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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 417-428
REVIEW ARTICLE
Chromatin Remodeling and Leukemia: New Therapeutic Paradigms
By
Robert L. Redner,
Jianxiang Wang, and
Johnson M. Liu
From the Division of Hematology/Oncology, Department of Medicine,
University of Pittsburgh Medical Center, Pittsburgh, PA; and the
Hematology Branch, National Heart, Lung and Blood Institute, Bethesda
MD.
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INTRODUCTION |
LOCAL REMODELING OF chromatin is a key
step in the transcriptional activation of genes. Dynamic changes in the
nucleosomal packaging of DNA must occur to allow transcriptional
proteins contact with the DNA template. The realization that the
proteins that regulate the modification of chromatin are themselves
disrupted in many leukemic chromosomal rearrangements has generated new excitement in the study of chromatin structure. Recent reports have
kindled the hope that pharmacological manipulation of chromatin remodeling might develop into a potent and specific strategy for the
treatment of these leukemias. In this review, we will discuss the
structure of chromatin and the mechanisms by which cells remodel chromatin, alteration of these pathways in leukemias, and therapeutic approaches.
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CHROMATIN: BEADS ON A STRING OR STRING ON BEADS? |
The condensation of DNA into an ordered chromatin structure allows the
cell to solve the topological problems associated with storing huge
molecules of chromosomal DNA within the nucleus. DNA is packaged into
chromatin in orderly repeating protein-DNA complexes called
nucleosomes.1,2 Each nucleosome consists of approximately
146 bp of double-stranded DNA wrapped 1.8 times around a core of 8 histone molecules (Fig 1). Two molecules
each of H2A, H2B, H3, and H4 comprise the histone ramp around which the
DNA superhelix winds. Stretches of DNA up to 100 bp separate adjacent
nucleosomes. Multiple nuclear proteins bind to this linker region, some
of which may be responsible for the ordered wrapping of strings of
nucleosomes into higher-order chromatin structures.3,4
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| Fig 1.
Cartoon of nucleosomal structure. (A) represents the
random-coiled tails of the histone octamer intertwined with DNA. (B)
represents the nucleosome with histones acetylated (acetyl groups drawn
as lollipop structures). The acetylated histone tails do not bind the
DNA strands. This allows the DNA to assume a more open configuration
that is accessible to the transcriptional machinery.
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Nucleosomal structure has been well characterized by x-ray
crystallographic studies, most recently to a resolution of 2.8 Å.1 The histones are arranged as heterodimers of H2A and
H2B, H3 and H4; the heterodimers, in turn, form a tetrameric structure known as the octamer core. Each histone heterodimer binds approximately 30 bp of DNA through electrostatic contacts with the phosphate backbone
in the minor groove of the DNA. The interaction with histones causes
the DNA to become distorted and bend and bulge at several positions:
this twisting of the helix results in a deviation of the periodicity of
the basepairs as they spiral along the DNA superhelix. The change in
periodicity leads to alignment of the DNA minor grooves to form
channels, through which pass the random-coil histone tails. The histone
tails are thus able to contact the DNA on the exterior of the
nucleosomal particle to further stabilize DNA/histone interactions.
Although nucleosomal architecture depends primarily on nonspecific
interactions between histones and the phosphate backbone of DNA, the
thermodynamic stability of the interaction is influenced by the
basepair composition of the DNA. A-T-rich sequences in particular
impart the DNA with flexibility that allows it to form the tertiary
structures necessary for optimal nucleosomal packing.1,5 In
addition, histone leucine residues bound to the minor groove of DNA are
able to interact with nearby thymidine residues; arginines form
hydrogen bonds with neighboring pyrimidines. These sequence-specific characteristics likely contribute to variations in the number of bases
in each nucleosome particle, the length of internucleosomal spacer DNA,
and the phasing of adjacent nucleosomes.
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NUCLEOSOMAL REMODELING |
Both distortion of the periodicity of the superhelix and electrostatic
shielding by the positively charged histones act to constrain the
access of nonhistone proteins to nucleosomal DNA.6,7 Extensive remodeling of chromatin structure, particularly at cis-acting promoter sites necessary for transcriptional initiation, must take
place for the DNA template to become accessible to the transcriptional machinery. A complete picture of the way in which DNA separates from
nucleosomes during transcription is not yet available, but most likely
only partial dissociation from the core histones is necessary.8
Several mechanisms have been identified that contribute to chromatin
remodeling. They vary from those that alter histone-DNA interaction to
those that physically dissociate the DNA from histones in an
ATP-dependent manner to those that destabilize histone-histone tetramerization (see the recent review by Tsukiyama and Wu9 for a more detailed discussion). All of these processes likely act
simultaneously and in concert to regulate access to the DNA template.
Two of the mechanisms are particularly relevant to the discussion of
leukemic fusion proteins (see below): histone acetylation and
ATPase-mediated DNA-histone dissociation.
