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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3167-3215
REVIEW ARTICLE
Deconstructing a Disease: RAR , Its Fusion Partners, and Their
Roles in the Pathogenesis of Acute Promyelocytic Leukemia
By
Ari Melnick and
Jonathan D. Licht
From the Derald H. Ruttenberg Cancer Center and Department of
Medicine, Mount Sinai School of Medicine, New York, NY.
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INTRODUCTION |
IN THE LATE 1980s and early 1990s, the
elucidation of the molecular basis of acute promyelocytic leukemia
(APL) emerged as a paradigm for the connection between the bench and
bedside. At that time, it became apparent that APL was, among the forms of acute myeloid leukemia, uniquely sensitive to all-trans
retinoic acid (ATRA)1,2 and clinical trials indicated
that ATRA induced complete remissions by differentiation and eventual
elimination of the malignant clone (reviewed
previously3-8). In 1991, it was discovered that the
consistent chromosomal translocation of APL, t(15:17),9
fused the retinoic acid receptor  (RAR) gene to the promyelocytic
leukemia (PML) gene on chromosome 15, yielding the fusion protein
PML-RAR.10-15 These data suggested that disruption of
RAR function was the major cause of APL. According to this line of
reasoning, retinoic acid in pharmacological doses could then overcome
this pathology, leading to in vivo differentiation and clinical
remission. Although this hypothesis is essentially correct, 7 years of
intense investigation of the APL model have begun to uncover a more
complicated picture.
APL is now associated with four different gene rearrangements, fusing
RAR to the PML, promyelocytic leukemia zinc finger (PLZF),
nucleophosmin (NPM), or nuclear matrix associated (NuMA) genes
(Fig 1), leading to the formation of reciprocal
fusion proteins (N-RAR and RAR-N). This again highlights the
importance of retinoid metabolism, but also suggests that partner genes
with RAR could also play important roles. In this review, we will
deconstruct the APL problem by evaluating the role of RAR in normal
and neoplastic myeloid development. We will examine each of the genes
fused to the RAR in APL, searching for similarities and differences
among the four partner proteins that may explain the distinct clinical outcome some patients with variant forms of APL. Finally, we will reconstruct the disease of APL and examine the leukemogenic functions of the RAR fusion proteins in cell culture models, animal models, and patients. We will also examine how the recent explosion of knowledge in APL has led to the development of new therapeutic agents
such as arsenic trioxide16,17 and sodium butyrate.
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| Fig 1.
The four chromosomal translocations associated with APL
result in fusion proteins in which the B through F domains of RAR ,
including the DNA binding and ligand binding domains of protein, are
linked C-terminal to four different nuclear proteins containing
self-association domains. The t(11;17) APL syndrome linking PLZF and
RAR is unique among these forms of APL in its resistance to
differentiation therapy with ATRA or conventional chemotherapy.
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THE RETINOIC ACID RECEPTOR |
Retinoids may be key for myeloid differentiation. Vitamin A-deficient
mice and humans were noted to have defects in
hematopoiesis18,19 and retinoids can preferentially
stimulate granulopoiesis.20,21 In the early 1980s, it was
noted that retinoic acid (ATRA) could induce differentiation of myeloid
cell lines such as HL6022 and of primary cells from
patients with APL.23 The cloning of the RARs and other
members of the nuclear receptor superfamily24-26 allowed
for further detailed studies into the mechanism of action of ATRA.
Among the genes encoding RARs (reviewed previously24-26), RAR is identified with myeloid development.27-29
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TRANSCRIPTIONAL FUNCTION OF RAR |
RAR structure parallels that of other nuclear receptors and is
divided into 6 evolutionarily conserved domains (A through F; Fig 2).
The most highly conserved domain among nuclear receptors and retinoid
receptors is the C domain, which contains two
C2C2 zinc finger motifs (reviewed in
Chambon30). Through this domain, RAR binds to retinoic
acid response elements (RARE) located in the promoters of many genes,
including those of RAR,31,32 RAR ,33,34
and RAR .35 RAREs consist of a direct repeat
(A/G)G(G/T)TCA separated by 2 or 5 nucleotides. RAR binds as a
heterodimer to this site along with the related retinoid X receptor
protein (RXR).36-38 Heterodimerization is mediated both by
the DNA binding and ligand binding domain of RAR.39,40
RAR is a ligand-dependent transcription factor stimulated by ATRA,
whereas its partner, RXR, responds to ATRA or 9-cis retinoic
acid.41 RAR and other nuclear receptors contain two
domains, AF-1 (A/B domains) and AF2 (E domain), which can cooperate to
activate transcription.42 AF-1, contained within the
N-terminal A/B domain, is a ligand-independent transcriptional activation domain that works in a promoter context-dependent
manner.42,43 Through alternative promoter usage, the RAR
protein can have two different A domains (A1 or A2). The C-terminal E
domain of RAR contains the AF2 ligand-dependent transcriptional
activation domain as well as a dimerization interface for
RXR.39,40,43
RARs modulate transcription through interaction with cofactors. The
AF-2 domain of the protein associates with corepressor molecules in the
absence of ligand. These corepressors, N-CoR and SMRT,44,45
were recently shown to be part of a multiprotein repressor complex also
containing the Sin3A corepressor and histone deacetylases (Pazin and
Kadonaga46 and references therein). This suggests that RARs
may silence certain promoters by alterations in chromatin
configuration. Structural studies of RXR and RAR indicate that,
in the presence of ligand, the AF2 changes its conformation, making new
residues available30,47-49 to bind to coactivator proteins.
Nuclear receptor coactivators include TIF150,51 related to
the PML protein associated with t(15;17)-associated APL (see below),
Trip1/sug1,52 Tif2,53 ACTR,54
Src-1,55 TAFII135,56 and
CBP.57-59 The functions of these coactivators are beginning
to be elucidated. TIF150 interacts with the TATA binding
protein (TBP), TBP-associated factors (TAFs),60 and the
basal factor TFIIE.61 Murine and yeast Trip1/sug1 have DNA helicase activity,62 which could unwind DNA, whereas the
CBP and ACTR cofactors have histone acetylase activity and associate with P/CAF, another histone acetylase.54,63-66 It is
believed that histone acetylation leads to alterations in the
conformation of chromatin and stimulation of gene
transcription.67 Hence, the ligand-activated RAR can
best be imagined as a multiprotein complex bound to DNA in association
with RXR and a number of coactivator proteins. The ligand bound complex
might then stimulate transcription though interaction with basal
factors, alteration of chromatin, and unwinding of DNA.
The use of synthetic ligands specific for RAR and RXR indicate that
RAR/RXR complexes that stimulate gene transcription are responsible for
the pro-differentiation effect of ATRA,68-71 whereas RXR/RXR complexes cannot induce differentiation of APL cells. RARs can
also repress transcription through cross-talk with other transcriptional activators, including the AP1 family of activator proteins,72 probably due to competition for limiting
coactivators such as CBP.73-75 However, the most important
action of RAR in myeloid differentiation is its ability to activate
transcription through RAREs, because artificial ligands that inhibit
AP1 activity but fail to stimulate RARE-mediated transcription fail to
induce myeloid differentiation.68,76 Which key genes are
stimulated to affect myeloid differentiation remain to be determined.
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RAR AND MYELOID DIFFERENTIATION |
The importance of RAR in myeloid differentiation was underscored
when Collins et al77-79 developed a HL60 cell line
resistant to differentiation by ATRA. This cell line harbored a
dominant negative mutant RAR with a truncation within the C-terminal
AF-2 domain. Differentiation of these cells under the influence of ATRA
was restored by infection with a retrovirus expressing wild-type RAR, RAR, or RAR.77,80 Furthermore, RXR
expression in the resistant cells restored myeloid differentiation,
suggesting that the mutant receptor may have acted in a dominant
negative mode by heterodimerizing with wild-type RXR and forming an
inactive transcriptional complex. Overexpression of RXR overcame this
block, perhaps by recruiting other isoforms of RAR to mediate the
transcriptional response required for differentiation.
