Impact of TET2 mutations On Responsiveness to Demethylating Agents in MDS.

Abstract Abstract 1606 Poster Board I-632 Aberrant epigenetic silencing of tumor suppressor and differentiation genes constitutes an important mechanism in the pathogenesis of MDS and related myeloid malignancies. Demethylationg agents such as azacitidine and decitabine lead to degradation of DNMT1 and may reverse aberrant methylation. While the drugs demonstrate efficacy in MDS, response rates are variable. Thus, many primarily refractory patients are exposed to these therapies unnecessarily. While search for markers of responsiveness included study of methylation status of potential marker promoters or global methylation patterns, to date predictive tests have not been developed. Similarly, mechanisms of epigenetic instability responsible for wide-spread promoter methylation have not been clarified, preventing development of diagnostic markers. TET2 mutations are frequent events in a variety of MDS subtypes, particular chronic myelomonocytic leukemia (CMML) and other MDS/MPN, as well as AML derived from those conditions. It is likely that TET2 alterations are important in the pathogenesis of myeloid malignancies, but little is known regarding the function of TET2. A recent report indicated that a related family member, TET1, converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (hmC). Hydroxylation of 5mC prevents DNMT1 from homologous methylation of daughter DNA strands during cell division, thus preventing maintenance methylation (Tahiliani et al. Nature 324, 2009). Consequently, closely related TET2 may play a role in epigenetic regulation. As a consequence, TET2 mutations may lead to accumulation of aberrantly methylated CpG islands. Of utmost importance is whether TET2 mutations or resultant epigenetic silencing of specific gene or gene groups affects response to hypomethylating agents. We hypothesized that TET2 mutations play a role in epigenetic instability and may serve as markers of responsiveness/refractoriness to the therapy with demethylating agents. We have determined TET2 mutational status by sequencing all exons in 32 patients with myeloid malignancies (MDS (N=18) and MDS/MPN (N=14)) who underwent therapy with the demethylating agents azacitidine (N=27) or decitabine (N=5). For definition of response we applied International Working Group Criteria in patients who received a sufficient dose and number of cycles to allow assessment of response. Overall response rate (complete+partial responses (CR+PR) + hematologic improvement (HI)) was achieved for 9/32 patients (28%) after 35 cycles, including 4 patients who achieved CR, 2 PR, and 3 CI. In total, 12 TET2 mutations were identified in 9/32 patients (28%), of whom 5 had MDS/MPN (3 with CMML-1/2) and 4 had sAML. Unique compound heterozygosity was found in 3 patients; consequently biallelic inactivation of TET2 was found in 4 patients. For analyses, patients with partial and complete responses were compared with refractory patients and response was correlated with the presence of TET2 mutations. Among TET2-mutated patients, only 1 patient responded to therapy, whereas 8 additional patients, including 3 patients with biallelic inactivation of the TET2 gene, did not show any improvement (11%). In contrast, among patients with WT TET2 gene, responses were seen in 8/23 patients (35%; p=.19). While the cohort of treated patients was small, our preliminary results indicate that the presence of TET2 mutations may represent a negative predictor of response to demethylating agents. Disclosures No relevant conflicts of interest to declare.

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Involvement of TET2 in Regulation of Epigenetic Silencing in MDS and Related Myeloid Malignancies.

Blood ◽

10.1182/blood.v114.22.2908.2908 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2908-2908 ◽

Cited By ~ 2

Author(s):