The best understood mechanism by which cells regulate chromatin
structure is posttranslational modification of histones by acetylation.10-15 Change in electrostatic attraction for
DNA and steric hindrance introduced by the hydrophobic acetyl group
leads to destabilization of the interaction of histones with
DNA.1,11-13,15,16 Lysine residues in the N-terminal
extensions of H2B, H3, and H4 that bind to the exterior surface of
nucleosomes are particularly accessible to acetylation. Acetylation of
residues within the octamer core interferes with formation of the
histone heterodimers and tetramers around which the DNA winds.
Furthermore, posttranslational modification of histones interferes with
interactions with nonhistone, chromatin-associated proteins, such as
MeCP2 (which binds to methylated regions of DNA17) and the
high-mobility group proteins (HMG,18,19 so named for their
properties in electrophoretic fields), which also contribute to
higher-order nucleosomal packing. In summary, acetylation of histones
disrupts nucleosomes and allows the DNA to become accessible to
transcriptional machinery. Removal of the acetyl groups allows the
histones to bind more tightly to DNA and to maintain a
transcriptionally repressed chromatin architecture. Acetylation is
mediated by a series of enzymes with histone acetylase (histone acetyl
transferase [HAT]) activity; conversely, acetyl groups are removed by
specific histone deacetylase (HDAC) enzymes.
The ability to modulate histone acetylation in a gene-specific fashion
presents a challenge for cells. The state of histone acetylation is
determined by the balance between competing enzymatic activities of
histone acetyltransferases and deacetylases. Fine regulation of the
catalytic activities of these enzymes likely involves cooperative
subunit interaction and posttranslational modification. However, our
limited understanding of the regulation of gene-specific histone
acetylation is best captured in the rather simplistic model of local
recruitment of acetylases or deacetylases by sequence-specific DNA
binding proteins.
The paradigm for local remodeling of chromatin through gene-specific
recruitment of HAT activity is the retinoic acid receptor (RAR).20 Because RAR-mediated chromatin remodeling is
perturbed in acute promyelocytic leukemia (APL; see below), it is
appropriate to consider this model in some depth; similar models have
been developed for other transcriptional activators and
repressors.21 RAR is a ligand-dependent transcriptional
activator.22 Through its zinc (Zn) finger domain, it binds
as a heterodimer with a related protein, RXR,23 to a
well-defined consensus DNA sequence found in the promoters of retinoic
acid-responsive genes.24 In the absence of ligand (retinoic
acid [RA]), the RXR/RAR heterodimer binds to DNA and actively
represses transcription below the basal level expected from random
initiation by the transcriptional machinery.25a It does
this through an indirect mechanism. The RXR/RAR heterodimer binds a
nuclear corepressor molecule, either N-CoR26,27
(Nuclear Receptor Co-Repressor) or
SMRT28,29 (Silencing Mediator of Retinoid and Thyroid Receptors), through specific
interaction domains in the ligand-binding region of RAR. N-CoR, the
better analyzed of the 2, binds to many sequence-specific DNA-binding transcriptional repressor proteins (Table
1). N-CoR (or SMRT) itself binds another intermediary protein, Sin3,
which serves as a bridge to HDAC1, a histone
deacetylase30-32 (there have been 3 homologous HDACs
identified in human cells33). Thus, the end result of the
association of unliganded RAR/RXR with N-CoR is to recruit HDAC1 to the
local environment of the promoter. By removing acetyl groups from
histones and restructuring the chromatin into a repressive
configuration, HDAC1 serves as the effector molecule in this pathway.
N-CoR, Sin3, and HDAC1 function in many repressive pathways, including
transcriptional silencing by the MAD/MAX members of the MYC
family,30,31,34,35 ETO36-38 (see below), and
other members of the steroid hormone receptor
superfamily.26,32
Binding of ligand to RAR initiates a conformational change in the
C-terminus of RAR, which contains the N-CoR binding
site.39-41 An amphipathic helix within RAR (helix 12, which
is highly conserved within the steroid receptor superfamily) swings
into a position that both locks the ligand into its binding pocket and
creates a new hydrophobic domain. N-CoR does not bind to this altered conformation of RAR, and hence the N-CoR/Sin3/HDAC1 complex dissociates itself from liganded RAR. In its place, the newly created hydrophobic domain within RAR binds a large multimolecular complex that enhances transcriptional activation.21,42,43 Several components of this coactivator complex have been identified
(Table 2), including the adenovirus
E1A-associated protein p300, pCAF (CBP-associated factor44), and a family of homologous 160-kD proteins,
including p/CIP (p300/CBP cointegrator-associated
protein),43,45 NCoA-146 (nuclear coactivator-1,
also known as SRC-142), and NCoA-2 (also known as
TIF-246-48). p300, or the homologous cyclic-AMP
response-element-binding protein cofactor
CBP,21,43,49,50 has been shown to interact through
nonoverlapping domains with at least a dozen DNA-binding transcriptional activators, including other members of the steroid hormone receptor family, members of the STAT family, Jun, Fos, AML1,
and Myb (see review by Giles et al51). In this regard, p300/CBP serves as a bridge between multiple transcriptional
activators. Several members of this complex have HAT activity,