RAR may help program normal hematopoietic development. Erythroid
induction of multipotent FDCP mixA4 cells by erythropoietin was
correlated with complete downregulation of RAR expression, whereas
myeloid differentiation induced by granulocyte colony-stimulating factor (G-CSF) was correlated with upregulation of RAR, particularly the RAR2 isoform.81 Introduction of an RAR mutant,
with a deletion in the ligand-binding domain, into a multipotential
hematopoietic cell line resulted in a switch in cell fate from the
granulocyte/monocyte to the mast cell lineage.82
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated
myeloid differentiation of these cells was blocked at the promyelocyte
stage, an effect that could be overcome by high doses of
ATRA.83 Although truncation of the RAR within the ligand
binding domain has a profound effect on myeloid differentiation, this
type of mutation was not identified in a series of 118 specimens of
human cancer, including a number of fresh APL specimens.84
In leukemia, the RAR gene is only disrupted by the formation of
chromsomal translocations yielding fusion genes (see below). The notion
that the dominant negative RAR functions by sequestration of RXR
into inactive complexes was supported by the finding that
overexpression of wild-type RAR in murine bone marrow
cultures85 led to the accumulation of promyelocytic
colonies. Upon addition of ATRA, the RAR-expressing marrow colonies
consisted mainly of more differentiated granulocytes. Hence,
overexpression of wild-type RAR, C-terminal truncated forms of
RAR and fusion proteins consisting of partners fused to the
N-terminus of RAR (eg, PML-RAR; see below) can all lead to the
blockade of myeloid differentiation at the promyelocyte stage when
cells are grown at physiological levels (~10 8
mol/L) of ATRA. Only pharmacological levels of ATRA
(107 to 106 mol/L) can overcome
this block. How might this blockade occur at the molecular level? The
fact that wild-type RAR as well as mutant forms of RAR can cause
the block suggests a squelching mechanism.86 At low ATRA
concentrations, coactivators may bind loosely to nuclear receptor/DNA
complexes and be easily sequestered by high levels of free normal or
aberrant receptor in the nucleoplasm. Only at pharmacological ATRA
concentrations would the cofactors be drawn to target genes along with
RAR and the basal transcriptional machinery. In support of this
notion, in vivo footprinting of the RAR promoter shows occupancy of
the RARE only under pharmacological ATRA concentrations.87
It might be predicted that forced expression of RXR and/or RAR
coactivators would rescue the block by dominant negative RAR and
allow differentiation to proceed at physiological ATRA concentrations.
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RAR TARGET GENES |
ATRA treatment of myeloid precursor cells and other cells drives the
expression of multiple classes of genes
(Table 1) expressed either immediately
after ATRA treatment or after some delay. ATRA-induced changes in
myeloid gene expression are accompanied by inhibition of cell growth
and induction of terminal differentiation and production of a mature
cell ready to fight infection. The retinoic acid syndrome encountered
during treatment of APL with ATRA, characterized by an increase in
leukocyte count, fever, and pulmonary infiltrates, may be due to the
increased adhesive characteristics of the differentiating granulocytes
and secretion of cytokines.88 ATRA also downregulates the
expression of procoagulants found in the undifferentiated promyleocyte,
explaining the ability of differentiation therapy to treat APL without
inducing hemostatic disorders (reviewed in Barbui et al89).
