Rodolphe Taby

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.

TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites.

A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome

Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+=not reached, CIMP-=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+=2.34, A-CIMP-=1.00; P=0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP+ was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.

Hypomethylation of TET2 Target Genes Identifies a Curable Subset of Acute Myeloid Leukemia

BACKGROUND Acute myeloid leukemia (AML) is curable in a subset of cases. The DNA methylation regulator TET2 is frequently mutated in AML, and we hypothesized that studying TET2-specific differentially methylated CpGs (tet2-DMCs) improves AML classification. METHODS We used bisulfite pyrosequencing to analyze the methylation status of four tet2-DMCs (SP140, MCCC1, EHMT1, and MTSS1) in a test group of 94 consecutive patients and a validation group of 92 consecutive patients treated with cytarabine-based chemotherapy. Data were analyzed with hierarchical clustering, Cox proportional hazards regression, and Kaplan-Meier analyses. All statistical tests were two-sided. RESULTS In the test cohort, hierarchical clustering analysis identified low levels of tet2-DMC methylation in 31 of 94 (33%) cases, and these had markedly longer overall survival (median survival 72+ vs 14 months, P = .002). Similar results were seen in the validation cohort. tet2-DMC-low status was shown to be an independent predictor of overall survival (hazard ratio = 0.29, P = .0002). In The Cancer Genome Atlas (TCGA) dataset where DNA methylation was analyzed by a different platform, tet2-DMC-low methylation was also associated with improved outcome (median survival = 55 vs 15 months, P = .0003) and was a better predictor of survival than mutations in TET2, IDH1, or IDH2, individually or combined. CONCLUSIONS Low levels of tet2-DMC methylation define a subgroup of AML that is highly curable and cannot be identified solely by genetic and cytogenetic analyses.

Abstract B22: Genome-wide methylation analysis reveals an independently validated CpG island methylator phenotype associated with favorable prognosis in acute myeloid leukemia.

Background: Acute myeloid leukemia (AML) accounts for the most leukemia-related deaths in the United States and its incidence has been rising as the population ages. Although certain molecular aberrations are prognostic and have come into mainstream clinical practice, the genetic and epigenetic determinants of curability in AML remain incompletely understood. Our study examines the role of DNA methylation patterns in AML prognosis and expands on our preliminary work showing DNA hypermethylation may associate with improved overall survival. Methods: To quantitatively interrogate genome-wide CpG methylation we used Digital Restriction Enzyme Analysis of Methylation (DREAM) on a cohort of 102 AML patient samples and 25 normal control samples. We validated our findings using DNA methylation data from 194 patient samples from The Cancer Genome Atlas (TCGA) on the Illumina Infinium HumanMethylation450 platform. Statistical analysis was done using R. Results: Preliminary analysis by our group of DNA methylation levels at promoter CpG islands (CGI) of OSCP1, NPM2, OLIG2, SCGB3A1, and SLC26A4 showed significant hypermethylation in a small group of long-surviving AML patients compared to a short-surviving cohort (median OS = 2,694 days vs. 207 days). We expanded on this observation using DREAM to measure genome-wide DNA methylation in clinical AML samples and found that hierarchical clustering based on 2,537 CpG sites with a standard deviation above 20% stratified patients into three groups with significant differences in overall survival. The hypermethylated cluster had the best prognosis and seemed to be defined by hypermethylation at promoter CGIs, suggesting that a CGI methylator phenotype (CIMP) in AML may be a favorable prognostic factor (median OS: CIMP = 5,110 days, Cluster 2 = 380 days, Cluster 3 = 555 days; log-rank p=0.0162). We then validated these findings using TCGA data from AML patient samples. Hierarchical clustering on the basis of CGI promoter sites revealed three distinct groups with the CIMP cluster having significantly improved overall survival compared to the other clusters (median OS: CIMP = 761 days, Cluster 2 = 306 days, Cluster 3 = 365 days; log-rank p=0.0013). There was also a trend in overall survival when non-CGI non-promoter sites were used to cluster samples (median OS: CIMP = 593 days, Cluster 2 = 245 days, Cluster 3 = 456 days; log-rank p=0.1530). Consistent with our DREAM data, combining CGI promoter sites with non-CGI, non-promoter sites revealed a hierarchical clustering pattern of three major clusters with significant differences in overall survival (median OS: CIMP = 822 days, Cluster 2 = 365 days, Cluster 3 = 365 days; log-rank p=0.0295). Despite technical differences between platforms, there was significant overlap in the genes most proximal to differentially methylated sites between the DREAM and TCGA analyses. These common genes were significantly enriched in transcription factors, pyrimidine metabolism genes, and development genes. Interestingly, the presence of IDH1 R140 mutations was significantly greater in the CIMP clusters in both the DREAM and TCGA analyses (p Conclusions: We propose that the CIMP methylation pattern is associated with favorable prognosis in AML. We have identified a subset of methylation sites that, when interrogated, predict overall survival independent of other clinical factors. Citation Format: Andrew D. Kelly, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjian, Jean-Pierre J. Issa, Jaroslav Jelinek. Genome-wide methylation analysis reveals an independently validated CpG island methylator phenotype associated with favorable prognosis in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B22.