Hadrian Szpurka ◽

Anna M. Jankowska ◽

Kwok Peng Ng ◽

Joshua R. Clevenger ◽

Yogen Saunthararajah ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Line ◽

Fold Increase ◽

Epigenetic Silencing ◽

Luciferase Reporter ◽

Aberrant Methylation ◽

Global Methylation ◽

Myeloid Malignancies ◽

Cpg Sites ◽

Epigenetic Instability

Abstract Abstract 2908 Poster Board II-884 Genomic (chromosomal gain losses and UPD) and genetic damage (mutations) are hallmarks of MDS and related myeloid malignancies. In addition, aberrant hypermethylation of CpG islands, leading to epigenetic silencing of tumor suppressor and differentiation genes, appears to play an important pathogenic role in evolution to AML. While various mechanisms leading to chromosomal instability have been identified, the pathogenesis of epigenetic instability leading to aberrant methylation remains unexplored. The recent discovery of mutations in TET2 gene in MDS/MPN, sAML and especially CMML provided an intriguing possibility that dysfunction of this bona fide tumor suppressor gene may result in increased promoter methylation. This hypothesis is based on the description of the function of the highly related TET1 gene: the corresponding protein mediates hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which prevents the DNMT1-catalyzed maintenance methylation during cell division. Consequently, TET2 mutations could lead to epigenetic instability and accumulation of aberrantly hypermethylated CpG sites. Both homozygous and heterozygous TET2 mutations were found suggesting that the presence of WT allele or residual activity is not protective. A large proportion of these mutations results in introduction of frame shifts or stop codons resulting in loss of function whilst missense mutations may lead to decreased function. To clarify the function of TET2 and consequences of the mutations we transduced M5 THP-1 AML cell line with lentiviral vectors, containing partial TET2 cDNA (TET2+) and shRNA (TET2-), to achieve overexpression or knockdown of TET2 gene. Following selection, TET2+ cell line showed 3-fold TET2 mRNA overexpression while TET2- cells demonstrated 85% knockdown of TET2 gene as compared to control cells. Based on the putative function of TET2, we performed thin layer chromatography (TLC) comparing 5hmC DNA content in THP1, TET2+, TET2- and appropriate control cells. Parental THP1 cells revealed abundant level of 5hmC. Whilst a 1.5-fold increase of 5hmC content was found in TET2+ cells, it remained unchanged in TET2- cells. However, early results showed a 20% decrease in 5hmC in bone marrow of patients with myeloid malignancies and homozygous TET2 mutations. Illumina 27k methylation arrays were used to assess the effects of altered TET2 expression on methylation patterns. Although global methylation levels, as expressed by averaged β-values, did not significantly differ, the number of hypermethylated CpG sites, defined by individual β-values >0.9, was decreased by 25% (3756 vs. 2839) in TET2+ cells. No difference was seen in TET2- cells vs. controls. However, a number of promoter CpG in TET2- displayed >2-fold change in methylation level compared to TET2+, including MGMT, PCGF2, PTPRO and NOX1. Among differentially silenced promoter CpG we selected CDKN2B (p15) promoter as a marker for methylation activity. When p15 promoter controlling a luciferase reporter was transfected into TET2+ and control cells, a 2.4-fold increase of luciferase activity occurred, consistent with increased demethylating activity in cells expressing higher levels of TET2. Additionally, qPCR showed a 2.5-fold increase in p15 expression in TET2+ cells. Based on this result we also studied methylation status of p15 promoter in patients with WT (N=20) and mutant TET2 (N=27); patients with MDS and MDS/MPN who carried TET2 mutations presented consistent methylation of various promoters including p15 in 2/3 mutant cases vs. 1/3 WT cases. Whereas proliferation kinetics was not significantly altered by changes in TET2 levels, 72h treatment with decitabine (0.5 μM) resulted in a differential effect on TET2+ and TET2- cells. TET2+ cells were not significantly affected however survival of TET2- cells decreased by 30%. In addition, TET2+ cells showed a relative resistance to HDAC inhibitor Entinostat (1μM) with 26% inhibition of proliferation vs. 60% seen in TET2- cells measured after 72h by cell proliferation assay (MTS). In sum, our results suggest that TET2 gene may be involved in epigenetic regulation of promoter CpG that are relevant to proliferation/differentiation of myeloid cells and changes in the expression/activity of this gene may result in altered methylation and thereby epigenetic silencing patterns. Disclosures: No relevant conflicts of interest to declare.

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Hypermethylation-Associated Silencing of miR-125a and miR-125b: A Potential Marker in Colorectal Cancer

Disease Markers ◽

10.1155/2015/345080 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 23

Author(s):

Hui Chen ◽

Zhiying Xu

Keyword(s):

Colorectal Cancer ◽

Cpg Island ◽

Methylation Status ◽

Pcr Analysis ◽

Aberrant Methylation ◽

Dna Hypermethylation ◽

Expression Levels ◽

Qrt Pcr ◽

Potential Marker ◽

Potential Biomarker

Background. MicroRNAs (miRNAs) have been found to be downregulated in human colorectal cancer (CRC), and some of them may function as tumor suppressor genes (TSGs). Aberrant methylation triggers the inactivation of TSGs during tumorigenesis.Patients and Methods. We investigated the methylation status of miR-125 family in CRC tissues and adjacent nontumor tissues by using bisulfite sequencing PCR (BSP). The expression levels of the two miRNAs were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).Results. The methylation frequency of miR-125a and miR-125b was higher in CRC tissues. QRT-PCR analysis showed that miR-125a and miR-125b were significantly downregulated in CRC tissues. Moreover, the expression levels of miR-125a and miR-125b were inversely correlated to CpG island methylation in CRC.Conclusions. Our results suggest that DNA hypermethylation may be involved in the inactivation of miR-125a and miR-125b in CRC, and hypermethylation of miR-125 is a potential biomarker for clinical outcome.

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Methylation Analysis of the Wnt Antagonist in Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.5048.5048 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5048-5048

Author(s):

Hongyan Tong ◽

Chen Mei ◽

Kongfei Li ◽

Jie Jin

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Hematological Malignancies ◽

Conflicts Of Interest ◽

Methylation Status ◽

Wnt Signaling Pathway ◽

Aberrant Methylation ◽

Methylation Frequency ◽

Kaplan Meier ◽

Close Relationship

Abstract Abstract 5048 Background and Objectives: Myelodysplastic syndrome (MDS) is one of the most threatening hematological malignancies. Recently, the epigenetic changes have been recognized in the MDS, and some research found that the aberrant DNA methylation had close relationship with the MDS. Meanwhile, some studies showed that the activation of the Wnt signaling pathway and abnormal methylation of the Wnt antagonists had close relationship with hematological malignancies, such as AML AALL Aand CLL, but less is known in MDS. In our research, we studied the methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in the patients with MDS and evaluated the role of them in the pathogenesis and progression of MDS, with providing a new theory support for clarifying the complicated pathogenesis and progression of MDS. Methods: The methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in pretreatment bone marrow samples from 53 patients with MDS was measured by methylation-specific polymerase chain reaction (MSP). On the other hand, we collected the clinical materials of the patients with MDS and follow up the patients. Then the correlation between methylation and clinical features as well as prognosis of MDS patients was analyzed byχ2 test and Kaplan-Meier method. Results: In 53 bone marrow samples, the methylation frequencies of the Wnt antagonists were as follows: SFRP4 for 62.3 % (33/53), DKK1 for 45.3 % (24/53), HDPR1 for 34.0 % (18/53), SFRP1 for 15.1 % (8/53), DKK3 for 9.4 % (5/53), and WIF-1 for 5.7 % (3/53). After analyzing individual tumor suppressor gene, clinical parameters and prognostic information, it was found that the patients with the percentage of BM blast above 5% had higher methylation frequency of HDPR1 than the patients with the percentage of BM blast bellow 5% (44.4%∼a13.3%, P=0.034); besides, the methylation frequency of HDPR1 exhibited significant differences in the MDS subtypes (P=0.019), and was significantly correlated with the WPSS (P=0.037); In addition, Kaplan-Meier survival curve indicated that the mean overall survival of patients with methylation was significantly shorter than that of patients without HDPR1 methylation (457.3 days vs. 919.6 days, P=0.037) (Fig.1). Conclusion: The methylation of the Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) were observed in patients with MDS and their methylation frequencies were different. The aberrant methylation of HDPR1 may play an important role in the progression and prognosis of MDS. Disclosures: No relevant conflicts of interest to declare.

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Somatic Genetic and Epigenetic Architecture of Myelodysplastic Syndromes Arising from GATA2 Deficiency

Blood ◽

10.1182/blood.v126.23.299.299 ◽

2015 ◽

Vol 126 (23) ◽

pp. 299-299 ◽

Cited By ~ 5

Author(s):

Victor Bengt Pastor Loyola ◽

Shinsuke Hirabayashi ◽

Sandra Pohl ◽

Emilia J Kozyra ◽

Albert Catala ◽

...

Keyword(s):

