Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact

We performed a genome-wide analysis of aberrant DNA methylation in chronic lymphocytic leukemia (CLL) using methylated CpG island amplification (MCA) coupled with a promoter microarray. We identified 280 potential targets of aberrant DNA methylation in CLL. These genes were located more frequently in chromosomes 19 (16%, p=0.001), 16 (11%, p=0.001), 17 (10%, p=0.02) and 11 (9%, p=0.02) and could be grouped in several functional networks. Methylation status was confirmed for 22 of these genes (SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCY5, HAND2,SPOCK, MLL, ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4, KLK10, TFAP2 and APP) in 78 CLL patients by pyrosequencing. As a proof of principle, we analyzed the expression of 2 genes, PRIMA1 and APP, in primary cells and of GALGT2, TFAP2C and PRIMA1 in leukemia cells. There was an inverse association between methylation and gene expression. This could be reversed by treatment with 5-aza-2’-deoxycytidine in cell lines. Treatment in a clinical trial with 5-azacitidine resulted in decreased methylation of LINE, DLX4 and SALL1 in the peripheral blood B-cells of patients with CLL. IgVH mutational status or ZAP-70 expression were not associated with specific methylation profiles. By multivariate analysis, methylation of LINE and APP was associated with shorter overall survival (p = 0.045 and 0.0035, respectively). This study demonstrates that aberrant DNA methylation is common and has potential prognostic and therapeutic value in CLL.     

A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.     

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.     

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.     

Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact

We performed a genome-wide analysis of aberrant DNA methylation in chronic lymphocytic leukemia (CLL) using methylated CpG island amplification (MCA) coupled with a promoter microarray. We identified 280 potential targets of aberrant DNA methylation in CLL. These genes were located more frequently in chromosomes 19 (16%, p = 0.001), 16 (11%, p = 0.001), 17 (10%, p = 0.02) and 11 (9%, p = 0.02) and could be grouped in several functional networks. Methylation status was confirmed for 22 of these genes (SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCY5, HAND2, SPOCK, MLL, ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4, KLK10, TFAP2 and APP) in 78 CLL patients by pyrosequencing. As a proof of principle, we analyzed the expression of 2 genes, PRIMA1 and APP, in primary cells and of GALGT2, TFAP2C and PRIMA1 in leukemia cells. There was an inverse association between methylation and gene expression. This could be reversed by treatment with 5-aza-2′-deoxycytidine in cell lines. Treatment in a clinical trial with 5-azacitidine resulted in decreased methylation of LINE, DLX4 and SALL1 in the peripheral blood B-cells of patients with CLL. IgV_H mutational status or ZAP-70 expression were not associated with specific methylation profiles. By multivariate analysis, methylation of LINE and APP was associated with shorter overall survival (p = 0.045 and 0.0035, respectively). This study demonstrates that aberrant DNA methylation is common and has potential prognostic and therapeutic value in CLL.Key words: chronic lymphocytic leukemia, DNA methylation, MCA/promoter microarray, epigenetics     

Methylation markers identify high risk patients in IGHV mutated chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) exhibits a very variable clinical course. Altered DNA methylation of genes has shown promise as a source of novel prognostic makers in a number of cancers. Here we have studied the potential utility of a panel of methylation markers (CD38, HOXA4 and BTG4) in 118 CLL patients. Each of the three loci assessed exhibited frequent methylation, as determined by COBRA analysis, and individually correlated with either good (CD38, BTG4 methylation) or poor (HOXA4 methylation) prognosis. Using a combined approach to produce an overall methylation score, we found that methylation score was significantly associated with time to first treatment in CLL patients. Multivariate Cox regression analysis revealed that methylation score was the strongest predictor of time to first treatment, and was independent of IGHV gene mutational status and CD38 expression. This study provides proof of principle that a panel of methylation markers can be used for additional risk stratification of CLL patients.     

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.     

Downregulation of death-associated protein kinase 1 (DAPK1) in chronic lymphocytic leukemia

The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.     

B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients

On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-mu in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.     

Circadian Clock-Regulated Expression of Phytochrome and Cryptochrome Genes in Arabidopsis

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.     

Methylation markers identify high risk patients in IGHV mutated chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) exhibits a highly variable clinical course. Altered DNA methylation of genes has shown promise as a source of novel prognostic makers in a number of cancers. Here we have studied the potential utility of a panel of methylation markers (CD38, HOXA4 and BTG4) in 118 CLL patients. Each of the three loci assessed exhibited frequent methylation, as determined by COBRA analysis and individually correlated with either good (CD38, BTG4 methylation) or poor (HOXA4 methylation) prognosis. Using a combined approach to produce an overall methylation score, we found that methylation score was significantly associated with time to first treatment in CLL patients. Multivariate Cox regression analysis revealed that methylation score was the strongest predictor of time to first treatment and was independent of IGHV gene mutational status and CD38 expression. This study provides proof of principle that a panel of methylation markers can be used for additional risk stratification of CLL patients.Key words: DNA methylation, prognostic marker, leukemia, CD38, HOXA4, BTG4Chronic lymphocytic leukemia (CLL) is the most commonly diagnosed form of leukemia in the UK and exhibits a highly variable clinical course.¹ While some patients rapidly succumb to the disease, others can survive for well over ten years, often without the need for treatment. This extreme variability in clinical course among patients presenting with early CLL emphasizes the need for good prognostic markers to help direct patient treatment.^1,2 A number of molecular markers are already in frequent use, most notably IGHV mutational status.¹ In addition, other markers, such as protein expression of ZAP70 and CD38 and genomic abnormalities are also commonly used.² However, the group of patients defined as having a good prognosis through the use of current markers is still large (about 60% of CLL patients³) and these patients still exhibit a wide variety in outcome. Thus, additional markers are needed to aid clinical management in these cases.Altered DNA methylation, especially at promoter associated CpG islands, is one of the hallmarks of cancer.⁴ The high frequency of DNA methylation changes, associated with the relative ease and sensitivity with which DNA methylation can be detected, have led to increasing interest in the use of methylation based markers for diagnosis and prognosis.⁵ A number of methylated genes have already been shown to correlate with clinical features of CLL. For example, TWIST2 has been reported to exhibit frequent hypermethylation in CLL with mutated IGHV genes, which have a good prognosis. It is, however, rarely methylated in unmutated CLL cases, which have a poorer prognosis.⁶ Methylation of ZAP70 was strongly correlated with lack of ZAP70 expression and, similarly to TWIST2 methylation, was also strongly correlated with mutation of IGHV.⁷ In contrast, we recently showed that the HOXA4 gene was a frequent target for hypermethylation in CLL but, in this case, methylation of the gene was associated with the poor prognosis IGHV unmutated cases.⁸ While in many cases the functional role of the altered methylation still remains to be clearly demonstrated, the association between differential methylation and specific subsets of CLL patients raises the possibility that altered gene methylation could be used to identify patient populations with differing clinical outcome.In this report we have more closely examined the relationship between methylation of specific candidate marker genes and patient outcome, using a panel of genes we have identified as frequently methylated in CLL. In addition to the previously described methylation of the HOXA4⁸ gene, we have also found frequent methylation, as determined by COBRA analysis, of two further genes (CD38 and BTG4). All three genes independently exhibit a correlation with patient outcome. However, a combined approach using all three markers produced the strongest correlation with patient outcome. This combined marker was independently prognostic in multivariate analysis and identified a subset of patients with IGHV mutated genes (i.e., those regarded as good prognosis) who had a significantly greater likelihood of disease progression.