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168 Part IV: Molecular and Cellular Hematology Chapter 12: Epigenetics 169

TET PROTEINS AND ACTIVE N-terminus to the ALL1-fused (AF) genes (AF9) and AF4 proteins
(partial internal tandem duplications are also leukemogenic, which
DNA DEMETHYLATION likely also affect interactions with chromatin modifiers). Interestingly,
the AF9 and AF4 partners are themselves members of more than one
DNAme is chemically highly stable, and therefore very useful in stable chromatin and transcription complex, 38,39 and therefore capable of
propagation of epigenetic states, even through germline inheritance. recruiting a range of chromatin modifiers, including a H3 methyltrans-
Although stable, DNA demethylation can occur, and does so mainly ferase, DOT1 (which methylates histone H3K79), 40–42 or TIP60, CBP,
through two routes: (1) “passive” demethylation (dilution of DNAme by and EP300 (which acetylate histones H3 and H4). AF4 is also a member
the failure to conduct maintenance DNAme after DNA replication), and of a complex important for transcriptional elongation, 38,39 which inter-
(2) “active” demethylation, mainly involving proteins of the TET family. acts with acetylated histone tails via a bromo and extraterminal (BET)-
TET proteins are dioxygenase enzymes that oxidize the methyl group of family bromodomain present in the BRD4 subunit. MLL fusions also
5-methylcytosine (5mC) to 5-hydroxymethylcytosine 33,34 (5hmC) and involve direct fusion to a chromatin modifier, including fusion to the
additional oxidized intermediates (not addressed further). Here, 5hmC HAT enzymes CBP or EP300. (Fusion of MLL to TET proteins are cov-
and other intermediates cause DNA demethylation by one of two modes: ered separately in the context of DNAme in the section “Misregulation
first, following replication the maintenance DNMT1 and its partner, of Dna Methylation/Demethylation In Hematologic Malignancies”).
UHRF1, do not recognize these oxidized products as 5mC, leading to Regarding mechanism, current thinking supports the targeting
passive demethylation. Second, glycosylases with roles in DNA repair of MLL fusions to constitutively activate key genes involved in blood
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(e.g., thymine DNA glycosylase [TDG]) can remove 5-hydroxymeth- development, causing a block in differentiation. This block (and contin-
ylcytosine (5hmC) and similar oxidized intermediates, which are then ued proliferation) provides the opportunity for other genetic and epige-
replaced with an unmodified cytosine by base-excision repair systems. netic events that enhance proliferation and survival. Confirmed targets
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An important aspect of TET-family dioxygenases is their use of iron for MLL fusions include HoxA9 and the Meis1 gene in mouse, where
and 2-oxoglutarate (2OG) as cofactors during the oxidation reaction; a the fusion contributes to their transcriptional activation. However, it
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feature that renders TET enzymes sensitive to concentrations of these is likely that a larger repertoire of target genes is involved, as ectopic
metabolites and related compounds, which can act as inhibitors during expression of HoxA9 and Meis1 in mice is effective at inducing leuke-
metabolic dysregulation (see section “Misregulation of Dna Methylation/ mias only under certain contexts. Finally, it is important to note that
Demethylation In Hematologic Malignancies”).
MLL fusions represent one particular class and mechanism; in contrast,
fusions involving the retinoic acid receptor (RAR) (e.g., RAR-PLZF) are
EPIGENETICS AND HEMATOLOGIC known to block differentiation through the constitutive recruitment of
repressive chromatin modifiers (e.g., HDACs), conferring repression
MALIGNANCIES of genes important for activation. 43,44 Thus, as illustrated in Fig. 12–3,
proper differentiation involves waves of transcription that involve acti-
CONCEPTS IN CANCER EPIGENETICS vating a new program and silencing the former program.
Misregulation of epigenetic factors is common in cancer, and a higher The involvement of multiple enzymes in MLL fusions has inspired
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level of understanding is achieved by recognizing recurring themes. therapeutic approaches based on enzyme inhibition. For example,
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 Cell Type A Cell Type B
impacts the genome and transcriptome in a particular manner, as (Hematopoie (Erythroid
described below: tic stem cell) Differentiation Progenitor)
Signals
Theme 1: Fusion Proteins
Fusion proteins are commonly observed in hematologic malignancies,
and are often the product of reciprocal chromosomal translocations. Switch in Transcription Factor
Common configurations involve fusions of DNA-binding proteins to Transcription Factor A Transcription Factor B
(e.g GATA1)
(e.g. GATA2)
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, Inhibit alternative program
or survival. A well-studied and conceptually informative example Chromatin Factor A Chromatin Factor B
involves fusion of the aminoterminal portion of the HMT mixed-lineage (e.g. EZH2) (e.g. EZH1)
leukemia (MLL) protein to other proteins that interact with chromatin Switch in Chromatin Factor
modifiers. 36,37 Oncogenic MLL fusions are typically driven by the endoge-
Conduct Transcription Program for Cell Type A, and
nous MLL promoter and retain the DNA-binding domain and additional Modify/Poise Enhancers and Promoters for Cell Type B
regions present in the MLL aminoterminus, but omit the catalytic HMT
domain normally present at the MLL C-terminus. MLL is normally part Figure 12–3. Conceptual model for a developmental switch involv-
of a large complex that methylates histones (H3K4me3) at the promot- ing transcription factors, chromatin modifiers, and a feedback loop.
ers of active genes. Oncogenic MLL-fusion proteins can dimerize with Here signals for differentiation alter transcription factor and chromatin
normal full-length MLL, and retain the ability to bind DNA binding and modifier abundance and activity. This collaboration defines the current
also the ability to interact with chromatin and DNA-binding factors. chromatin and transcription state and helps prepare the enhancers and
promoters of genes needed for future states/cell types, with an exam-
As previewed above, the fusion partner of MLL is often a protein ple given related to HSC-to-erythroid transition. Furthermore, the tran-
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that interacts with and recruits chromatin modifiers, providing a route scription factor-chromatin modifier interactions of the new state (cell
to aberrant/constitutive recruitment of chromatin modifiers to partic- type) can feed back to inhibit the prior program, ensuring the proper
ular loci. The most common MLL fusions involve fusion of the MLL developmental trajectory.

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