Somatic Mutations ◽

Conflicts Of Interest ◽

Hair Follicles ◽

Global Methylation ◽

Monosomy 7 ◽

Myeloid Malignancies ◽

Biological Studies ◽

European Working ◽

Targeted Ngs ◽

Gata2 Deficiency

Abstract The emergence of GATA2 deficiency as a germline predisposition to myeloid malignancies raises questions about the nature of acquired secondary genetic and epigenetic events facilitating leukemogenesis. Previously, mutations in ASXL1 were implicated as a possible somatic driver in single cases of GATA2-related MDS. However the landscape of secondary changes had not yet been systematically examined in larger MDS cohorts, and accounting for confounding factors. In this study, we used next-generation genomic platforms to investigate targeted mutational landscape and global epigenetic profiles in patients with GATA2 deficiency. In a large cohort of consecutively diagnosed children with MDS we had initially established that GATA2 deficiency accounts for 7% of primary MDS cases. Exploring the known association between GATA2 mutated (GATA2mut) cases and monosomy 7 (-7), the prevalence of GATA2 deficiency was very high in patients with -7 (37%), reaching its peak in adolescence (>70%). We next tested 60 GATA2-deficient patients with MDS for the presence of secondary mutations using targeted NGS for genes involved in myeloid malignancies. Somatic status was confirmed by matched analysis of fibroblasts, hair follicles or T-cells. Single hematopoietic CFU colonies were sequenced to identify subclonal patterns. For comparison, a GATA2 wildtype (GATA2-WT) cohort of 422 children and adolescents with MDS enrolled in the studies of the European Working Group of Childhood MDS were analyzed by targeted NGS. Somatic mutations were detected in 45% (27/60) of GATA2mut as compared to 19% (82/422) GATA2-WT MDS cases (p<0.0001). Recurrently mutated genes in the GATA2mut group included SETBP1, ASXL1, STAG2, RUNX1, CBL, EZH2, NRAS/KRAS, JAK3, and PTPN11. No mutations were found in TP53, BCOR/BCORL and a number of other oncogenes. Because -7 karyotype was significantly overrepresented in GATA2mut cases with somatic mutations (78%), we next focused on this cytogenetic category. Within the -7 subgroup the rate of somatic mutations was the same in GATA2mut (56%) and GATA2-WT (58%) subgroups. However, hotspot SETBP1 mutations were overrepresented in GATA2-deficient patients with -7 (50%) vs. GATA2-WT MDS cohort (22%, p<0.05). Furthermore, STAG2 mutations were found frequently in the GATA2mut group (10%, 6/60) as opposed to only 0.2% (1/422) of the total GATA2-WT cohort (p<0.0001). Next, we aimed to define the clonal hierarchy of concurrent mutations by longitudinal NGS-analysis during disease course in selected patients. Our results indicate that somatic SETBP1 lesions precede the development of ASXL1 mutations. Remarkably, this model of clonal evolution does not depend on preexisting germline GATA2 lesion, as confirmed by sequencing of single CFU colonies cultivated from the bone marrow of 3 GATA2mut and 3 GATA2-WT MDS patients. Finally, to elucidate the epigenetic effects, we compared methylation patterns using methyl-CpG-immunoprecipitation and Illumina-NGS in 25 GATA2mut to 17 GATA2-WT patients and 10 healthy controls. Based on the degree of global methylation, there were no significant alterations allowing for the discrimination of GATA2-deficient patients from the total MDS cohort, when accounted for bias arising from cytogenetic and morphologic subgroups. In summary, somatic SETBP1 and STAG2 mutations are associated with MDS arising from GATA2 deficiency. The remaining targeted clonal landscape is essentially determined by the presence of monosomy 7. Similarly, the global epigenetic changes correlate with morphological and cytogenetic subgroups, rather than with germline GATA2 status. The prospect of potential drug targetability of mutations frequently found in children, particularly in the SETBP1 oncogene, and in histone modifiers ASXL1 and EZH2, warrants further biological studies. Disclosures No relevant conflicts of interest to declare.

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Global Methylation Analysis of Cyclin D1vs D2 Dysregulated Primary Myeloma Samples

Blood ◽

10.1182/blood.v124.21.3389.3389 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3389-3389

Author(s):

Jonathan I Sive ◽

Andrew Feber ◽

Dean Smith ◽

John Quinn ◽

Stephan Beck ◽

...

Keyword(s):