The initial waves of leukocytes found in APL patients are derived from
the malignant clone90 and function normally in vitro to
kill pathogens,91 despite some abnormalities of secondary
granules involved in immune function.92,93
Many of the target genes of RAR that induce rapidly after ATRA are
themselves transcription factors.38 There are RAREs in the
promoters of the RARs (see above) that may help explain the ability of
ATRA to induce differentiation in APL. ATRA treatment of fresh APL
cells upregulates the mRNA for RAR, correlating with the presence of
a RARE in the second promoter of the RAR gene.32,94
Therefore, one way that ATRA may induce myeloid differentiation may be
to upregulate the RAR/RXR complexes, overcoming the dominant negative
PML-RAR protein. This hypothesis is supported by recent data that
indicate that PML-RAR is selectively degraded in APL cells by
treatment with ATRA,95,96 further shifting the balance
towards the wild-type receptor. Other candidates for directly
regulation by the retinoid receptors during myeloid development include
members of the hox family of homeobox-containing transcription factors (reviewed previously97,98). Hox genes are expressed in myeloid cell lines in a coordinated, dynamic manner.99
Enforced expression of the hox genes100-103 or disruption
of their expression104 as in leukemia-associated
translocations105,106 is associated with altered myeloid
growth and differentiation.107 RAREs were identified in the
promoters and enhancers of hoxb1108-110
hoxa1,111,112 and hoxd4.113
Furthermore, specific RARs differentially regulate the homeobox genes.
In embryonic carcinoma cells null for RAR, ATRA fails to induce the
hoxb1,38,114 whereas RAR null cells fail to
express hoxa1. This information suggests that in APL the disruption of RAR function may alter expression of a subset of ATRA-inducible genes critical for myeloid differentiation.
Recent data indicate an interplay between retinoic acid and interferon
(IFN)-mediated signaling. ATRA can rapidly induce transcription of the
IFN regulatory factor-1 (IRF-1) gene. IRF-1 expression is associated
with the expression of IFN and IFN-inducible genes,115 cessation of cellular growth, and induction of apoptosis. Thus, IFN may
potentially mediate some of the antiproliferative effects of
ATRA.116,117 ATRA induction of the IRF-1 promoter is
mediated by a GAS (-IFN activation sequence) rather than an
RARE,118 signifying a role for retinoic acid in the STAT
(signal transducer and activator of transcription) pathway for IFN
signaling.119 ATRA rapidly induces the expression of
STAT1 at the mRNA level and increases tyrosine phosphorylation of
STAT1, together leading to a large increase in DNA binding activity
of the STAT1 complex to an IFN-responsive element
(IRE).120 RAR and STAT1 synergized to stimulate
transcription from an IRE-containing reporter plasmid, whereas
PML-RAR did not, suggesting that cross-talk between the two
signaling pathways may be aberrant in APL and play a role in disease pathogenesis.
Recently C/EBP- , a newly identified basic-zipper transcription
factor that recognizes CCAAT DNA sequences, was found to be rapidly
upregulated during ATRA-mediated differentiation. C/EBP- is the only
member of this family of transcription factors expressed in the APL
cell line NB4,121 suggesting that the gene may play a role
in the promyelocyte stage of differentiation.122-125 HL60 cells engineered to express PML-RAR downregulate C/EBP-
expression in the absence of retinoic acid; the C/EBP- gene is then
upregulated when pharmacological doses of ATRA are added to the
cell.126 Hence, C/EBP- may be a model target gene of the
PML-RAR fusion protein that is inhibited in expression at ambient
physiological concentrations of ATRA and stimulated in expression when
cells are treated with ATRA.
ATRA is known to alter cell cycle kinetics, because it induces the
differentiation of APL and other myeloid cells.127,128 ATRA
treatment is associated with G1 arrest and the accumulation of
hypophosphorylated forms of the retinoblastoma protein.129 ATRA induces the expression of the p21WAF1/Cip1
cyclin-dependent kinase inhibitor in myeloid cell
lines.130,131 ATRA-mediated p21 induction may also depend
on the PML protein disrupted in t(15;17) APL.132 RAR in
combination with RXR binds to an imperfect RARE within the human p21
promoter and RAR can activate the p21 promoter in a ligand-dependent
manner. Therefore, p21 meets criteria for a bona fide RAR target
gene whose expression could decrease leukemic cell proliferation.