including pCAF,44,52 the 160-kD proteins,53 and
p300/CBP itself.50 Thus, recruitment of the coactivator
complex brings several histone acetylases to the proximity of the
promoter to which RAR binds, so that the deacetylated histones can be
efficiently acetylated. The coactivator complex serves to reverse the
suppressive effects of the N-CoR/Sin3/HDAC1 complex and, by decreasing
the affinity of histones for DNA, remodel nucleosomal configuration so
that the DNA template can be accessed for transcription. It is as yet
unclear which of the proteins in the complex directly interact with
histones, which acetylate other proteins in the complex (or the
proteins that comprise the basal transcriptional machinery), which
stabilize the nascent basal transcriptional machinery or, which, as has
been proposed for p300/CBP, function in all 3 capacities.50
An interesting twist to this paradigm has recently arisen through
studies of DNA methylation, which has been a long-accepted mechanism of
transcriptional suppression54,55. A possible contributor to
the transcriptional-silencing effect of DNA-methylation may be the MeCP
family of proteins, which specifically bind to regions of methylated
DNA between adjacent nucleosomes.56 The MeCP2 protein
directly binds Sin3/HDAC1.17 Thus, by recruiting a histone deacetylase, MeCP2-binding leads to a change in the state of histone acetylation and in the chromatin structure of methylated DNA. Whether
this mechanism directly mediates the transcriptional silencing effects
of DNA methylation or whether it serves a synergistic or supporting
role is not yet clear. However, it leads to the interesting speculation
that DNA-methyltransferase inhibitors such as
5-azacytidine57 might alter transcriptional activity by
indirectly remodeling regions of methylated chromatin.
A second mechanism for alteration of chromatin that is less well
understood than acetylation of histones involves an ATP-dependent enzymatic activity that directly acts on nucleosomal structure. Based
on analogous protein complexes in yeast and Drosophila (known by the acronyms SWI/SNF,58-62 NURF,63 and
RSC64,65), it has been proposed that these complexes act as
ATP-dependent motors that track along the DNA strands and pull them
away from the histone octamer cores.59-62,66 During this
shift of histone-DNA contact points, the DNA would presumably become
accessible to the transcriptional machinery. However, there is
conflicting evidence to suggest that these complexes serve to maintain
chromatin in a repressive configuration, perhaps through dissociating
other chromatin-associated proteins from the DNA, such as errant
TATA-binding protein.67 Reinforcing the concept of
interplay between the chromatin remodeling mechanisms is the finding
that 2 members of the human p300-associated coactivator complex68,69 have the predicted ATPase activities requisite for a SWI/SNF type of engine.
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DISRUPTION OF CHROMATIN REMODELING MECHANISMS IN LEUKEMIA |
APL: Abnormal histone deacetylation.
Chromatin remodeling is fundamental to transcription. The models
presented above outline the normal control of chromatin remodeling during gene-specific transcription. Disruption of these mechanisms gives rise to transcriptional chaos and leukemic transformation. The
best understood example of this is in APL (French-American-British [FAB] M3).
APL holds a unique position in the study of leukemias in that it is the
only form of leukemia and the only malignancy described to date that
responds to differentiation therapy. First published in Blood
by Huang et al70 from the Shanghai Institute of Hematology, APL blasts undergo terminal differentiation in response to all-trans retinoic acid (ATRA). Differentiation therapy with ATRA has become the
mainstay of therapy for this disease.71,72 Although
relapses uniformly occur when used by itself, in combination with
conventional chemotherapy, ATRA has revolutionized the treatment of
APL, generating response rates of close to 90%, with 3-year
disease-free survival greater than 75%.73
The explanation for the restriction of the success of ATRA therapy to
the M3 subtype of leukemias likely lies in the chromatin alterations
induced by the RAR-fusion proteins expressed uniquely in APL cells
(Fig
2). RAR (there are 3 homologous RAR proteins, called ,  , and
 ) is a transcriptional activator that binds to specific DNA
sequences and inducibly recruits corepressor or coactivator complexes
to regulate transcription of retinoic acid-responsive genes (see
above). Ordered expression of retinoic acid-responsive genes is
necessary for myeloid development; dominant-negative mutants of RAR
inhibit myeloid maturation at the promyelocytic stage.74,75
All patients with APL have a chromosomal translocation within the
second intron of the RAR locus on chromosome 17q12 to
produce a chimeric protein comprised of all but the first 30 AA of
RAR.76 The N-terminal fusion partners are
PML77,78 on chromosome 15q21,
PLZF79 on chromosome 11q23,
NPM80 on chromosome 5q31, or
NUMA81 on chromosome 11q13. The PML-fusion is the
most common: only 8 patients with PLZF-RAR,82 3 with NPM-RAR,80 and 1 with NUMA-RAR81 have
been described to date. All of these RAR fusion proteins contain the
DNA-binding, heterodimerization, ligand-binding, corepressor-binding,
and coactivator-binding motifs of RAR. Like wild-type RAR, they
bind to retinoic acid-response elements in DNA and, under appropriate
conditions, can activate transcription of RA-target
genes.77,80,83,84
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| Fig 2.
Fusion proteins in acute promyelocytic leukemia. (A)
indicates the unliganded interactions of the RXR/RAR heterodimer
with an N-CoR/Sin3/HDAC1 complex. Upon binding retinoid acid, the
RXR/RAR heterodimer releases the corepressor complex and binds a
coactivator complex with histone acetylase activity. (B) indicates the
analogous interactions of the RXR/PML-RAR heterodimer with the
corepressor complex. Release of the corepressor complex occurs only in
the presence of pharmacological levels of retinoic acid. (C) depicts
the ligand-independent binding of the corepressor complex to
PLZF-RAR. (It has been proposed, but not yet been formally
demonstrated, that liganded RXR/PLZF-RAR binds both coactivator and
corepressor complexes.) Chromatin remodeling occurs only in the
presence of both RA and an HDAC inhibitor.
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| Fig 3.
AML-ETO fusion protein. (A) depicts the association of
the AML1 transcriptional activator with a coactivator complex; (B)
indicates the binding of a corepressor to the AML-ETO fusion protein.
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| Fig 4.
MLL-CBP. One of several models for MLL-CBP function. (A)
MLL binds to DNA through interactions between its AT hooks and the
minor groove of DNA. (B) MLL-CBP alters chromatin structure at
MLL-target sites through the action of the histone acetyltransferase
domain of CBP.
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However, at physiological levels of retinoic acid, PML-RAR represses
rather than activates transcription.85-88 This is
apparently the consequence of enhanced interaction between PML-RAR
and the N-CoR/Sin3/HDAC1 corepressor complex. PML-RAR binds N-CoR at levels of ligand that are otherwise sufficient to release the corepressor complex from wild-type RAR. By maintaining the promoters of RA-responsive genes in a repressive deacetylated configuration, PML-RAR suppresses transcription and produces the identical
phenotype to that experimentally produced by a dominant-negative
inhibitor of RAR.74 Only at pharmacological levels of
ligand does PML-RAR release N-CoR, recruit a coactivator complex,
and allow histone acetylation and chromatin remodeling to proceed.
Thus, for PML-RAR-expressing APL cells, pharmacological levels of
RA are needed to induce differentiation.
If the end result of PML-RAR binding to the corepressor complex is
active repression of transcription through a HDAC1-dependent pathway,
then one would predict that the APL phenotype might be overcome by
inhibitors of HDAC. This prediction has been validated by the finding
that RA and inhibitors of HDAC1 (see below) synergize to induce
differentiation of the APL cell line, NB4, or U937 cells engineered to
express PML-RAR.85-87 The molecular mechanism underlying the strong interaction of PML-RAR with N-CoR is not yet known: there
is apparently no direct binding between PML and N-CoR. Fusion with PML
presumably inhibits the ligand-induced conformational changes necessary
for release of the N-CoR complex. A similar mechanism may also be at
play with the NPM-RAR t(5;17) fusion, which is also retinoic
acid-responsive.89
The t(11;17)(q23;q12) chromosomal translocation of APL fuses the same
sequences of RAR to the N-terminus of PLZF.79 PLZF is
itself a DNA-binding transcription factor, capable of binding N-CoR via
the 120 AA N-terminal POZ motif90, 91 (conserved between
poxvirus and zinc finger proteins) retained in the
PLZF-RAR fusion.92-94 As a result, PLZF-RAR interacts with N-CoR through 2 binding sites: a ligand-dependent site in the
RAR domain and a ligand-independent site in the PLZF
N-terminus.85-88,93 When retinoic acid binds to the
ligand-binding domain of PLZF-RAR, the RAR domain of the fusion
protein loses its attraction for N-CoR, but the PLZF ligand-independent
domain continues to bind N-CoR. As a result, even in the presence of
pharmacological levels of retinoic acid, N-CoR/Sin3/HDAC1 remains
tethered to RA-responsive promoters, permanently suppressing
transcription and blocking differentiation. This model explains the
lack of response of t(11;17) patients to ATRA differentiation
therapy95 and may explain in part the observation that
t(11;17) blasts show morphologic features indicating less
differentiation than classical APL cells.82 Based on this
model, one would predict that inhibitors of HDAC might partially
overcome the suppressive effects of PLZF-RAR. This is indeed the
case: several groups have recently demonstrated that inhibitors of
HDAC1 synergize with retinoic acid to induce differentiation of
otherwise nonresponsive PLZF-RAR cells.85-88
AML1-ETO: Exchanging a coactivator for a corepressor.