Cyclin D1 ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cohort Analysis ◽

Methylation Status ◽

Progression Free Survival ◽

Methylation Analysis ◽

Cyclin D2 ◽

Global Methylation ◽

Ccnd1 Gene

Abstract Multiple myeloma (MM) may be classified according to D-type cyclin dysregulation. Amongst nonhyperdiploid cases, those with IgH translocations t(4;14) or t(14;16) express high levels of cyclin D2, and those with t(11;14) express elevated cyclin D1. Although translocation-based subgroups may behave differently, cyclin D2 dysregulated disease tends to progress with more proliferative disease and poorer outcomes compared to cyclin D1. The importance of methylation status has been described in MM, with the transition from MGUS to symptomatic MM characterised by global hypomethylation, and gene-specific hypermethylation differences between cytogenetic subtypes. To investigate the utility of methylation profiling between D1 and D2-dysregulated MM, we carried out a pilot study of global methylation changes between these groups. Primary bone marrow samples were collected from eight cyclin D1 patients with t(11;14), and eight D2 patients (four t(4;14) and four t(14;16)), and CD138+ cells purified by magnet-assisted selection. Following bisulfite conversion, samples were processed on llumina Infinium human methylation27 arrays. Methylation was classified with a beta score between 0 (unmethylated) and 1 (methylated). Initial analysis was performed using Illumina GenomeStudio, and subsequently with the Limma package in R. Differentially methylated probes were corrected for false discovery rate (FDR), with a threshold of 0.01 considered significant. Survival analyses were calculated from the date of sampling. Unsupervised clustering split the samples into two groups (group 1 and group2), which did not match with D-cyclin status, but did show clear differences in clinical course. Survival analysis between groups 1 and 2 showed trends toward differences in median overall survival (61.7 vs 11.9 months, p=0.06) and progression free survival (24.4 vs 7.2 months, p=0.34). Although not significant at the p<0.05 threshold, the survival curves suggest a difference that may become clearer in a larger cohort. Analysis of the probes between these groups revealed 1379 methylation variable positions (MVPs) which were significantly different. Interestingly, almost all of these (1376/1379) were hypermethylated in the poorer outcome Group 2. This suggests a difference in methylation status between two prognostic groups which warrants further investigation. We then went on to perform supervised clustering between the samples, splitting them into two groups (D1 and D2) based on their cyclin D status. This analysis did not reveal any MVPs even at a less stringent FDR-adjusted threshold of 0.05. However, we did observe that of the top 20 differentially methylated probes three were for the CCND1 gene, which in all cases showed relative hypomethylation in the cyclin D1 dysregulated samples. Other genes of potential interest with relative hypomethylation in the cyclin D1 group were DAB2 which has previously been reported to be hypermethylated in the t(4;14) OPM2 cell line, and AK3L1 – a kinase which interestingly, has been reported as showing vulnerability to targeted RNAi inhibition in two cyclin D2 dysregulated cell lines (KMS11 and JJN3). In this small cohort, although cyclin D1 vs cyclin D2 classification does not appear to be sufficient to define distinct methylation profiles, a group of genes with hypermethylation appears to be associated with poorer prognosis. The hypomethylation of CCND1 in cyclin D1 dyregulated samples, although below the significance threshold in this dataset, is consistent with previously described findings of CCND1 hypomethylation in t(11;14) cell lines, but our results in the D2 MM samples differ from previous reports of hypomethylation in all nonhyperdiploid primary samples. We intend to investigate this further and extend this analysis by prospective sampling and methylation analysis on a larger cohort of patients treated on a standard protocol. Disclosures No relevant conflicts of interest to declare.

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UTX Mutations and Epigenetic Changes In MDS/MPN and Related Myeloid Malignancies

Blood ◽

10.1182/blood.v116.21.121.121 ◽

2010 ◽

Vol 116 (21) ◽

pp. 121-121 ◽

Cited By ~ 3

Author(s):

