Jean-Pierre Issa

Decitabine improves patient outcomes in myelodysplastic syndromes: Results of a phase III randomized study

Aberrant DNA methylation, which results in leukemogenesis, is frequent in patients with myelodysplastic syndromes (MDS) and is a potential target for pharmacologic therapy. Decitabine indirectly depletes methylcytosine and causes hypomethylation of target gene promoters.

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.

Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer

Colon cancer has been viewed as the result of progressive accumulation of genetic and epigenetic abnormalities. However, this view does not fully reflect the molecular heterogeneity of the disease. We have analyzed both genetic (mutations of BRAF, KRAS, and p53 and microsatellite instability) and epigenetic alterations (DNA methylation of 27 CpG island promoter regions) in 97 primary colorectal cancer patients. Two clustering analyses on the basis of either epigenetic profiling or a combination of genetic and epigenetic profiling were performed to identify subclasses with distinct molecular signatures. Unsupervised hierarchical clustering of the DNA methylation data identified three distinct groups of colon cancers named CpG island methylator phenotype (CIMP) 1, CIMP2, and CIMP negative. Genetically, these three groups correspond to very distinct profiles. CIMP1 are characterized by MSI (80%) and BRAF mutations (53%) and rare KRAS and p53 mutations (16% and 11%, respectively). CIMP2 is associated with 92% KRAS mutations and rare MSI, BRAF, or p53 mutations (0, 4, and 31% respectively). CIMP-negative cases have a high rate of p53 mutations (71%) and lower rates of MSI (12%) or mutations of BRAF (2%) or KRAS (33%). Clustering based on both genetic and epigenetic parameters also identifies three distinct (and homogeneous) groups that largely overlap with the previous classification. The three groups are independent of age, gender, or stage, but CIMP1 and 2 are more common in proximal tumors. Together, our integrated genetic and epigenetic analysis reveals that colon cancers correspond to three molecularly distinct subclasses of disease.

Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system

OBJECTIVE Methylation of the promoter region of the estrogen receptor gene alpha (ER alpha) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. METHODS We used Southern blot analysis to determine the methylation state of the ER alpha gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. RESULTS An age related increase in ER alpha gene methylation occurs in the right atrium (range 6 to 19%, R = 0.36, P < 0.05). Significant levels of ER alpha methylation were detected in both veins and arteries. In addition, ER alpha gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10 +/- 2% versus 4 +/- 1%, P < 0.01). In endothelial cells explanted from human aorta and grown in vitro, ER alpha gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER alpha gene methylation (19-99%). CONCLUSIONS Methylation associated inactivation of the ER alpha gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.

p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3

For several human tumour types, allelic toss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5′ flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of C418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Changes in DNA methylation in neoplasia : Pathophysiology and therapeutic implications

