Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia

Dkk-3 is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a ‘hot spot’ for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P¼0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P¼0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease

Reversion of epigenetically mediated BIM silencing overcomes chemoresistance in Burkitt lymphoma

In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2(-/-)γc(-/-) mice and of murine B220(+)IgM(+) B-cell lymphomas generated in Eμ-MYC and Eμ-MYC-BIM(+/-) transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL

Epigenetic Signatures Associated with Different Levels of Differentiation Potential in Human Stem Cells

The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles

Identification of importin (IPO-8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Abstract Background: The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results: We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RTPCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion: Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples

Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia

Promoter hypermethylation plays an important role in the inactivation of cancerrelated genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). To determine the extent of hypermethylation in ALL, we analyzed the methylation status of the CDH1, p73, p16, p15, p57, NES-1, DKK-3, CDH13, p14, TMS-1, APAF-1, DAPK, PARKIN, LATS-1, and PTEN genes in 251 consecutive ALL patients.Atotal of 77.3% of samples had at least 1 gene methylated, whereas 35.9% of cases had 4 or more genes methylated. Clinical features and complete remission rate did not differ among patients without methylated genes, patients with 1 to 3 methylated genes (methylated group A), or patients with more than 3 methylated genes (methylated group B). Estimated disease-free survival (DFS) and overall survival (OS) at 11 years were 75.5% and 66.1%, respectively, for the nonmethylated group; 37.2% and 45.5% for methylated group A; and 9.4% and 7.8% for methylated group B (P < .0001 and P .0004, respectively). Multivariate analysis demonstrated that the methylation profile was an independent prognostic factor in predicting DFS (P < .0001) and OS (P .003). Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in AL

Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia

Aberrant genome-wide hypomethylation is thought to be related to tumorigenesis by promoting genomic instability. Since DNA methylation is considered an important mechanism for the silencingof retroelements, hypomethylation in human tumors may lead to their reactivation. However, the role of DNA hypomethylation in chronic myeloid leukemia (CML) remains to be elucidated. In this study, the methylation status of the LINE-1 (L1) retrotransposon promoter was analysed in CML samples from the chronicphase (CP, n¼140) and the blast crisis (BC, n¼47). L1 hypomethylation was significantly more frequent in BC (74.5%) than in CP (38%) (Po0.0001). Furthermore, L1 hypomethylation led to activation of both ORF1 sense transcription (Po0.0001) and c-MET gene antisense transcription (Po0.0001), and was significantly associated with high levels of BCR–ABL (P¼0.02) and DNMT3b4 (P¼0.001) transcripts. Interestingly, in CP-CML, extensive L1 hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (P¼0.004) or imatinib (P¼0.034) and progression-free survival (P¼0.005). The above results strongly suggest that activation of both sense and antisense transcriptions by aberrant promoter hypomethylation of the L1 elements plays a role in the progression and clinical behavior of the CML

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML