Characterizing DNA methylation alterations from The Cancer Genome Atlas (original) (raw)
In 1999, Issa and colleagues first described a distinct subset of human colorectal cancers with extensive DNA hypermethylation of a subset of CpG islands that remained unmethylated in the remaining colorectal tumors (30) and are therefore distinguished from general cancer-specific DNA methylation for a specific tumor type (Figure 1). These tumors were classified as positive for a CIMP. TCGA Research Network and others have identified CIMPs in breast, colorectal, and endometrial tumors as well as in glioblastomas and acute myeloid leukemias, but not in serous ovarian, lung squamous, or kidney renal cell cancers. TCGA has unveiled similar and unique characteristics between CIMPs of different tumor types that have potential implications for the development of novel cancer diagnostics and therapeutic agents.
CIMP in human cancer. Eight individual methylomes are listed (numbered 1–8). Each row indicates an individual cancer methylome, in which clusters indicate individual CpG islands. CIMP-specific DNA hypermethylation is specific only for a proportion of tumors, while cancer-associated CpG islands are frequently methylated in both CIMP and non-CIMP tumors.
Colorectal CIMP is tightly associated with the BRAFV600E mutation. Colorectal CIMP has been identified and characterized using candidate and genome-scale approaches in numerous reports after Issa first identified CIMP in colorectal cancer (30). Colorectal CIMP tightly associates with mutation of the v-raf murine sarcoma viral oncogene homolog B (BRAF) gene correlating with the V600 amino acid (BRAFV600E), DNA methylation of the mutL homolog 1 (MLH1) promoter region, microsatellite instability (MSI), location in the proximal colonic region, and female gender (31–35). Ogino et al. first described a CIMP-low (CIMP-L) subgroup as having an attenuated CIMP phenotype, and showed an association with mutations in the kirsten rat sarcoma viral oncogene homolog (KRAS) gene rather than BRAF (36). Similarly, Shen et al. identified the CIMP2 subgroup as displaying CIMP-associated DNA methylation, with enrichment in KRAS mutations (32). KRAS mutations are also enriched in non-CIMP tumors, but are strikingly absent in CIMP-high (CIMP-H) tumors (34–37). TCGA confirmed CIMP-H, CIMP-L, and two non-CIMP subgroups of colorectal cancer (34, 35). CIMP-H was present in approximately 15% of colorectal tumors, the majority of which also showed elevated mutation rates (hypermutated) and showed few SCNAs in contrast to the majority of colorectal tumors, which are non-CIMP, non-hypermutated, and microsatellite stable but which show substantial SCNAs (34).
The molecular mechanisms that explain the tight correlation between CIMP-H and the _BRAF_V600E mutation are not well understood. TCGA did not identify driver events, such as specific mutations or SCNAs in a _trans_-acting factor in CIMP-H and _BRAF_V600E colorectal tumors. While both the _BRAF_V600E mutation and DNA methylation changes are thought to occur early in colorectal tumorigenesis, it is unclear whether CIMP or _BRAF_V600E mutation is the initiating event. Hinoue and colleagues did not observe CIMP after introducing exogenous _BRAF_V600E into a wild-type BRAF, non-CIMP colorectal cancer cell line (38). However, Hinoue et al. did identify CIMP-specific epigenetic silencing of IGF-binding protein 7 (IGFBP7), which mediates BRAFV600E-induced apoptosis and cellular senescence. As _BRAF_V600E has been implicated in oncogene-induced senescence in melanomas and colorectal cancers (39), CIMP-specific silencing of IGFBP7 may create a favorable context for the generation of the _BRAF_V600E mutation in CIMP-positive tumors (38). However, additional experiments are needed to determine the molecular mechanism linking CIMP and _BRAF_V600E in colorectal cancer.
Isocitrate dehydrogenase mutations and DNA hypermethylation in glioma and AML. Glioblastoma multiforme (GBM) was the first cancer selected by TCGA Research Network for molecular characterization (40). The majority of GBM tumors are considered primary or de novo, and approximately 5% are secondary GBMs which progress from lower-grade tumors. Parsons and colleagues used a comprehensive sequencing strategy and identified specific heterozygous somatic point mutations in the isocitrate dehydrogenase 1 (IDH1) gene (most often at amino acid residue R132) in 12% of GBM patients (41). Somatic mutations in IDH2 (amino acid R172) were also identified in gliomas. These mutations occur at a low frequency and are mutually exclusive with IDH1 mutations (42, 43).