Potentially relevant to the treatment of APL, the cytosolic retinoic
acid binding protein II (CRABPII) promoter contains an RARE and can be
transcriptionally induced by ATRA.133,134 CRABPII may
sequester ATRA, contributing to therapy resistance.135
Tissue glutaminase II, which plays a role in differentiation and
apoptosis, is induced rapidly by ATRA136 and contains
functional RAREs in its promoter.137,138
Identification of further direct target genes of RAR relevant to
normal and malignant myelopoiesis has accelerated, using techniques
such as subtractive cloning,139,140 differential
screening,141 differential display (DD),142,143
and representative difference analysis (RDA).144 These
studies focused on genes induced 24 hours after ATRA treatment and
tended to identify indirect targets of ATRA action. These included the
IFN-inducible RIG-G145 and the related RI58
gene,146 RIG-E, encoding a GPI-linked cell surface molecule,147 and Jem1,148 a basic/leucine
zipper transcription factor gene. In contrast, using RDA, a
calcium/calmodulin kinase was isolated from ATRA-treated murine
promyelocyte MPRO cells harboring a dominant negative RAR molecule.
This gene was activated within a few hours after ATRA treatment in a
cycloheximide-resistant fashion and therefore is a reasonable candidate
as a direct target of RAR.149 Another report also using
the MPRO cell line isolated, by subtractive hybridization, several
genes rapidly induced by ATRA treatment.150 One transcript
induced threefold by ATRA treatment was identical to that encoding
LAPTM5, a lysosomal protein expressed preferentially in hematopoietic
cell lines.151 The LAPTM5 promoter contained RAREs, and an
LAPTM5-luciferase reporter gene was inducible by retinoic acid. The
importance of this gene in differentiation is unknown. Current efforts
towards the identification of ATRA target genes are using microarrays
of immobilized cDNAs152,153 or
oligonucleotides,154 which can monitor expression of
thousands of gene simultaneously.155,156
Table 2 summarizes some of the salient
points regarding myeloid biology, RARs, and APL.
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PML |
PML structure.
The t(15;17) rearrangement affecting the PML gene on chromosome 15q22
is the molecular basis for approximately 98% of all cases of
APL.5 The PML gene locus spans 35 kb and contains 9 exons
encoding mRNAs of 4.6, 3.0, and 2.1 kb. Alternative splicing of
C-terminal exons yields up to 20 different isoforms of the protein5; however, most cell lines express a similar
pattern of isoforms.11-13,157-159 The longest cDNA open
reading frame encodes a 560 aa polypeptide with a predicted molecular
weight (MW) of 70 kD.10,159
The PML protein has a modular structure with several domains
(Fig 3). These include the
following:
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| Fig 3.
Functional domains of the PML protein and structure of
the PML-RAR and reciprocal RAR-PML proteins generated in
t(15;17)-APL. In all patients, the RING finger, B boxes, and at least
the first two coiled coil domains of PML are included in the fusion
protein. Heterogeneity in the breakpoint within the PML gene leads to a
long and short form of PML-RAR depicted, as well as the rarer
intermediate form (not shown). The RAR-PML fusion is detected in the
majority of cases, but no evident function can be ascertained from its
structure.
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| Fig 4.
2D-NMR structure of the PML RING finger.166
Spheres representing Zn2+ ions coordinately bind to
histidine and cysteine residues allowing loops of protein to extend in
a spherical configuration (courtesy of K. Borden).
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(1) A cystein-rich region (aa 57-222) composed of three zinc-finger
like structures. The first is a RING (Really Interesting New Gene)
finger,160 a Zn2+ binding motif with the
configuration C3HC4 (aa 57-91). The following two are called B box zinc fingers (aa 140-161 and aa
189-222).12,13,157,159 The RING finger motif161
is found in more than 80 proteins involved in oncogenesis, regulation
of gene expression, mRNA processing, and DNA recombination and repair
(reviewed previously160-162). RING finger/B Box proteins
often are linked to a coiled-coil domain and comprise a subfamily of
RING proteins (RBCC for RING-B Box-Coiled-Coil).
