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Blood, 1 November 2000, Vol. 96, No. 9, pp. 3200-3208
NEOPLASIA
Altered ligand binding and transcriptional regulation by
mutations in the PML/RAR ligand-binding domain arising in retinoic
acid-resistant patients with acute promyelocytic leukemia
Sylvie Côté,
Dacheng Zhou,
Andrea Bianchini,
Clara Nervi,
Robert E. Gallagher, and
Wilson
H. Miller Jr
From the Lady Davis Institute for Medical Research, Sir
Mortimer B. Davis Jewish General Hospital, and the McGill University
Department of Oncology and Medicine, Montreal, Quebec, Canada;
Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, NY;
and Dipartimento di Istologia ed Embriologia Medica, Università
di Roma "La Sapienza," Rome, Italy.
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Abstract |
Acute promyelocytic leukemia (APL) is characterized by a specific
translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RAR .
Pharmacologic doses of retinoic acid (RA) induce differentiation in
human APL cells and complete clinical remissions. Unfortunately, APL
cells develop resistance to RA in vitro and in vivo. Recently,
mutations in PML/RAR have been described in APL cells
from patients clinically resistant to RA therapy. The mutations cluster
in 2 regions that are involved in forming the binding pocket for RA.
These mutant PML/RAR proteins have been expressed in vitro,
which shows that they cause a diversity of alterations in binding to
ligand and to nuclear coregulators of transcription, leading to varying
degrees of inhibition of retinoid-induced transcription. This contrasts
with the nearly complete dominant negative activity of mutations in
PML/RAR previously characterized in cell lines developing RA
resistance in vitro. Current data from this study provide additional
insight into the molecular mechanisms of resistance to RA and suggest
that alterations in the ability of mutants to interact with
coregulators can be determinant in the molecular mechanism of
resistance to RA. In particular, ligand-induced binding to the
coactivator ACTR correlated better with transcriptional
activation of RA response elements than the ligand-induced release of
the corepressor SMRT. The diversity of effects that are seen in
patient-derived mutations may help explain the partial success to date
of attempts to overcome this mechanism of resistance in patients by the
clinical use of histone deacetylase inhibitors.
(Blood. 2000;96:3200-3208)
© 2000 by The American Society of Hematology.
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Introduction |
Acute promyelocytic leukemia (APL) accounts for
approximately 10% of all cases of acute myeloid leukemia. APL is a
unique subtype of leukemia characterized by a distinct chromosomal
translocation, t(15;17), with breakpoints within the retinoic acid
receptor alpha (RAR) gene on chromosome 17q21 and
a gene termed PML (promyelocytic leukemia), on
chromosome 15q22. This translocation generates a chimeric
PML/RAR gene and a PML/RAR fusion
protein.1-4 The resulting fusion protein is found in all
APL patients and contributes to the pathogenesis of the disease. The
oncogenic potential of PML/RAR derives from its ability to inhibit,
in a dominant negative fashion, both PML- and RAR-signaling
pathways. This inhibition of PML and RAR functions leads to a block
of myeloid differentiation and ultimately to the APL
phenotype.5,6
The fusion protein consistently retains both the DNA-binding C-domain
of the RAR and the E-domain, which is required for ligand binding
and receptor dimerization.7,8 When dimerized with a
retinoid X receptor (RXR), RARs bind to the retinoic acid response
elements (RAREs) on target genes and activate transcription in the
presence of all-trans retinoic acid (RA). In the absence of
ligand, RXR/RAR heterodimers interact with nuclear receptor corepressors (termed SMRT and N-CoR) that
recruit histone deacetylases to induce chromatin modifications
and transcriptional repression.9,10 In APL, the PML/RAR
fusion protein can heterodimerize with RXR, bind RA, and activate
transcription through the RAREs.11,12 All-trans
RA can dissociate SMRT from both RAR and PML/RAR, but higher
concentrations are required for the fusion protein, suggesting a more
tightly associated PML/RAR-SMRT complex. The differentiation block
by PML/RAR may be due to the association of the corepressor complex
and the inability to dissociate this complex with physiological
concentrations of RA.13 The release of corepressor induced
by pharmacological concentrations of RA may underlie the
cytodifferentiation of APL cells.14
RA therapy induces complete remission in a high percentage of
patients with APL.15 Unfortunately, the duration of
response is short, and further therapy with this agent is less
effective, suggesting the development of drug
resistance.16-19 Explanations for this resistance include
progressive reduction of RA plasma concentration, which may be
explained by an increased level of cellular RA-binding protein