The AML1-ETO oncoprotein has recently been shown to alter gene
expression through an analogous mechanism of errant recruitment of an
N-CoR repressor complex. AML1-ETO is derived from the fusion of
the AML1 gene on chromosome 21q22 with the ETO (also
called MTG8) locus on chromosome 8q22 (see review by Hiebert et
al96). Accounting for over 10% of acute myeloid leukemias
(AML), t(8;21) is seen exclusively in FAB M2. Patients with t(8;21)
have a better response rate to chemotherapy and a higher remission rate
than M2 patients with normal karyotype.97,98
AML1 is a sequence-specific DNA binding protein that complexes with
core binding factor (CBF) to activate transcription of target
genes.99 Many of the AML1-dependent genes have been implicated in myeloid maturation, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), myeloperoxidase, and neutrophil elastase.96 Unlike the RAR model discussed in detail
above, AML1/CBF binds no ligand; regulation of AML1 activity occurs as a result of transcriptional and posttranslational modulation of AML1
(as yet, these mechanisms have been poorly defined). Recently, it has
been shown that the C-terminus of AML1 interacts with a p300-containing
coactivator complex,100 suggesting that one of the
mechanisms by which AML1 activates transcription is through local
histone acetylation and nucleosomal remodeling.
AML1-ETO, on the other hand, inhibits transcription of AML1-responsive
genes.99,101 AML1-ETO knock-in mice102
have similar embryonic-lethal phenotype as aml1 knock-out
mice,25 which show absent fetal liver hematopoiesis. The
AML1-ETO protein contains the N-terminal 177 AA of AML1, including the
runt homology domain that mediates sequence-specific DNA
binding,99 fused to near full-length ETO
protein.103 ETO was originally identified through its
involvement in the t(8;21) translocation. Expressed in CD34 cells and
in the brain,104 ETO shares regions of homology with the
Drosophila Nervy protein.103 It contains 2 putative
C-terminal Zn-finger domains, but has not yet been shown to directly
interact with DNA. Little is known about its normal function. It acts
as a transforming oncogene when overexpressed in NIH3T3
cells.105 In tests of its ability to modulate
transcription, ETO acts as a repressor, although it is not yet clear
how this relates to the function of wild-type ETO.36
Recently, our group demonstrated that ETO's ability to repress
transcription is mediated through its interaction with N-CoR and
recruitment of an N-CoR/Sin3/HDAC1 complex.36 These results have been confirmed by 2 other laboratories.37,38 Because
the N-CoR interaction domain maps to the Zn-finger regions of ETO, a
region that is retained in the AML1-ETO fusion, the same domain likely
mediates AML1-ETO's repressive effects. AML1-ETO mutants that lack
this domain lose their ability to recruit N-CoR and lose their ability
to repress transcription and inhibit differentiation.36,38 The model that has developed is the following (Fig 3): AML1-ETO binds
to AML1 consensus sequences in DNA. Unlike wild-type AML1, the fusion
protein does not initiate p300/CBP-directed histone acetylation and
transcriptional activation. Rather, the opposite occurs: through its
ETO domain, AML1-ETO recruits N-CoR/Sin3/HDAC1. By permanently
tethering this repressive complex to AML1-responsive promoters,
AML1-ETO actively suppresses transcription by maintaining the histones
in a deacetylated conformation and making DNA inaccessible to the
transcriptional apparatus. Inhibition of the expression of
AML1-responsive genes leads to a block in myeloid development and
leukemic transformation of the maturing hematopoietic progenitors. One
might predict that HDAC inhibitors could overcome the effects of
AML1-ETO and lead to reversal of the leukemic phenotype; preliminary data suggests that this strategy of chromatin-remodeling therapy holds
promise for the treatment of t(8;21) acute myeloid leukemia (see below).
MLL fusions: A SET of alterations.
The MLL locus is involved in a greater assortment of
chromosomal rearrangements in leukemias than any other gene (see recent reviews by Downing and Look106 and Cimino et
al107). Translocations or inversions of this gene on
chromosome 11q23 are associated with a variety of FAB subtypes and some
lymphoid malignancies as well (thus the name, Mixed
Lineage Leukemia). Because of its involvement in
the t(4;11) pediatric B-cell acute lymphoblastic leukemia
(ALL),108 MLL is also called
ALL1. MLL rearrangement is found in both de novo
leukemias and chemotherapy-associated (most often
topoisomerase-inhibitor) secondary leukemias. All of the 11q23
leukemias are aggressive, respond poorly to chemotherapy, and have a
poor prognosis. More than 40 translocation sites have been identified
for MLL, and nearly 20 fusion partners have been cloned.