Hadrian Szpurka ◽

Anna M Jankowska ◽

Bartlomiej Przychodzen ◽

Zhenbo Hu ◽

Yogen Saunthararajah ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

Epigenetic Regulation ◽

Aberrant Methylation ◽

Mrna Levels ◽

Epigenetic Changes ◽

Myeloid Malignancies ◽

Cpg Sites ◽

Epigenetic Instability ◽

Tumor Suppressor Gene Inactivation

Abstract Abstract 121 Balanced and unbalanced chromosomal lesions and genetic mutations are hallmarks of myeloid malignancies. In addition, aberrant methylation of CpG islands, leading to epigenetic silencing appears to play a significant role in tumor suppressor gene inactivation and malignant progression. While various mechanisms of chromosomal instability have been identified, the pathogenesis of epigenetic instability remains unexplored. ASXL1, EZH2 and TET2 mutations, found in myeloid disorders provide a potential link between genetic and epigenetic events. UTX is a histone H3K27 demethylase belonging to the polycomb group of proteins. Methylation at lysine 27 correlates with gene silencing and repression. A recent mutational screen of cancer cell lines has identified mutations in UTX in AML THP-1 cells. We screened a series of myeloid disorders for UTX gene mutations and identified an index case in a CMML patient with a somatic UTX missense mutation. This sample demonstrated × chromosome copy neutral loss of heterozygosity (CN-LOH) that included UTX on a whole genome SNP-A array. UTX was then sequenced in 49 additional patients with MDS/MPN and 24 with secondary AML. Two of the MDS/MPN (2/49, 4%) and 3 of the sAML patients (3/24; 12%) showed UTX mutation. Three previously unreported missense polymorphisms were noted (present in paired CD3+ cells and detected with a frequency of <1/400 of controls). No mutations were found in MDS (N=15). The identified lesions were not associated with loss of sex chromosomes. Both homozygous (UPD) and heterozygous UTX mutations were identified and included frame shift or stop codons, as well as missense mutations. Inactivating or hypomorphic mutations may result in similar functional consequences with decreased expression of UTX. We quantitated UTX mRNA abundance in 15 hematopoietic cell lines using TaqMan PCR, and THP1 cells (UTX null) showed the lowest UTX mRNA expression. In primary patient samples, decreased UTX mRNA levels were found in the bone marrow of MDS/MPN patients (N=21) as compared to controls. To examine UTX expression in normal hematopoietic differentiation, CD34+ cells, along with myeloid CD33+ or monocytic CD14+ cell fractions (N=5) were tested and expressed easily quantifiable UTX transcripts, while the highest UTX expression was found in Gly-A+ erythroid precursors. UTX mutation and dysfunction might be predicted to lead to altered histone repression marks and alterations in epigenetic regulation. Consistent with this hypothesis, we tested for H3K27-Me3 methylation by ELISA and observed a 45% methylation increase in a UTX mutant patient sample. To further investigate the function of UTX, consequences and treatment implications of the corresponding mutations we transduced THP-1 cells with a lentiviral vector containing the UTX cDNA (UTX+) or an empty vector. Following clone selection, the resultant UTX+ cell line showed 1000-fold UTX mRNA increase and distinct protein overexpression by western blot (WB), as compared to the baseline UTX null cells. Proliferation kinetics up to 120h indicated that overexpression of UTX results in increased cell proliferation by 20% compared to controls. Treatment with decitabine (120h; 0.5μ M) resulted in a differential effect on UTX+ cells: proliferation increased 4.6-fold vs. 1.3-fold in UTX null cells. Based on the hypothesis that mutation in UTX may lead to epigenetic instability and accumulation of aberrant methylated CpG sites, we further investigated epigenetic changes in UTX+ and UTX null cells. WB showed comparable levels of global H3K27-Me3 and H3 acetylation levels in UTX+ and UTX null cells, and only modest decreases (9%) of trimethylation in a more precise ELISA assay. Similarly, when methylation array (14K genes; 27K CpG sites) was used to assess the effects of UTX overexpression, global methylation levels expressed by averaged β-values did not significantly differ. However, 153 specific CpG sites were found to be differentially methylated on the array: with 68 genes hypo- and 85 hypermethylated. In particular SSRP1, NCOR2 and DIRAS3 genes showed hypomethylated promoters in the UTX+ cells, suggesting that UTX is involved in gene specific demethylation. In sum, our results suggest that UTX gene may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutation may compromise this function, thereby promoting repression of tumor suppressor genes. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Profiling of Primary CLL Reveals Novel DNA Methylation-Based Clusters and Novel Mechanisms of Leukemogenesis

Blood ◽

10.1182/blood.v120.21.3877.3877 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3877-3877

Author(s):

Fong Fong ◽

Haim Y Bar ◽

Kerby Shedden ◽

Kamlai saiya-Cork ◽

Peter Ouillette ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

B Cells ◽

Conflicts Of Interest ◽

Critical Role ◽

Methylation Status ◽

Snp Array ◽

Aberrant Methylation ◽

Unequal Variance ◽

Genomic Deletions

Abstract Abstract 3877 Introduction: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world with nearly 15,000 new cases diagnosed every year in the USA. The characterization of CLL has resulted in the identification of important disease biomarkers: these include the recurrent genomic deletions del17p and del11q, genomic complexity, TP53 mutations, the expression level of ZAP70 and the mutational status of IgVH. While genomic and transcriptional profiling of CLL identified clinically and biologically relevant markers, there is still significant uncertainty about the pathobiology and the origin of CLL. It is increasingly clear that epigenetic deregulation plays an important role in the biology of all lymphomas/leukemias including CLL. Methods: We hypothesized that DNA methylation profiling would allow us to identify new, biologically significant CLL subtypes and yield greater insight into the biology of this disease. We therefore examined the DNA methylation of over 240 patients with CLL using the HELP assay and hybridization to high density custom microarray that reports on the methylation status of more than 250,000 CpGs corresponding to 20,401 genes. Gene expression profiling and SNP array-based copy number assessments and targeted gene resequencing were available on most of these cases. We performed unsupervised analysis on the most variable probesets (standard deviation > 1.3) using K-means consensus clustering. Results: The experimental approach reproducibly identified three robust CLL subtypes based on epigenetic profiles. To identify the genes that define these three subtypes we next performed unequal variance t-test of the CLL subtypes comparing them to Peripheral Blood CD19+ B cells as a normal control, and identified that clusters are defined by differential methylation of 3719, 6145 and 3349 genes (selected probes displayed changes in methylation of at least 30% and FDR corrected p-value < 0.05), The three clusters featured respectively i) aberrant methylation of MYC and WNT target genes, ii) aberrant methylation of NOTCH1 targets and iii) aberrant methylation of bcl6 and inflammatory cytokines. There was inverse correlation between gene expression and cytosine methylation, suggesting that DNA methylation had an impact on the transcriptional programming of these CLL cases. Strikingly the CLL MYC/WNT cluster displayed poorer prognosis as opposed to the CLL BCL6 cluster (HR=0.14 95% CI: 0.07–0.30). The CLL NOTCH1 cluster had an intermediate prognosis. It was also notable that all CLL patients exhibited deregulation of the B-cell receptor pathway as compared to normal CD19+ B-cells, consistent with the notion that this pathway plays a critical role in CLL pathogenesis. Finally, we divided the cohort into training and testing cohorts and used a machine learning BDVAL algorithm to identify DNA methylation outcome classifiers. This procedure identified a 40-probeset classifier that accurately predicted outcome (Area Under the ROC Curve of 0.77; performance was assessed with 10 fold cross-validation in a training set with 76 patients; validation on an independent set of 105 samples). Conclusion: This large epigenetic profiling study in CLL identifies aberrant epigenetic regulation as a core part of the pathobiology of CLL and identifies novel CLL clusters with distinct effects on survival. MYC-WNT pathway inhibitors are warranted for use in clinical trials for patients belonging to this aggressive epigenetically defined subtype. Disclosures: No relevant conflicts of interest to declare.