Our increasing knowledge of the molecular pathophysiology of cancer is beginning to find applications in the diagnosis and treatment of various neoplastic diseases. In particular, new therapeutic approaches such as targeted agents, differentiation therapy, and immunotherapy promise to yield substantial clinical benefits with relatively few side effects. Recently, aberrant methylation of the cytosine base within the regulatory area of selected genes was shown to be a very common event in neoplasia; it is thought to contribute to the molecular pathogenesis of the disease through inactivation of tumor suppressor genes (1, 2). This finding has increased interest in use of drugs that can inhibit the process of DNA methylation and restore tumor suppressor gene function as a potential strategy to treat various malignant diseases. Hematopoietic neoplasms in particular have a high degree of aberrant methylation (3), and clinical trials have demonstrated significant activity for hypomethylating drugs in this setting. We discuss the importance and prevalence of DNA hypermethylation in cancer and review the potential value of hypomethylating agents in the treatment of human neoplasms. DNA Methylation The presence of 5-methylcytosine in human DNA (4) has genetic and epigenetic effects on cellular development, differentiation, and neoplastic transformation. 5-Methylcytosine differs from cytosine by the presence of a methyl group at the 5 position of the pyrimidine ring (Figure 1). Methylcytosine is formed after replication by addition of a methyl group to a cytosine already present in the DNA strand. Dramatic changes in overall methylation of DNA occur at different periods of embryogenesis, development, and differentiation to adult cells (5). A wave of demethylation initially erases preset methylation patterns in the first days of embryogenesis. This is followed by several waves of de novo methylation that eventually establish adult patterns of gene methylation. In differentiated cells, methylation patterns change relatively little and are perpetuated after DNA replication through the high affinity of DNA methyltransferase for hemimethylated DNA (6) (Figure 2). Unlike cytosine, 5-methylcytosine is a relatively unstable base because its spontaneous deamination leads to uracil. Through evolution, such mutations have resulted in a relative depletion of 5-methylcytosine in human DNA, and they are a major cause of germ-line mutations in inherited disease and of somatic mutations in neoplasia (7). Figure 1. Structure of cytosine, 5-methylcytosine, and hypomethylating 5-methylcytidine analogues. Figure 2. The maintenance methylation process. Top. Middle. Mtase Bottom. left 5-Aza right The functions of DNA methylation in mammalian cells remain poorly defined. Early speculation that attributed a global transcriptional regulation role to cytosine methylation (8) has not yet been confirmed experimentally. In bacteria, methylation plays a role in defense against genomic invasion by foreign DNA sequences (9). In mammalian cells, most normal methylation takes place within highly repeated transposable elements, and it has been proposed that such methylation also plays a role in genome defense by suppressing the potentially harmful effects of expression at these sites (10). This hypothesis was questioned recently (11). Regardless of its global functions, one unequivocal role for DNA methylation is in irreversible gene inactivation in selected cases, such as imprinted genes (12) and genes on the inactivated X chromosome (13). CpG Island Methylation and Gene Silencing In mammalian DNA, normal methylation is restricted to cytosine followed by guanosine (the CpG dinucleotide). These CpG sites are rarer in the human genome than their predicted frequency, presumably because they are eliminated during evolution through C to T mutations of methylcytosine (14). The human genome, however, also contains small regions of DNA called CpG islands, in which the frequency of CpG is normal or higher than expected (14). About half of all human genes (including most housekeeping genes) have CpG islands in their 5-promoter regions. Of note, the promoter regions containing CpG islands are in fact usually unmethylated in normal tissues, regardless of the transcriptional status of the gene. CpG island methylation is associated with changes in chromatin organization and consequent repression of gene transcription (1). In normal tissues, CpG island methylation is limited to exceptional situations, such as imprinted alleles (12) and genes on the inactive X chromosome [13]. These well-studied exceptions to the rule of absent methylation at CpG islands suggest that, once established, gene silencing by CpG island methylation is physiologically irreversible during the lifetime of affected cells. A direct correlation between CpG island methylation and inhibited gene transcription is supported by the facts that 1) cells in which silencing occurs are usually transcriptionally competent for the affected genes [as demonstrated by normal expression of the unmethylated alleles and exogenously inserted unmethylated promoters], 2) demethylation by pharmacologic (15) or genetic [16] manipulation results in reactivation of gene expression, and 3) in vitro methylation substantially reduces gene expression in reporter experiments (1). The mechanism of CpG islandassociated gene silencing appears to involve binding of specific methylated DNA binding proteins, followed by recruitment of a silencing complex that includes histone deacetylases (Figure 3) (17, 18). Figure 3. Effects of methylation and histone deacetylation on gene expression and silencing. top black boxes ovals arrows (left m MBP bottom HDAC right top Aberrant CpG Island Methylation in Cancer Neoplastic cells often have simultaneous global DNA hypomethylation, localized hypermethylation that involves CpG islands, and increased levels of DNA methyltransferase activity (1). Hypomethylation was initially postulated to play a role in carcinogenesis through activation of oncogenes (19), but this hypothesis has not been experimentally confirmed. Hypomethylation has been linked to chromosomal instability in vitro (20), and it may play such a role in neoplasia. Aberrant CpG island hypermethylation in cancer, in contrast, is clearly associated with transcriptional silencing of gene expression, and increasing experimental data suggest that it plays an important role as an alternate mechanism by which tumor suppressor genes are inactivated in cancer (1, 2). Aberrant CpG island methylation in cancer was initially described for the calcitonin (21) and MyoD (22) genes. These two genes are not thought to play a tumor suppressive role in cancer, but these findings prompted additional investigations into the process. The first tumor suppressor gene shown to be inactivated by hypermethylation was the RB1 gene, in which methylation appeared to be a clear alternate to mutations and deletions for eliminating expression of functional protein (23). Several additional tumor suppressor genes have since been shown to be similarly inactivated in some cancers, including VHL (24), P16 (25), E-cadherin (26), and hMLH1 (27). For most of these genes, hypermethylation appears to provide a similar selective advantage as genetic inactivation and is usually associated with absence of coding region or promoter mutations of involved alleles. The list of genes that display hypermethylation-associated inactivation in some sporadic cancers has grown long (Table 1). Multiple cellular systems can be affected by this process, including cell growth and differentiation, cell cycle control, and DNA repair, as well as angiogenesis and invasion. However, hypermethylation in cancer is not invariably associated with repressed transcription. In some cases, the involved CpG island is not in the promoter of the genes (28). In other cases, methylation involves genes that are not normally expressed in the diseased tissues (29). In still others, methylation is relatively sparse, and although it can easily be detected experimentally, it does not lead to substantial decreases in gene expression. Aberrant methylation in cancer therefore functions as a mechanism of generating molecular diversity in neoplasia. In a manner analogous to mismatch repair defects in cancer, methylation defects affect many different loci, only some of which are pathophysiologically relevant to the neoplastic process (30). Table 1. Genes That Are Hypermethylated in Sporadic Cancers The causes of aberrant methylation in cancer remain poorly defined. Both hypomethylation and methyltransferase activation can occur in cells that are induced to proliferate (31), and it is not clear whether the observed changes in malignant cells simply reflect cell cycle deregulation. De novo CpG island methylation, however, is not a feature of proliferating cells, and it appears to represent a true pathologic event in neoplasia. For many genes, hypermethylation begins in normal tissues during the process of aging (32), which may partially explain the dramatic increase in cancer incidence associated with aging. Other genes are methylated exclusively in malignant cells and are presumed to arise from rare chance events that lead to gene inactivation and a selective advantage for affected cells (1, 2). Recent data in multiple neoplasms suggest that some cancers have a high degree of de novo methylation compared with others (33), and specific genetic defects or exposure events may explain these differences. In particular, acute and chronic leukemias have a high degree of aberrant CpG island methylation; genes involved include the cell-cycle regulator p15 (34), the p53 homologue p73 (35), the drug-resistance gene MDR1 (36), ER (37), and HIC1 (38). Therefore, hematologic malignant conditions present unique opportunities for studying the clinical implications of aberrant methylation. Rationale for Use of Methylation Inhibitors in Neop

Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System.

Recent studies have highlighted issues with the International Prognostic Scoring System (IPSS) model in relation to the exclusion of many subgroups that now represent a large proportion of patients with myelodysplastic syndrome (MDS) (eg, secondary MDS, chronic myelomonocytic leukemia [CMML] with leukocytosis, prior therapy) and its lack of applicability to most patients on investigational programs, because many would have received prior therapies and would have had MDS for a significant length of time.

Widespread and tissue specific age-related DNA methylation changes in mice

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer.

ABSTRACT The mechanism of DNA hypermethylation-associated tumor suppressor gene silencing in cancer remains incompletely understood. Here, we show by chromatin immunoprecipitation that for three genes (P16, MLH1, and the O6-methylguanine-DNA methyltransferase gene, MGMT), histone H3 Lys-9 methylation directly correlates and histone H3 Lys-9 acetylation inversely correlates with DNA methylation in three neoplastic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in moderately increased Lys-9 acetylation at silenced loci with no effect on Lys-9 methylation and minimal effects on gene expression. By contrast, treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5Aza-dC) rapidly reduced Lys-9 methylation at silenced loci and resulted in reactivation for all three genes. Combined treatment with 5Aza-dC and TSA was synergistic in reactivating gene expression through simultaneous effects on Lys-9 methylation and acetylation, which resulted in a robust increase in the ratio of Lys-9 acetylated and methylated histones at loci showing dense DNA methylation. By contrast to Lys-9, histone H3 Lys-4 methylation inversely correlated with promoter DNA methylation, was not affected by TSA, and was increased moderately at silenced loci by 5Aza-dC. Our results suggest that reduced H3 Lys-4 methylation and increased H3 Lys-9 methylation play a critical role in the maintenance of promoter DNA methylation-associated gene silencing in colorectal cancer.