Partial tandem duplication of MLL with an abnormal
MLL/MLL fusion occurs in AML patients with the (+11)
karyotype109 and is sometimes seen in leukemias with normal
karyotype. Possibly relating to a fragile site in the MLL
locus, all of the rearrangements cluster within exons
5-11.110
The MLL gene111-114 is more than 100 kb long,
encodes an 11.7-kb transcript with 23 exons,110 and gives
rise to a protein of 3972 AA. Clues to the function of MLL have come
from the identification of domains that share homology with other known
proteins. The most significant match is seen in 4 domains that are
conserved with the Drosophila protein Trithorax (TRX), giving
rise to the alternative name for MLL as the human trithorax
gene (HRX or HTRX).111-114 In
Drosophila, TRX positively regulates homeotic gene
expression,115-118 which in turn directs development of
embryonic structures. In mammalian hematopoiesis, MLL is thought to
regulate expression of homeobox (HOX) genes,119
which serve a similar critical role as transcriptional activators of
developmentally expressed gene families. It follows that dysregulation
of HOX gene expression would have global consequences for
hematopoietic cells and give rise to aggressive malignant
transformation. As expected, murine mll  /
knockouts are embryonic lethal.119 Mll +/
animals have numerous developmental skeletal abnormalities, as well as
abnormal hematopoiesis.119
MLL has a series of N-terminal domains known as AT-hooks and a
Zn-finger motif in its C-terminus.111-115 As with other
AT-hook containing proteins, it is thought that the AT-hooks allow MLL to bind to the minor groove of DNA (Fig 4A). Although it has not been
shown to recognize specific DNA sequence motifs, TRX does localize to
discrete areas of polytene chromosomes in
Drosophila.120 Several of these sites have been
identified as regulatory regions of target genes, supporting the
hypothesis that TRX and MLL associate physically with cis-acting
elements of target genes to facilitate their expression during
appropriate stages of development. Unlike TRX, MLL has an N-terminal
region that shares homology with the regulatory region, but not the
catalytic domain, of DNA methyltransferases112; the
significance of this is not yet clear, although one could speculate
that this region might participate in chromatin structure recognition.
Perhaps key to their function, MLL and TRX share a highly conserved
C-terminal 150 AA protein interaction domain, known as the SET
domain.120 This region is also found in a number of other
proteins that have transcriptional regulatory roles. Through its SET
domain, MLL has been shown to bind INI1 and SNR1, 2 proteins that are
homologous to members of the SWI/SNF complex.120 SWI/SNF
complexes alter chromatin structure in an ATP-dependent fashion (see
above). To date, coprecipitation experiments have failed to identify
MLL or TRX as part of a stable SWI/SNF-like complex, indicating that
the interaction may be a transient one.120
It is reasonable to speculate that MLL serves as a chromatin-binding
protein that serves a regulatory function necessary for expression of
target genes. Through its SET domain, MLL recruits an SWI/SNF-like
ATPase-engine that changes the nucleosomal structure to maintain an
open chromatin conformation. Supporting this hypothesis is the
observation that initial transcription of downstream targets of MLL
occurs normally in mll-null mice; however, in the absence of
MLL, transcription of MLL-target genes cannot be
maintained.119
It is difficult to propose a single model that would suggest a common
mechanism for all of the MLL fusion proteins, including the
partial-tandem duplication of MLL itself (Table
3). In all of the MLL fusions (with the
exception being the partial-tandem duplication), the N-terminal AT
hooks and methyltransferase homology domains are retained, but the
C-terminal Zn-finger and SET domains are lost.121 One
simple model is that loss of the ability to recruit the SWI/SNF complex
disrupts MLL function as a regulator of gene expression. In the tandem
MLL duplication, in which the SET domain is preserved, one might
postulate that the altered MLL conformation, and the abnormal distance
of the SET domain from the N-terminal AT-hooks, might render the MLL
protein nonfunctional.
However, the picture for 11q23 rearrangements is likely not so simple.
Two of its fusion partners,122 AF9 in t(9;11)(p22;q23) and
ENL in t(11;19)(q23;p13.3), are homologous to proteins that are
themselves associated with the SWI/SNF complex.123 Fusion with the DNA-binding AT-hook domain of MLL might result in permanent tethering of a SWI/SNF-like complex to MLL targets, resulting in
constitutive activation of the SWI/SNF chromatin remodeling complex.
Another speculative model is that the fusions disrupt MLL-target
chromatin structure through recruitment of histone acetylase enzymes.
For example, ENL contains a transcriptional activation domain that is
retained in the MLL-ENL fusion. Through its activation motif, ENL might
tether an HAT-containing coactivator complex to MLL target genes.
Switching HATs.
Two other MLL-fusions potentially act through a similar
gain-of-function mechanism involving histone acetylase complexes: t(11;16)(q23;p13) and t(11;23)(q23; q13) fuse the upstream sequences of
MLL to CBP124-126 and
p300,127 respectively. Most of the domains of CBP
and p300 are retained, including those that bind other components of
the coactivator complex, as well as the catalytic domain that encodes
histone acetylase activity (Fig 4B). For these fusions it is likely
that abnormal chromatin remodeling occurs either though constitutive
activation of HAT activity or through recruitment of other
acetyltransferase components of the coactivator complex.
Histone acetylases are also mutated in the
t(8;16)(p11;p13)128 and inv(8)(p11;q13)48
chromosomal rearrangements associated with M4 and M5 AML. In the first,
CBP is fused to upstream elements of a gene called
MOZ.128 MOZ itself has predicted acetyltransferase activity, although its targets and biological function have not yet
been determined. MOZ contains a variant Zn-finger but has not been
shown to bind DNA. In the MOZ-CBP fusion the Zn-finger and catalytic
domain of MOZ are fused to almost the entire CBP protein. The
breakpoint in CBP is similar to that seen in the MLL-CBP fusion (see
above). MOZ-CBP therefore has 2 HAT activities.
