CONCEPTS IN CANCER EPIGENETICS
Misregulation of epigenetic factors is common in cancer, and a higher level of understanding is achieved by recognizing recurring themes. Epigenetic factors are often misregulated by one of three modes: fusion, loss-of-function (via mutation or expression changes), or gain-of-function (via mutation or expression changes). Notably, each mode impacts the genome and transcriptome in a particular manner, as described below:
Fusion proteins are commonly observed in hematologic malignancies, and are often the product of reciprocal chromosomal translocations. Common configurations involve fusions of DNA-binding proteins to chromatin modifiers, or proteins that interact with chromatin modifiers. This creates a dominant gain-of-function protein that targets chromatin-modifying activity to genes important for proliferation, development, or survival. A well-studied and conceptually informative example involves fusion of the aminoterminal portion of the HMT mixed-lineage leukemia (MLL) protein to other proteins that interact with chromatin modifiers.36,37 Oncogenic MLL fusions are typically driven by the endogenous MLL promoter and retain the DNA-binding domain and additional regions present in the MLL aminoterminus, but omit the catalytic HMT domain normally present at the MLL C-terminus. MLL is normally part of a large complex that methylates histones (H3K4me3) at the promoters of active genes. Oncogenic MLL-fusion proteins can dimerize with normal full-length MLL, and retain the ability to bind DNA binding and also the ability to interact with chromatin and DNA-binding factors.
As previewed above, the fusion partner of MLL is often a protein that interacts with and recruits chromatin modifiers, providing a route to aberrant/constitutive recruitment of chromatin modifiers to particular loci. The most common MLL fusions involve fusion of the MLL N-terminus to the ALL1-fused (AF) genes (AF9) and AF4 proteins (partial internal tandem duplications are also leukemogenic, which likely also affect interactions with chromatin modifiers). Interestingly, the AF9 and AF4 partners are themselves members of more than one chromatin and transcription complex,38,39 and therefore capable of recruiting a range of chromatin modifiers, including a H3 methyltransferase, DOT1 (which methylates histone H3K79),40,41,42 or TIP60, CBP, and EP300 (which acetylate histones H3 and H4). AF4 is also a member of a complex important for transcriptional elongation,38,39 which interacts with acetylated histone tails via a bromo and extraterminal (BET)-family bromodomain present in the BRD4 subunit. MLL fusions also involve direct fusion to a chromatin modifier, including fusion to the HAT enzymes CBP or EP300. (Fusion of MLL to TET proteins are covered separately in the context of DNAme in the section “Misregulation of Dna Methylation/Demethylation In Hematologic Malignancies”).
Regarding mechanism, current thinking supports the targeting of MLL fusions to constitutively activate key genes involved in blood development, causing a block in differentiation. This block (and continued proliferation) provides the opportunity for other genetic and epigenetic events that enhance proliferation and survival. Confirmed targets for MLL fusions include HoxA9 and the Meis1 gene in mouse, where the fusion contributes to their transcriptional activation.41 However, it is likely that a larger repertoire of target genes is involved, as ectopic expression of HoxA9 and Meis1 in mice is effective at inducing leukemias only under certain contexts. Finally, it is important to note that MLL fusions represent one particular class and mechanism; in contrast, fusions involving the retinoic acid receptor (RAR) (e.g., RAR-PLZF) are known to block differentiation through the constitutive recruitment of repressive chromatin modifiers (e.g., HDACs), conferring repression of genes important for activation.43,44 Thus, as illustrated in Fig. 12–3, proper differentiation involves waves of transcription that involve activating a new program and silencing the former program.
Conceptual model for a developmental switch involving transcription factors, chromatin modifiers, and a feedback loop. Here signals for differentiation alter transcription factor and chromatin modifier abundance and activity. This collaboration defines the current chromatin and transcription state and helps prepare the enhancers and promoters of genes needed for future states/cell types, with an example given related to HSC-to-erythroid transition.21 Furthermore, the transcription factor-chromatin modifier interactions of the new state (cell type) can feed back to inhibit the prior program, ensuring the proper developmental trajectory.