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Spectrum of Gene Mutations and Clinical Significance in Myeloid Malignancies Patients with ASXL1 Mutations

Blood ◽

10.1182/blood-2018-99-114471 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3906-3906

Author(s):

Jie Bai ◽

Guangshuai Teng ◽

Yingshao Wang ◽

Jing Xu ◽

Chenxiao Du ◽

...

Keyword(s):

Overall Survival ◽

Disease Progression ◽

Clinical Significance ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Specific Gene ◽

Ras Pathway ◽

Myeloid Malignancies ◽

The Impact

Abstract Abstract Introduction: With the development of next generation sequencing (NGS), the interrelation between genetic and epigenetic abnormality in myeloid malignancies has attracted significant attention. Clinical reports provide strong evidence that while the specific gene mutations are the initial event for the myeloid malignancies, the concomitant gene mutations contribute to the disease progression. Although ASXL1 mutations have been found in myeloid malignancies, the impact of co-mutation with ASXL1 on the disease progression remains largely unknown. In the current study, we aim to investigate the clinical significance of the association between ASXL1 mutations and a spectrum of gene mutations in a large cohort of patients with myeloid malignancies. Methods: Targeted sequencing including 112 hematopoietic malignancy-related genes was used to analyze the gene mutations in patients with ASXL1 mutations. The impact of gene mutations on clinical characteristics and prognosis was further analyzed. The correlation between clinical/laboratory features and the gene mutations was performed by the χ2 test, and differences in values and in ranks were assessed by Student t-tests. Overall survival rate was assessed by the Kaplan-Meier method and calculated by the Log-rank test. Results: A cohort of 138 myeloid malignant patients harboring ASXL1 mutations was recruited to the current study, including patients with myelodysplastic syndromes (MDS) (37.68%, n = 52), myeloproliferative neoplasms (MPN) (21.01%, n = 29), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (7.25%, n = 10), and acute myeloid leukemia (AML) (34.06%, n = 47). In addition, to ASXL1 mutations, 89 genes were mutated in these patients, and 96.4% (133) of the patients were accompanied by at least one gene mutation. Among those mutated genes, 55.8% (77/138) was epigenetic genes, 65.9% (91/138) was signal transduction pathway genes, 28.2% (39/138) was spliceosome related genes, 36.9% (51/138) was transcription factor genes, and 18.8% (26/138) was cell cycle and apoptosis related genes. The most common co-mutated genes were RAS pathway related genes (25.4%, 35/138) and SETBP1 (21.7%, 30/138). Patients with ASXL1 and RAS pathway co-mutations (ASXL1mutRASmut) had significantly lower levels of hemoglobin and platelets compared to ASXL1 mutated patients without RAS pathway mutation (ASXL1mutRASwt) (hemoglobin 81 (33-152) g/L vs. 96 (18-195) g/L, P=0.012; and platelets (51 (8-695)×109/L vs. 75 (3-3149) × 109/L, P=0.032, respectively). Importantly, MDS patients with ASXL1mutRASmut were more likely to be associated with high International Prognostic Scoring System (IPSS) scores (P=0.016). Moreover, the median survival time of these ASXL1mutRASmut patients (mean = 17 months, 1-35 months) was significantly shorter than that of ASXL1mutRASwtpatients (mean = 21 months, 2-75 months) (P=0.031). Conclusions: Our study provides a comprehensive overview of the association between the clinical features and prognosis with genes co-mutated with ASXL1 in patients with myeloid malignancies. We conclude that concomitant mutations of ASXL1 with RAS pathway genes associate with high risk of myeloid transformation and lower overall survival rates. Disclosures No relevant conflicts of interest to declare.