Similarly, the inv(8) rearrangement fuses the upstream sequences of
MOZ to TIF2.48 TIF2 is a homologue of
p/CIP, a component of the CBP/p300 coactivator complex. The domain of
p/CIP that mediates direct interaction with nuclear hormone receptors
is lost in the MOZ-fusion, but the CBP-interaction domain is retained. TIF2 itself is predicted to have acetyltransferase activity, and, like
MOZ-CBP, MOZ-TIF2 retains the acetyltransferase homology domains from
each of the fusion partners.48 Like the MOZ-CBP rearrangement, such fusion proteins might result in altered chromatin remodeling of MOZ targets either through a dominant-negative effect, altered substrate specificity of the fusion enzyme, or recruitment of a
HAT-containing coactivator complex to MOZ targets. Any of these
mechanisms might impair normal chromatin remodeling, leading to
aberrant transcription. It is worth noting that the MOZ-CBP t(8;16) and MOZ-TIF2 inv(8) leukemias have virtually identical FAB M5 phenotypes,48,128 suggesting that the 2 fusions have a similar final common pathway.
| |
CHROMATIN THERAPY |
Excitement in the field of chromatin structure has been generated with
the realization that remodeling mechanisms might be targeted in
therapeutic strategies. Preliminary studies, mostly in the in vitro
setting, have focused on inhibition of histone deacetylase, in part
because HDAC1 was one of the first enzymes identified in nucleosomal
remodeling, because its function is best understood, and because it is
the only candidate for which specific inhibitors have been identified.
Butyric acid, or butyrate, a physiologic byproduct of colonic bacterial
fermentation, was the first identified of the HDAC inhibitors.129-131 It functions as a competitive inhibitor
of HDAC, perhaps by mimicking the normal substrate (butyrate is a
4-carbon molecule, whereas acetyl groups are 2-carbon). In micromolar
concentrations, butyrate is not specific for HDAC: it also inhibits
phosphorylation and methylation of nuclear proteins as well as DNA
methylation. Its analog phenylbutyrate132,133 acts in a
similar manner.
More specific for HDAC than butyrate or its analogs are trichostatin
A134,135 (TSA) and trapoxin136,137 (TPX). TSA,
a product of Streptomyces hygropicus, was originally isolated
as an antifungal agent. TPX, a cyclic tetrapeptide containing 2 L-phenylalanines, was identified in screens of fungal metabolites that
induced morphological reversion of transformed NIH3T3
cells.138 TPX and TSA have emerged as potent inhibitors of
the histone deacetylases. TSA reversibly inhibits, whereas TPX
irreversibly binds to and inactivates the HDAC enzyme. TSA inhibits
histone deacetylation with a Ki of 3.4 nmol/L,135 about 1,000-fold less than butyrate; TPX is even
more potent. Unlike butyrate and its analogs, nonspecific inhibition of
other enzyme systems has not yet been reported for TSA or TPX. To date,
no data are available on the pharmacokinetics or pharmacodynamics of
TSA or TPX. Besides TSA, TPX, and butyrate and its derivatives, a
number of hybrid polar compounds139,140 have been found to potently inhibit HDAC enzymes, such as suberoylanilide hydroxamic acid
and m-carboxycinnamic acid bishydroxamide. Tributyrin,141 a
triglyceride with butyrate molecules esterified at the 1, 2, or 3 positions, holds potential as a long-acting, orally administered prodrug.
A major issue concerning the use of such HDAC inhibitors is the
potential for modulating chromatin of genes that are not involved in
the leukemia or genes in nonleukemic bystander cells. As discussed above, the translocations that cause HDAC to function in inappropriate ways do not truly alter the enzyme itself, but rather bring the normal
enzyme into a chromatin environment that it would not otherwise contact. Would HDAC inhibitors have unwanted nonspecific effects that
disrupt chromatin of all genes in a cell? Although the explanations are
not yet apparent, experience in multiple tissue culture systems has
suggested that the effects of HDAC inhibitors are limited. Butyrate, at
doses that should inhibit HDAC enzymatic activity, does not kill
cells.129 However, the nonspecific effects of butyrate on
phosphorylation and methylation make it difficult to draw definite conclusions as to the contribution of HDAC inhibition to its biological effects. TSA has been used in a number of in vitro settings: it was
first shown to induce differentiation of MEL cells,142
without having significant apoptotic or transforming effects on the
cells. TSA inhibition of HDAC has been shown to increase acetylation of
only a subset of promoters,143 suggesting that a minority of genes are regulated through HDAC-dependent chromatin remodeling mechanisms. The observation of normal ordered differentiation in APL
model systems with a variety of HDAC inhibitors85-88
supports the notion that these agents do not have global effects on
chromatin structure and gene expression.
HDAC inhibitors in leukemia.