TCGA Research Network sequenced 601 candidate genes for somatic mutations but did not sequence IDH1 or IDH2 in their initial report (40); however, in a recent follow-up report, TCGA identified IDH1 mutations through whole exome and whole genome sequencing approaches (44). In 2010, Noushmehr and TCGA network colleagues (45) convincingly showed an extremely tight correlation between GBM tumors with the _IDH1_R132H mutation and a glioma CIMP (G-CIMP). All primary GBM tumors with an IDH1 mutation were G-CIMP, but there were also a very small number of WT IDH1 (IDH1WT) G-CIMP tumors. G-CIMP tumors also showed attenuated SCNAs and TP53 alterations and correlated with younger patient age and improved survival. Moreover, G-CIMP is highly prevalent in recurrent and secondary GBM tumors and is inversely correlated with glioma stage (45).
Brennan and TCGA colleagues (44) confirmed the G-CIMP subgroup and its association with IDH1 and TP53 alterations. Including the G-CIMP subgroup, TCGA identified a total of six DNA methylation subgroups using unsupervised clustering analyses and showed enrichment of some individual DNA methylation groups with gene expression–based subgroups (neural, proneural, mesenchymal, and classical) identified previously (46), with G-CIMP tumors tightly associated with the proneural subgroup. Interestingly, one subgroup (cluster M6) was generally DNA hypomethylated and enriched for proneural non–G-CIMP tumors with IDH1WT. G-CIMP patients displayed longer survival times, whereas non–G-CIMP patients had shorter survival outcomes. Interestingly, even though the M6 tumors were proneural, patients with this tumor type did not show survival advantages, which suggests that the aberrant molecular features of G-CIMP may be important in conferring the survival advantage in G-CIMP patients.
Because of the expanded GBM tumor collection and exome sequencing depth, TCGA identified amplifications of MYC (v-myc avian myelocytomatosis viral oncogene homolog) in G-CIMP tumors. MYC is a transcription factor that is frequently altered in cancer and is involved in cell cycle progression, transformation, and apoptosis (reviewed in ref. 47). In addition, TCGA also identified ATRX (α-thalassemia/mental retardation syndrome X-linked) somatic mutations in G-CIMP tumors, which are highly correlated with IDH1 mutations. ATRX belongs to the SWI/SNF family of chromatin remodelers and functions as an ATPase and helicase that facilitates the substitution of variant histone H3.3 into chromatin at telomeres (48).
ATRX mutations are predominant in the alternative lengthening of telomeres (ALT), a process by which telomere length is maintained independent of telomerase in cancer cells (48, 49). Interestingly, TCGA identified telomerase (TERT) promoter mutations in 21 of 25 GBM tumors sequenced, and these mutations correlated with increased TERT gene expression. Notably, all four tumors without TERT mutations did not display elevated TERT gene expression, but instead contained ATRX alterations. Therefore, it is possible that GBM tumors maintain telomere length by either TERT mutations to reactivate TERT gene expression or via ATRX mutations in ALT.
The link between the _IDH1_R132H mutation and G-CIMP has generated tremendous attention from basic and translational scientists. Recent studies have shown that introducing exogenous _IDH1_R132H into immortalized cell lines with endogenous IDH1WT was sufficient to drive G-CIMP–based DNA methylation events and increased occupancy of histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3), and H3K36me3, which correlate with methylated DNA regions in the cancer genome (50, 51).
These epigenomic changes may occur as a result of the function of the mutant IDH proteins. IDH1WT functions as a dimer to catalyze the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH by converting of isocitrate to α-ketoglutarate (α-KG) (reviewed in ref. 52). However, the IDH mutant catalyzes the conversion of α-KG to D-2-hydroxyglutarate (2-HG) (53–55), resulting in elevated 2-HG levels (53, 56, 57). IDH1 mutant-mutant (IDH1MUT-MUT) homodimers or mutant-WT (IDH1MUT-WT) heterodimers have been identified in vitro (58). However, recent in vitro experiments have shown that the presence of the IDH1WT protein is associated with increased 2-HG levels (54), and that IDH1MUT-WT dimers show more enzymatic activity toward α-KG than IDH1MUT-MUT alone (59), suggesting that both forms may be required for 2-HG production.
2-HG inhibits the TET family of enzymes and Jumonji-C domain containing histone lysine demethylases, which normally utilize α-KG as a co-substrate (60, 61). Thus, the production of 2-HG by mutant IDH1-containing enzymes effectively inhibits TET activity (Figure 2A). TETs catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and ultimately to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (62, 63). 5fC and 5caC are substrates for the thymine DNA glycosylase-mediated base excision repair pathway that ultimately results in replacement with an unmethylated cytosine, effectively demethylating the locus (64–67) (Figure 2B).