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Global Methylation Array Analysis of Multiple Myeloma Samples Indicate An Alteration of Epigenetics During the Transition From MGUS to Myeloma and An Increased Frequency of Gene Methylation in t(4;14) Myeloma.

Blood ◽

10.1182/blood.v114.22.121.121 ◽

2009 ◽

Vol 114 (22) ◽

pp. 121-121

Author(s):

Brian A Walker ◽

Emma M Smith ◽

Nicholas J Dickens ◽

Fiona M Ross ◽

Faith E Davies ◽

...

Keyword(s):

Dna Methylation ◽

Conflicts Of Interest ◽

Clinical Stage ◽

Methylation Status ◽

Differential Methylation ◽

Fold Change ◽

Global Methylation ◽

Genome Methylation ◽

Relapsed Myeloma ◽

Average Beta

Abstract Abstract 121 The methylation status of genes in myeloma can change as the disease progresses and as such identifying genes deregulated by methylation that mediate disease aetiology and progression may offer epigenetically relevant therapeutic targets. We have analyzed 153 presenting myeloma samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. Initially, we compared global methylation of genes between MGUS, myeloma (n=153) and relapsed myeloma (n=18) in order to determine the effect of clinical stage on the general methylation state of the genome. There were 267 probesets showing an increase in methylation between presenting and relapsed myeloma. However, the largest changes in DNA methylation were between MGUS and myeloma with 4209 probesets showing a decrease in methylation and 879 probesets showing an increase in methylation as the pre-malignant stage progresses to myeloma. In order to address the potential for differential methylation between cytogenetic subtypes of myeloma we compared the translocation groups (t(4;14) n=14; t(11;14) n=32; t(14;16) n=7; t(14;20) n=3) and samples with no split IgH locus (n=66). When average beta-scores for each translocation are compared using a 1.5 fold-change cut-off we identified 8.7% of probes differentially methylated in t(4;14), 5.1% in t(14;20), 3.3% in t(14;16), and 2% in t(11;14), indicating that the t(4;14) translocation has the largest effect on genome methylation, and in addition there are significant methylation effects associated with deregulation of the MAF transcription factors. The t(4;14) translocation in myeloma results in the over-expression of two genes, MMSET and FGFR3, of which MMSET has histone methyltransferase properties and it has been shown that methylation of chromatin is associated with DNA methylation at CpG islands resulting in transcriptional repression. In this analysis the t(4;14) samples had a greater than 1.5-fold increase in methylation in 2410 probesets, corresponding to 1685 unique genes, when compared with non-translocation samples. On average the remaining translocation groups had only 746 probes with differential methylation, and with the exception of the t(14;20) group most were hypomethylated. Identifying the genes affected by these methylation changes is important. The gene with the largest fold-change in methylation in t(4;14) samples was APC. Clinically relevant changes in methylation may be characterised by associated changes in gene expression and when methylation and expression array data from the same samples are compared there are 23 genes with decreased expression and increased methylation in t(4;14) samples compared with non-translocation samples. These include potential tumor suppressor genes GLTSCR2 and NME4, as well as SEPTIN9. We also looked for differential methylation between common cytogenetic subgroups including hyperdiploidy (HRD n=64 vs. normal n=67), 1q+ (n=44 vs. n=83), del(1p32.3) (n=20 vs. n=104), del(13q) (n=66 vs. n=68), del(16q) (n=36 vs. n=96), and del(17p) (n=8 vs. n=126) but were unable to show that any gross differences in global methylation between samples with and without the abnormality. However, there were a limited number of genes that had a greater than 1.5-fold change in methylation between the analysis groups, indicating that there are genes of potential interest. We are also mapping the methylation of genes within these regions of copy number change. In summary, we have identified that the major influences on epigenetics occur at the transition between MGUS and myeloma. t(4;14) myeloma, characterised by deregulation of MMSET, along with the translocations that deregulate the transcription factor MAF have a higher frequency of genome methylation than the cases lacking these events. These analyses enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. Disclosures: No relevant conflicts of interest to declare.

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Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2868 ◽

2007 ◽

Vol 30 (4) ◽

pp. 90

Author(s):

Kirsten Niles ◽

Sophie La Salle ◽

Christopher Oakes ◽

Jacquetta Trasler

Keyword(s):

Dna Methylation ◽

Germ Cell ◽

Germ Cells ◽

Epigenetic Modification ◽

Methylation Pattern ◽

Chromosome 9 ◽

Specific Gene ◽

Global Methylation ◽

Dna Methylation Pattern ◽

Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

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