Abstract 2779: A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome

Background: Acute myeloid leukemia (AML) causes the most leukemia-related deaths in the United States, and has frequent epigenetic aberrations, including a CpG island methylator phenotype (CIMP). CIMP defines unique molecular subtypes of other cancers and has been linked to mutations in IDH1/2, however the clinical consequences of CIMP and the role of IDH1/2 mutations in AML remain unclear. Methods: To measure genome-wide CpG methylation we used Digital Restriction Enzyme Analysis of Methylation (DREAM) on AML bone marrow samples and normal peripheral blood controls. For validation we used methylation data from patient samples from The Cancer Genome Atlas (TCGA) on the Illumina Infinium HumanMethylation450 platform. We also used RNA-seq data from TCGA, and microarray data from GEO (GSE6891). Statistical analysis was done using R. Results: Genome-wide analysis of variably methylated CpG sites in 96 AML bone marrow samples using DREAM revealed two distinct CpG island methylator phenotypes by hierarchical clustering: IDH-CIMP (I-CIMP) in which 7/10 cases had oncogenic IDH1/2 mutations, and AML-CIMP (A-CIMP), which lacked any mutations in IDH1/2. At median follow-up of 6.16 years, A-CIMP cases, but not I-CIMP cases were associated with longer overall survival (median OS, years: A-CIMP = Not reached, P = 0.08; I-CIMP = 3.35, P = 0.50; CIMP-negative = 1.17). We validated and extended these findings using TCGA data. In this cohort A-CIMP cases also had significantly longer OS compared to CIMP-negative (median OS, years: A-CIMP = 2.34, P = 0.01; I-CIMP = 1.25, P = 0.89; CIMP-negative = 1.00). Aberrant hypermethylation in A-CIMP occurred preferentially at CpG islands by a factor greater than 3, while I-CIMP cases demonstrated a slight preference for hypermethylation at sites outside CpG islands. Interestingly, A-CIMP was enriched in CEBPA (19%) and WT1 mutations (14%), but inversely correlated with IDH, TET2, and NPM1 mutations. Functional pathway analysis revealed that genes hypermethylated in A-CIMP are associated with pluripotency maintenance - including PAX6, GBX2, and HOXA9 - and RNA-seq data largely, but not entirely, recapitulated methylation-based patterns. There was a strong correlation between promoter CpG island methylation and gene expression for many A-CIMP genes. Finally, the transcriptional program associated with A-CIMP was found to correlate with outcome and genetic backgrounds in both TCGA and additional independent datasets. Conclusions: Taken together, our data suggest that CIMP in AML is complex, multifactorial and cannot be explained solely by coding gene mutations (e.g. IDH1/2, TET2). There is an association between A-CIMP and curability in multiple AML datasets that cannot be recapitulated by mutational data alone and that may be worth validating in prospective studies. Citation Format: Andrew D. Kelly, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Sijia Yu, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjain, Jaroslav Jelinek, Jean-Pierre J. Issa. A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2779.

Abstract 5302: Genome-wide methylation analysis reveals multiple epigenetic subtypes of acute myeloid leukemia