DNA demethylation dynamics in human cancers. (A) Role of IDH1 in shaping the cancer methylome. WT IDH1 converts isocitrate to α-KG, but the mutant IDH1R132H enzyme catalyzes the conversion α-KG to 2-HG, which inhibits TET-mediated DNA demethylation. This mechanism is proposed to explain DNA hypermethylation in _IDH1_- and _TET_-mutated cancers. (B) Proposed mechanism of DNA demethylation by the TET family of DNA demethylases, followed by thymine-DNA glycosylase (TDG) base excision repair, resulting in unmethylated cytosines.
AML tumors also harbor IDH1 mutations (mostly at the R132 residue), IDH2 mutations (at residues R140 and R172) (68), and TET mutations. IDH1 and IDH2 mutations are mutually exclusive and occur in up to 30% of acute myeloid leukemias (69–74). Moreover, TET2 mutations are also mutually exclusive with IDH mutations. Figueroa and colleagues showed that AML tumors with TET2 mutations displayed a DNA hypermethylation signature that is similar to that of AML tumors with IDH mutations (69), suggesting that IDH and TET enzymes may have redundant roles in DNA demethylation. TCGA also demonstrated that AML tumors with IDH somatic mutations showed substantial gains in DNA methylation, and similarly, in _TET2_-mutated AML tumors (75).
The inhibition of DNA demethylation as a result of the IDH1 and TET mutations is consistent with the epigenomic landscapes identified in G-CIMP and AML tumors, but the basis for the target site specificity of the cancer-associated DNA methylation events observed in IDH1 mutant tumors is unclear. Two recent studies have identified IDAX (inhibition of the Dvl and Axin complex, also known as CXXC4) and early B cell factor 1 (EBF1) as potential TET2-interaction partners. IDAX can bind unmethylated promoter CpG islands as well as the TET2 catalytic domain, resulting in decreased 5hmC levels and TET2 degradation via caspase activation. EBF1, by binding to both DNA and TET2, may also regulate DNA demethylation in _IDH1_-mutant cancers in a tissue- and sequence-specific manner (76).
Clinical importance of MGMT DNA methylation in GBM. The standard of care chemotherapeutic agent for treating GBM patients is temozolomide (TMZ), which acts as a methyl donor for alkylation of the N-7, O-3, and O-6 positions of nucleotide bases (reviewed in ref. 77). TMZ treatment initiates a DNA repair response, but it is believed that the mismatch repair machinery cannot effectively incorporate the correct base opposite to O-6-methylguanine lesions after the initial DNA strand-nicking step in the repair pathway. The nicks accumulate and are thought to promote an apoptotic response that results in cell death.
O-6-methylguanine methyltransferase (MGMT) removes methyl groups from the O-6 position of guanines, thereby rendering TMZ ineffective. Promoter DNA hypermethylation–based silencing of MGMT sensitizes GBM tumors to TMZ, and as a result has been used as a diagnostic barometer for selecting TMZ as a treatment option for GBM patients. TCGA recently identified MGMT DNA methylation in nearly 50% of GBM patients, and MGMT DNA methylation was more prevalent in G-CIMP tumors than in non–G-CIMP tumors (44). MGMT DNA methylation correlated with patient response in GBMs belonging to the classical gene expression subgroup only, and not in the proneural, neural, or mesenchymal groups.
Breast cancer CIMP. Breast cancer is a complex and heterogeneous disease, and breast tumor subgroups have been proposed based on the expression status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2; also known as ERBB2). TCGA identified five DNA methylation subgroups of breast tumors (78), with one subgroup exhibiting a CIMP-like DNA methylation signature (B-CIMP), as previously described by Fang and colleagues (79). B-CIMP tumors were positive for ER, PR, and HER2 expression and were enriched for the luminal B gene expression subgroup as well as epigenetic silencing of genes in the Wnt-signaling pathway (78), as has also been described for colorectal tumors (34).
Endometrial carcinoma CIMP. Endometrial tumors are classified into two groups: the type I endometrioid tumors that are hormone receptor positive with good prognosis and the type II serous tumors that mostly occur in older women and correlate with poor outcome. TCGA identified four DNA methylation subgroups of endometrial tumors (80), with one subgroup displaying a CIMP-like (E-CIMP) DNA hypermethylation profile. E-CIMP was previously identified by Whitcomb and colleagues (81). Similar to colorectal CIMP tumors, the E-CIMP tumors were hypermutated, MSI positive due to MLH1 promoter DNA hypermethylation, and did not contain TP53 somatic mutations or extensive SCNAs (34, 80). However, E-CIMP tumors did not harbor _BRAF_V600E or IDH1 mutations, as described in colorectal and glioma CIMP tumors, respectively, pointing to an alternative mechanism of CIMP-specific DNA methylation in endometrial tumors.