The RING finger/B-box region is involved in localization of PML into
distinct nuclear domains known as nuclear bodies (NB), presumably
through interactions with protein partners (see
below).5,163,164 Biophysical studies, including 2-D NMR,
showed that the RING finger structure is spherically organized around
several loops of protein extending from two Zn2+ ions that
coordinately bind cysteine and histidine residues (Fig 4). This
positively charged structure precludes DNA binding, implying that the
RING domain is required for protein-protein
interactions.165,166 PML with mutations in critical RING
finger cysteine residues loses its characteristic nuclear body
localization and its biological activity as a growth suppressor (see
below),164,166-168 but other charged residues on the
surface of the globular RING structure may also affect NB
formation.169 The B box domains also bind zinc ions and any
mutation of the B-box cysteine residues disrupts NB
formation.164 Neither the RING finger nor the B-box motifs are required for PML to self-associate, suggesting that interactions with other proteins, through the Cys-rich motifs guide PML into the
multi-protein complex of the nuclear body (see
below).163,166,170
(2) A helical coiled-coil region (aa 229-360) consisting of eight
heptad repeats with hydrophobic amino acids at the first and fourth
positions. This region is responsible for multimerization of PML and
heterodimerization with PML-RAR and plays a role in NB localization.
This region also interacts with partner proteins and is required for
the growth suppression activity of PML as well as the ability of
PML-RAR to block differentiation.163,167,170,171 All PML
isoforms contain the RING Finger/B Box domains as well as at least the
N-terminal coiled-coil motifs.
(3) An N-terminal proline-rich sequence (aa 1-46) that can bind the
Arenavirus Z proteins involved in viral genome synthesis172 but is not required for growth suppression by PML.167
(4) A basic sequence, containing a nuclear localization signal (aa
476-490)167,170 required for the biological activity of the
protein.167 However, exact nuclear localization in NBs also requires the RING finger/B box and coiled-coil motifs.
(5) An acidic C-terminal Ser/Pro-rich domain of unknown
function, highly variable in length due to alternative splicing and rich in potential phosphorylation sites.11-13,157,159,167
The PML protein.
PML, when expressed after transfection into cells, is detected as a
series of 90- to 100-kD protein bands as well as a set of bands in the
70 kD range, as predicted from the amino acid sequence.173-175 Endogenous PML is detected as a 90-kD
species along with a variety of other protein species (150-50 kD) due
to alternative splicing and covalent
modifications.174,176-180 The PML sequence contains
potential casein kinase II and proline-directed kinase sites, and
32P-labeling studies demonstrated that PML is
phosphorylated on serine and to a lesser extent on tyrosine
residues.5,170,174,181 Some of the sites are constitutively
phosphorylated and others may be cell cycle dependent.182
In this regard, PML was found to be a substrate for phosphorylation by
Cyclin A/cdk2174,182 in vitro.
PML expression.
PML mRNA was widely expressed in all cell lines
tested.5,183 The pattern of PML protein expression in
tissues is complex and controversial, possibly due to differences in
techniques and antibodies used for immunodetection. In addition, PML
mRNA and protein expression are often not concordant, suggesting
posttranscriptional regulation. There are a few observations that
should be highlighted.
First, PML is highly expressed in inflammatory diseases such as
psoriasis and hepatitis, in inflammatory cells surrounding epithelial
cancers and Hodgkin's disease, in inflammatory lesions of
graft-versus-host disease, and in activated
fibroblasts.183-186 This suggests induction of PML
expression by soluble factors, probably IFNs (see below). Second, some,
but not all,185 groups found a correlation between the
level of PML expression and degree of dysplasia in atypical breast
hyperplasia and cervical intraepithelial neoplasia cells.
Interestingly, when the breast tumors became invasive, PML expression
decreased again. |
TOP
INTRODUCTION
THE RETINOIC ACID RECEPTOR
AND MYELOID...