Background: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy for which known molecular features, while useful, are inadequate for determining the likelihood of durable remission following chemotherapy. Our study examines multiple DNA methylation patterns associated with AML curability and clinical features, and expands on preliminary work relating epigenomic phenotype to survival. Methods: To measure genome-wide CpG methylation we used Digital Restriction Enzyme Analysis of Methylation (DREAM) on a cohort of 101 AML samples and 25 normal blood controls. We used methylation data from 194 patient samples from The Cancer Genome Atlas (TCGA) on the Illumina Infinium HumanMethylation450 platform as validation. Statistical analysis was done using R. Results: DREAM analysis of 3,003 CpG sites (with standard deviation > 20% across AML cases) revealed three distinct DNA methylation patterns by hierarchical clustering. A group of 22 AML cases demonstrated significant hypermethylation at many loci compared to normal blood, consistent with a CpG Island Methylator Phenotype (CIMP). Kaplan-Meier analysis demonstrated that CIMP-AML cases had significantly longer OS than either non-CIMP cluster (median OS, years: CIMP = 14, hypomethylator = 1.47, non-CIMP-2 = 1.04, log-rank p = 0.0021). In this dataset CIMP was prognostic independent of cytogenetic risk, and antecedent hematologic malignancy (AHD). We also found that CIMP-AML was significantly enriched in IDH1/2 mutations (32% of CIMP-AML vs. 9% of non-CIMP-AML, p = 0.02). Differential methylation analysis to characterize the specific CpG sites within the genome that define CIMP-AML demonstrated widespread hypermethylation predominantly at CGIs. Using a robust Cox regression approach, we identified candidate markers of CIMP in our DREAM dataset (sensitivity = 73%, specificity = 100%). We validated the presence of CIMP-AML in independent data from TCGA which demonstrated a consistent pattern regarding survival (median OS, years: CIMP = 2, non-CIMP = 1, log-rank p = 0.0109), and the enrichment of IDH1/2 mutations (30% of CIMP-AML vs. 14% of non-CIMP-AML, p = 0.02). Our DREAM analysis also revealed a distinct cluster of AML cases with a hypomethylator phenotype (HMP). This novel HMP-AML subtype was more common than CIMP, and was associated with intermediate OS. Differential methylation analysis revealed that HMP-AML is defined by loss of methylation predominantly at non-CGIs. HMP-AML demonstrated a relative enrichment for DNMT3A mutations (24% of HMP-AML vs. 7% of non-HMP-AML, p = 0.10). Conclusions: We propose that CIMP-AML is associated with CGI hypermethylation, IDH1/2 mutations, and favorable prognosis. Our Cox regression strategy identified a small group candidate biomarkers of CIMP-AML. In addition, we identified a novel HMP-AML phenotype associated with non-CGI hypomethylation, DNMT3A mutations, and an intermediate prognosis. Citation Format: Andrew D. Kelly, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjian, Jaroslav Jelinek, Jean-Pierre J. Issa. Genome-wide methylation analysis reveals multiple epigenetic subtypes of acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5302. doi:10.1158/1538-7445.AM2015-5302

Abstract 2302: CpG hypermethylation marks potentially curable acute myeloid leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Acute myeloid leukemia (AML) is a heterogeneous blood malignancy. Genetic markers identify distinct subgroups. Epigenetics can refine the classification and prognostic stratification. Our pilot data on a small subset of genes suggested that DNA hypermethylation was an indicator of prolonged survival. Experimental Design: To expand the analysis, we used Digital Restriction Enzyme Analysis of Methylation (DREAM) for high resolution quantification of CpG methylation at CCCGGG sequences across the genome. We analyzed pretreatment bone marrow samples from 100 AML patients and 19 control samples of normal blood leukocytes. Results: We quantified methylation at 22,576 CpG sites and identified 3004 variable CpG sites with the standard deviation of methylation in AML greater than 20%. Hierarchical clustering of methylation values at these variable sites divided the patients into three clusters. Cluster 1 showed extensive hypomethylation when compared to the remaining AML patients and normal controls. Mutations of DNMT3A in the R882 codon were observed in 8/32 (25%) patients while 2 patients (6%) had an IDH mutation. Cluster 2 had the methylation pattern similar to normal controls. IDH mutations were found in 5/45 (11%) patients. DNMT3A R882 mutations were detected in 3 patients (7%); in two of them they were associated with an IDH mutation. Cluster 3 had the highest proportion of IDH mutations - 7/22 (32%) patients; two of these patients also harbored the DNMT3A R882 mutation. This cluster displayed the CpG Island Methylator Phenotype (CIMP) with hypermethylation at multiple CpG sites. We identified significant hypermethylation at 785 CpG sites (P 20% when compared to Clusters 1 and 2). One third of the hypermethylated sites mapped within 1 kb of gene transcription start sites (TSS). We used DAVID Bioinformatics Resources 6.7 to characterize ontology of the genes associated with the hypermethylated sites. We found significant enrichment for negative regulators of transcription. Hypomethylation was rare in the CIMP Cluster 3. We did not find any significantly hypomethylated CpG sites when compared to Clusters 1 and 2. Kaplan-Meier survival analysis showed significant differences in the overall survival among the clusters of AML patients, all treated with standard ara-C + anthracycline chemotherapy. Median survival in Cluster 1 and 2 was similar, 1.5 and 1.0 years, respectively. The CIMP cluster of 29 patients showed a remarkably long median survival of 14 years (P=0.003). Conclusion: We propose that CIMP characterizes a subset of AML patients with a good response to chemotherapy and long survival. This work was supported by the NIH Leukemia SPORE grant CA100632 and by a Stand Up To Cancer-American Association for Cancer Research Dream Team Translational Cancer Research Grant, Grant Number SU2C-AACR-DT0109. Citation Format: Jaroslav Jelinek, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi-Kashani, Hagop M. Kantarjian, Jean-Pierre J. Issa. CpG hypermethylation marks potentially curable acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2302. doi:10.1158/1538-7445.AM2014-2302