ethical Committee of the University Hospital of Medical School of Ribeirão Preto, University of São Paulo, Brazil.
Immunomagnetic B-cell sorting
Cells from CLL patients and hematological healthy donors were isolated using Ficoll–Hypaque density gradient centrifugation (Sigma-Aldrich, St Louis, MO, USA). Isolated PBMCs from healthy donors were used to isolate B-cells by magnetic-activated cell sorting (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer's recommendations. Briefly, cells were incubated with an anti-human CD19 monoclonal antibody conjugated with micro beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). The cell suspension was passed through a LS column (MACS Separation Columns from Miltenyi Biotec, Bergisch-Gladbach, Germany) previously attached to the SuperMACS device for selection of labeled cells. After isolation, the purity rate of samples was higher than 90%.
For the immunophenotypic analysis, 1 × 106 mononuclear cells were used per tube. The initial flow cytometry evaluation was performed using the following antibodies: FITC-conjugated anti-CD20, PE-conjugated anti-CD79b, PE-Cy5-conjugated anti-CD19, APC-conjugated anti-CD5, FITC-conjugated anti-λ, PE-conjugated anti-κ, PE-Cy5-conjugated anti-CD19, APC-conjugated anti-CD5, polyclonal FITC anti-λ, polyclonal PE anti-κ, PE-Cy5-conjugated anti-CD19 and APC-conjugated anti-CD5. All monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA, USA), except the polyclonal anti-λ and anti-κ (Dako, Carpinteria, CA, USA).
Evaluation of ZAP-70 was performed calculating the percentage of positive cells compared to isotype control (cut off 20%), using PE-conjugated anti-ZAP-70 (Dako, Carpinteria, CA, USA). A total of 300.000 events per tube were acquired and the gate strategy was performed as previously described to identify the percentage of CD19 +/CD5 + B-lymphocytes  . All samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, USA), and the Cell Quest software (Becton Dickinson, USA) was used for data acquisition and analysis.
Classical cytogenetic (G-banding)
To induce metaphase we used 1 × 107 of PBMC cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal calf serum in the presence of interleukin-2 and CpG-oligonucleotide DSP30 (TIBMolBiol, Berlin, Germany). After 72 hours, colcemid (Sigma, Munich, Germany) was added before chromosome preparation. Standard procedures were followed for chromosome preparation and the subsequent cytogenetic analysis and interpretation were made according to ISCN 2009  .
A commercial panel of total RNA from twelve human normal tissues (lung, small intestine - SI, brain, colon, kidney, muscle, liver, spleen, heart, testis, stomach and placenta) obtained from Origene® (OriGene Technologies, Rockville, USA) was used in this study to determine EZH genes expression patterns in normal tissues.
RNA extraction and cDNA synthesis
Total RNA from leukemic samples and normal control samples was isolated using TRIzol LS Reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Single-stranded complementary DNA (cDNA) was generated from 1 μg of total RNA obtained from clinical samples and commercial panel of normal tissues using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer's recommendations.
Quantitative real-time PCR (qPCR)
Reactions of qPCR were performed on a StepOnePlus ™ Real-Time PCR System (Applied Biosystems) using TaqMan Universal PCR MasterMix and TaqMan Gene Expression Assays (Hs00940463_m1 for EZH1, Hs00544833_m1 for EZH2, Applied Biosystems), according to the manufacturer's instructions. Gene expression values are expressed as ratios between the amplification levels of genes of interest and that of endogenous control gene (Hs99999903_m1 for ACTB, Applied Biosystems), which provides a normalization factor for the amount of RNA isolated from a specimen.
qPCR data and statistical analysis
Relative expression was calculated using the formula 2−∆∆CT  . ACTB was used to normalize sample loading and the mean of Cq (quantification cycle) values of control samples as a reference. The amount of target gene, normalized to the endogenous control gene and relative to a reference sample, was converted into relative quantification. Based on the continuous distribution of the EZH gene expression on CLL samples, we adopted the median value of EZH1 and EZH2 expression as the cut-off to dichotomize CLL patients in “low” and “high” EZH1 and EZH2 expression, respectively. Clinical and laboratory information were then compared between groups. Prism 5 Software (GraphPad Software Inc., San Diego, CA, USA) was used to perform all statistical analyses. The level of significance was set to 5% (CI 95%). The Mann–Whitney U-test was used to examine differences between EZH expression profile (low or high) groups versus platelets, WBC count and ZAP-70 status and also to compare the groups of CLL patients with normal versus abnormal karyotypes. Karyotype stratification was done by Kruskal–Wallis followed by the Dunn's multiple comparison test. Association of EZH expression profile with karyotype was done using Fisher's exact test.