The involvement of multiple enzymes in MLL fusions has inspired therapeutic approaches based on enzyme inhibition.2 For example, DOT1 (a histone H3 methyltransferase) inhibitors have proven useful in mouse models of MLL-fusion–induced leukemia (and in cell lines),45 and have entered phase I clinical trials (NCT01684150). Notably, the involvement of BRD4 in this system, along with its known importance in transcriptional activation in MYC-driven cancers, provides additional therapeutic possibilities. Here, inhibitors of BET-family bromodomains (JQ1 and others) have proven very effective in cell lines from patients, laying the foundation for clinical trials.46,47,48,49
Theme 2: Loss-of-Function Mutations in Chromatin Modifiers
Loss-of-function mutations in chromatin modifiers are now very common in many cancers. The key concept in this theme is that the loss of epigenetic control confers both gene-specific and genome-wide epigenetic variation, eliciting transcriptome variation and plasticity. As a result, individual cells with transcriptomes that promote growth, survival, and/or metastasis can be selected from a diverse population. For example, this epigenetic variation can allow cells to sample a transcriptome that promotes invasion and later convert to a transcriptome that favors colonization. One mode involves the aberrant epigenetic silencing of tumor-suppressor proteins, either by the acquisition of “silencing” histone modifications, DNAme, or both. Epigenetic variation and selection are themselves powerful tools; however, they can also combine with genetic mutations to provide a further fitness benefit and reinforce oncogenic properties.
Examples of mutations in epigenetic factors in hematologic malignancies are numerous; even a partial list of factors and their impact is beyond the scope of this chapter.1 However, mutations in certain factors are found in many hematologic malignancies and help to illustrate the concepts above; consequently, they are treated further here. One example that builds on an earlier section “Epigenetics and Hematologic Malignancies” involves mutations in MLL genes. MLL is actually a family of five similar genes; however, whereas MLL1 is most commonly involved in leukemogenic fusion proteins (discussed earlier), mutations in MLL2 are very common in lymphomas, with mutation rates as high as 89 percent for follicular lymphoma.50 Other chromatin factors are also mutated at high frequency in BCLs, including the HAT complex members EP300 and CREBBP.50
Mutations that affect the addition or removal of the repressive histone modification H3K27me3, are increasingly common in hematologic malignancies. For example, mutations in the polycomb repressive complex 2 (PRC2) complex, which adds H3K27me3, are associated with myeloproliferative diseases, myelodysplastic syndromes, and T-cell ALL.51,52,53 In addition, mutations in the main enzyme that removes H3K27me3, known as X-chromosome encoded ubiquitously transcribed tetratricopeptide repeat (UTX), are common in multiple myeloma.54 Furthermore, mutations in these enzymes are known to synergize with mutations in other epigenetic enzymes, such as TET proteins, in myeloid disorders.51 Notably, many genes that become improperly methylated in cancer cells were marked by H3K27me earlier in their development and were DNA demethylated. Thus, proper regulation of H3K27me3—a modification present at many silent but “poised” developmental genes—appears critical for tumor prevention.
Theme 3: Gain-of-Function Mutations in Chromatin Modifiers
Gain-of-function of epigenetic enzymes typically occurs either through upregulation of expression (through copy number variation or promoter fusions) or via mutations that upregulate the activity of the enzyme. The main concept in this theme is that high levels and/or the inability to turn off an epigenetic enzyme can lead to sustained activation or silencing of target loci, depending on the main function of the modification. Among many examples is EZH2, the main enzyme for H3K27me addition, which is either greatly overexpressed or hyperactivated (via mutation) in various hematologic malignancies.55 For example, EZH2 is highly expressed in mantle cell lymphomas, whereas activating mutations (conferred by amino acid substitutions in the catalytic domain) are common in diffuse large B-cell and follicular lymphomas.56 This hyperactivity has led to therapeutic strategies involving selective competitive inhibitors that mimic the cofactor S-adenosyl-methionine, the methyl donor for the EZH2 enzyme, which have proven effective in mouse xenografts.56