We have already alluded to the relationship between RA responsiveness,
the HDAC complex, and the RAR-fusion proteins in APL. APL has become
the paradigm for the application of HDAC inhibitors. As described
above, HDAC inhibitors synergize with retinoic acid to overcome the
PML-RAR and PLZF-RAR induced maturation blockade in cell
models.85-88 Pandolfi's group87 has shown
that, similar to patients with APL, PML-RAR transgenic mice
responded to RA treatment, whereas PLZF-RAR transgenic mice
developed RA-resistant leukemia. In vitro, neither RA nor TSA had
significant effects on the PLZF-RAR blasts, but together they
induced differentiation. The interpretation of this data is that RA
alone failed to release the N-CoR/Sin3/HDAC1 complex from PLZF-RAR;
that TSA acted downstream to inhibit the HDAC complex, but did not
induce recruitment of a coactivator complex; and that only in the
presence of both molecules could the suppressive effects of PLZF-RAR
be overcome. These studies are consistent with other reports that
suggest that HDAC inhibitors might have a role in APL
therapy.85-88 The recent report of blast-cell
differentiation and successful remission induction in a patient with
PLZF-RAR treated with a combination of ATRA and granulocyte
colony-stimulating factor (G-CSF)144 raises speculation as
to whether G-CSF might alter HDAC activity.
Besides PLZF-RAR blasts, some PML-RAR-expressing APL cells do
not respond to RA alone but may differentiate in response to the
combination of RA and an HDAC inhibitor. Several patient samples have
been identified to have mutations in the C-terminal region of
PML-RAR, near the N-CoR binding domain.145 Warrell's group at Memorial Sloan-Kettering has attempted to capitalize on such
in vitro observations, reporting the use of the HDAC inhibitor phenylbutyrate in a PML-RAR patient who was in her third
relapse.146 She had previously been induced with ATRA and
chemotherapy, treated in first remission with ATRA and allogeneic bone
marrow transplantation, and treated in second relapse with arsenic
trioxide after failing reinduction with ATRA or standard chemotherapy.
After 11 days on ATRA at 45 mg/m2/d, no change was observed
in her bone marrow. At that time, phenylbutyrate was added to her
regimen at a dose of 150 mg/kg/d. Immunofluorescence and Western blot
analysis of blood and bone marrow mononuclear cells documented a
time-dependent increase in histone acetylation (presumably as a result
of antagonization of HDAC). Over the next 3 weeks, the doses of both
ATRA and phenylbutyrate were increased to 90 mg/m2/d and
210 mg/kg, respectively. A bone marrow performed 2 weeks after the
addition of the phenylbutyrate showed a decrease in the percentage of
leukemic cells from 23% to 9%. A bone marrow 10 days later showed
elimination of leukemic blasts. After a second course of therapy, she
achieved a molecular remission (PML-RAR negative) and continued to
be in remission after 6 months.
Aside from APL, HDAC inhibitors have a potential role in the treatment
of AML1-ETO AML (see above). We and others have proposed that
AML1-ETO's leukemic potential is mediated by the recruitment of an
HDAC-containing complex to AML1-responsive promoters.36-38 In our own experiments,147 we found that TSA or
phenylbutyrate was able to partially reverse transcriptional repression
mediated by the ETO moiety of AML1-ETO. In vitro treatment of an
AML1-ETO cell line (Kasumi-1) with clinically attainable levels of
phenylbutyrate induced partial differentiation and apoptosis. Such
results may herald the therapeutic application of HDAC inhibitors for
t(8;21) AML in the future.
The clinical use of HDAC inhibitors need not be limited to patients
with obvious abnormalities in histone deacetylation pathways. In 1983, continuous infusion of butyrate, at a dose of 500 mg/kg/d, was reported
to induce a partial remission in a patient with myelomonocytic leukemia.148 No chromosomal abnormalities were noted in
this case. It is possible that this patient did have an unrecognized rearrangement that would have aberrantly recruited a histone
deacetylase to an abnormal target or that inhibition of HDAC may have
compensated for an underactive HAT. Alternatively, the effects might
not have been related to fusion gene-specific effects. For example,
phenylbutyrate has been reported to upregulate the B7 costimulatory
molecule on AML blasts,149 suggesting a role in immune surveillance.
These exciting results with HDAC inhibitors invigorate the search for
other means of modulating chromatin remodeling. Despite the current
dearth of specific inhibitors of HAT and SWI/SNF enzymes, the
development of inhibitory peptides is a potential avenue that has yet
to be pursued. Thanks to insights gained from studies of the molecular
biology of leukemia, chromatin therapy may emerge as a potent
antileukemia strategy of the future.
| |
ACKNOWLEDGMENT |
The authors thank Daniel E. Johnson, Margaret V. Ragni, and Richard A. Steinman for critical reading of the manuscript and Neal Young for
encouragement and support.
| |
FOOTNOTES |
Submitted February 4, 1999; accepted April 19, 1999.
Supported by National Institutes of Health Grant No. CA67346 and
American Institute for Cancer Research Grant No. 98B039 to R.L.R.
Address reprint requests to Robert L. Redner, MD, E1058 Biomedical
Science Tower, University of Pittsburgh Medical Center, 211 Lothrop St,
Pittsburgh, PA 15213; e-mail: redner+{at}pitt.edu.
| |
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INTRODUCTION
CHROMATIN: BEADS ON A...