High EZH2 expression is significantly correlated with CLL poor prognostic factors
The summary of clinical and laboratory characteristics of CLL patients is shown in Table 1 . Among 59 patients analyzed, 66% were classified as Binet A, 22% as Binet B and 12% as Binet C  . According to G-banding cytogenetic analysis, 25% of the patients presented normal karyotype and 75% showed chromosomal abnormalities ( table 1 ). Initially, we compared EZH1 and EZH2 gene expression profiles between CLL patients and control samples, using the Mann–Whitney test. EZH2 gene expression was higher in CLL samples (P < 0.0001) ( Fig. 1 ), but no difference was detected regarding EZH1 gene expression (p = 0.13).
|Tumor stage (Binet system)|
|Binet A||39 (66%)|
|Binet B||13 (22%)|
|Binet C||7 (12%)|
|Cytogenetic analysis a|
|13q deletion||5 (8.3%)|
|17p deletion||4 (6.7%)|
|12 trisomy||16 (26.7%)|
|ZAP-70 expression b|
|> 20%||39 (67.2%)|
|< 20%||19 (32.8%)|
a One sample presenting 13q deletion and 12 trisomy was represented in both groups.
b One quantification was missed.
Differential EZH1 expression had no impact on platelets count (p = 0.15), ZAP-70 protein expression (p = 0.19) or cytogenetic abnormalities (p = 0.12); however, patients with EZH1 low expression had a higher WBC count compared to those with high EZH1 expression (p = 0.005) ( Fig. 2 A).
Overexpression of EZH2 was associated with higher WBC count, higher ZAP-70 expression and cytogenetic abnormalities. Patients with high EZH2 expression had higher ZAP-70 expression level (p = 0.02) ( Fig. 2 B) and higher WBC count, compared to those expressing low gene levels (p = 0.005) ( Fig. 2 C). EZH2 expression was higher in CLL patients with cytogenetic abnormalities when compared to those with normal karyotype (p = 0.0025) ( Fig. 3 A). Fisher's exact test also showed that high EZH2 expression had a significant association with cytogenetic abnormalities (relative risk: 6 (95% CI: 1.47– 24.48), p = 0.004). Kruskal–Wallis test followed by Dunn's multiple comparison test revealed that patients with any kind of cytogenetic abnormality showed higher expression of EZH2, when compared to patients with normal karyotypes (p = 0.0011) ( Fig. 3 B).
EZH genes are differentially expressed among normal adult tissues
EZH gene expression patterns in normal human tissues were analyzed using the relative quantification method ( Fig. 4 ). The lung tissue was chosen as reference (1X sample). The mean of Cq (quantification cycle) values from normal controls (purified B-cells from healthy donors) was included for the relative quantification. The RNA levels of EZH1 and EZH2 genes in normal tissues showed distinct patterns. EZH1 gene is more expressed in brain and slightly more expressed in kidney, muscle, heart and testis. EZH2 shows a very high expression in testis and are slightly more expressed in colon and spleen.
The clinical course of CLL is extremely heterogeneous. Accordingly, survival of patients with CLL ranges from less than 1–2 years to over 15 years  . The Rai and Binet clinical staging systems still remain the primary reference for identifying CLL patients with advanced disease. However, these staging systems do not provide risk stratification in early stage disease, which includes most cases of newly diagnosed CLL  . The elevated expression of ZAP-70 in patients with CLL has been extensively used to correlate to a worse prognosis. ZAP-70 is normally expressed in T and natural killer cells, where it is required for T-cell receptor signaling. It plays a major role in lymphocyte signal transduction, being a critical factor in the transition of pre-B to pro-B cells within the bone marrow resulting in cell differentiation and proliferation  . ZAP-70+ cells are more responsive to environmental signals and a favorable microenvironment could result in a more progressive form of the disease  . We showed that high EZH2 expression strongly correlated to elevated expression of ZAP-70 in patients with a worse prognosis (p = 0.02) ( Fig. 2 B). ZAP-70 usually is not mutated, but the mechanisms that regulate its expression are not completely understood. Methylation profiling of the ZAP-70 promoter identified regions responsible for transcriptional regulation, including a specific single CpG dinucleotide in its 5′ region, that when methylated is associated with decreased ZAP-70 expression and favorable clinical outcome in CLL  . In this context, and taking into account the heterogeneous expression of ZAP-70 among patients, it is reasonable that other epigenetic marks could influence its mRNA or protein expression. It was shown that genes marked by PRC2–EZH2 are major targets for DNA methyltransferases. In addition, the PRC2–EZH2 mediated H3K27me2/3 mark also facilitates the recruitment of PRC1, with ubiquitination function, and of histone deacetylases  . Although their expression levels are correlated, whether EZH2 influences expression of ZAP-70 is still to be investigated.
High white blood cell (WBC) count also has a prognostic significance, indicating disease progression  . In our study, patients with high EZH2 expression showed higher WBC count (p = 0.005) ( Fig. 2 C) and, inversely, those with high EZH1 expression revealed lower WBC count ( Fig. 2 A). Moreover, WBC count is higher in CLL patients with cytogenetic abnormalities than in patients with normal karyotype (data not shown). Blood count is essential to define the diagnosis of CLL, together with evaluation of blood smear and immune phenotype of the circulating lymphoid cells  . However, it measures an outcome of the disease process. Since CLL is a disease with a very heterogeneous clinical course, an ideal marker should predict a more aggressive form of the disease in early stage, allowing accurate risk stratification.
Genomic aberration in CLL is also used as a predictive mark of disease progression and patient survival, with 17p and 11q deletions and 12 trisomy related to worse prognosis  . We analyzed both EZH1 and EZH2 expression in patients with the most common cytogenetic abnormalities in CLL, such as 13q, 11q, 17p and 6q deletions, and trisomy of chromosome 12. EZH2 was more expressed in patients with cytogenetic abnormalities, especially patients harboring trisomy of chromosome 12. The small number of patients with other cytogenetic abnormalities did not allow a robust statistical analysis. Despite this limitation, all of them clearly showed higher expression of EZH2 when compared to patients with normal karyotypes ( Fig. 3 B).
EZH1 showed no significant differences between CLL and control groups. In the case of WBC count, the results revealed an inverse correlation between the expression levels of EZH1 and EZH2 genes. Indeed, although EZH1 and EZH2 share 96% sequence identity in their SET domains, they are not functionally redundant. PRC2–EZH1 has lower catalytic activity compared to PRC2–EZH2 and seems to compact chromatin in the absence of the methyltransferase cofactor SAM  . EZH1 and EZH2 have different patterns, with EZH1 being more abundant in non-proliferative adult organs while EZH2 is related to proliferative tissues  . Our results of mRNA levels of EZH genes in normal adult tissues are in accordance with these observations showing that EZH1 is more expressed specially in brain, while EZH2 is more expressed in testis.
EZH2 involvement in carcinogenesis is complex and seems to depend on numerous factors including the cellular context, the involvement of microRNA regulation, genetic mutations and the molecular partners involved. The complex mechanisms leading to EZH2 overexpression still have to be better understood. A few studies have linked its overexpression to the deletion of a multitude of microRNA. Deletions of microRNA-101, a negative posttranscriptional regulator of EZH2 expression, have been described in prostate cancer  . In natural killer/T-cell lymphoma (NKTL), the overexpression of EZH2 was related to Myc-mediated repression of miRNAs, such as the tumor-suppressors miR26 and miR101  . Interestingly, miR-101 downregulation was also associated with higher levels of EZH2 protein in aggressive CLL  . In addition to alterations in its expression, it was reported that both activating and inactivating mutations of EZH2 are associated with malignancy  , showing the complex role of PRC2–EZH2 in carcinogenesis and cell fate decision. Therefore, the oncogenic function of EZH2 might not depend solely on its enzymatic activities, since it was demonstrated that the ability to promote proliferation did not always require its histone methyltransferase activity, but the activation of transcription of Cyclin D1  .
The intricate scenario of EZH2 involvement in carcinogenesis will impose challenges to the development of therapeutic strategies, but studies on specific types of cancer can help to clarify the mechanisms and pathways deregulated in different diseases.
Our assessment on the correlation of EZH2 overexpression with classical poor prognostic factors in CLL may contribute to the development of better prognostic markers for this disease. Moreover, EZH2 could be an interesting therapeutic target to treat CLL.
Role of the funding source
The study sponsors had no involvement in the study design, collection, analysis or interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.
- - CNPq (National Council of Technological and Scientific Development): Number 482869/2009-7.
- - CAPES (Coordination for the Improvement of Higher Education Personnel): Number 23038.007324/2011-16.
- - FAPDF (Federal District Research Foundation): Number 0193.000.495/2009.
DAR: Conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing. ARLA: Conception and design, provision of study material or patients, collection and/or assembly of data, and manuscript writing. JCRAS, VBASE and MCCV: Conception and design, data analysis and interpretation. FMO: Conception and design, provision of study material or patients, collection and/or assembly of data. EMR: provision of study material or patients, collection and/or assembly of data. FSA: Conception and design, provision of study material or patients, data analysis and manuscript writing. FPS: Conception and design, provision of study material or patients, data analysis and manuscript writing. All authors read and approved the final manuscript.
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a Laboratory of Molecular Pathology of Cancer, University of Brasília, Av. L2 Norte, Brasília, DF 70.910-900, Brazil
b Department of Internal Medicine, Medical School of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14.048-900, Brazil
c Hospital de Base do Distrito Federal, Setor Hospitalar Sul, Área Especial, Quadra 101, Brasília, DF 70.330-150, Brazil
© 2014 Published by Elsevier B.V.