(CRABP),20 an increased oxidative catabolism of RA by
cytochrome P450 enzyme activity, or multidrug-resistance (MDR) gene
product.21,22 But additional genetic mechanisms of
retinoid resistance, such as mutations in nuclear retinoid receptors,
have previously been found in HL-60 myeloid leukemic cells.23-25
In vitro studies on APL cells are provided by a cell line, NB4, derived
from an APL patient.26 Our laboratory, and others, have
developed RA-resistant NB4 subclones to study cellular or molecular
mechanisms that mediate retinoid response or
resistance.27-29 We have reported RA-resistant subclones
that are highly resistant to natural and synthetic retinoids that do
not bind CRABP and are not metabolized by P450
enzymes.29,30 We identified a point mutation in the
ligand-binding domain (LBD) of the RAR portion of the PML/RAR
fusion protein in the RA-resistant NB4 subclone, NB4-R4.31
Relative to the RAR sequence,32 the mutation is located
at the amino acid 398, where the leucine (L) is replaced by a proline
(P) (L398P) in helix 11 (H11) of the LBD. The mutant PML/RAR
does not bind ligand but retains the ability to bind RXR and RAREs
and to block the transcription of RA-responsive genes in a dominant
negative fashion. We found that pharmacologic concentrations of RA
could not release the corepressor SMRT from this PML/RAR mutant,
suggesting that the phenotypic RA resistance may be directly coupled to
the inability to dissociate the corepressor complex and activate
retinoid target genes.13 There have been 4 additional
mutations reported in independently developed RA-resistant subclones.33-36 On the basis of these findings,
mutations in the E-domain of the RAR portion of the fusion protein
PML/RAR appear to be an important mechanism of developing RA
resistance in vitro.
Recently, mutations in the RAR/E-domain of the
PML/RAR chimeric gene were reported in APL cells
from patients who developed RA resistance, confirming the clinical
relevance of molecular aberrations in the retinoid receptors. In 2 patients who exhibited RA resistance at relapse, Imaizumi et
al37 identified mutations in the
PML/RAR chimeric gene. Both mutations were absent
at the onset of the disease, suggesting that the alterations
were acquired during clinical therapy. The 2 mutations result
respectively, in amino acid changes from arginine (R) to glutamine (Q)
at codon 272, and from methionine (M) to leucine at 297, according to
the sequence of the RAR.32 The mutation R272Q is
located in the H5, and the other one, M297L, in the H6 of the LBD of
the fusion protein.
At the same time, Ding et al38 found
PML/RAR mutations in 3 patients of 12 who
received RA in combination with chemotherapy, but none of 8 who
received chemotherapy alone. The mutations identified in these patients
resulted in the replacement of leucine with valine (V) at codon 290, arginine with tryptophan (W) at codon 394, and methionine with
threonine (T) at codon 413, respectively. The L290V mutation lies
between H5 and H6 in the central region of the LBD, whereas the R394W
and the M413T mutations are located at the carboxy-terminal region of
RAR LBD within H11 and H12, respectively. Very recently, additional
PML/RAR mutations have been reported in RA-resistant APL
relapsed patients.39,40 None of these studies found
mutations in the coexpressed nonrearranged RAR or
PML genes.
The goal of this study was to characterize the phenotype of these
clinically observed mutations and determine how these mutations, found
in RA-resistant APL patients, could impair the ligand-induced and
transcriptional functions of the RAR/E-domain of the fusion protein.
In vitro expression of these mutant PML/RAR proteins shows that
these amino acid changes cause a variety of abnormalities in the
ligand-binding, transactivation of RAREs and ligand-dependent binding to the nuclear corepressor SMRT and coactivator ACTR. The results of our analysis provide additional insight into the molecular mechanism of resistance to RA and suggest that the
inability of mutants to interact with nuclear coregulators of
transcription, in a ligand-dependent manner, can be determinant in the
development of resistance to RA.
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Materials and methods |
Cell culture
The Cos-1 cells were grown in RPMI-1640 medium (Life
Technologies, Inc [GIBCO BRL], Burlington, Ontario, Canada),
supplemented with 10% fetal calf serum (FCS) (Wisent Inc; St-Bruno,
Quebec, Canada) and incubated in a humidified chamber at 37°C with a
5% CO2 environment.
Plasmid constructs
The PML/RAR with either R272Q or M297L mutations were
cloned into the pCMX mammalian expression vector harboring the
short form (S-form) of the wild-type PML/RAR
complementary DNA (cDNA) by means of a QuikChange Site-Directed
Mutagenesis kit (Stratagene; La Jolla, CA). The PML/RAR with either
L290V or R394W mutations was derived from pSG5 mammalian expression
vector harboring the wild-type PML/RAR S-form cDNA by
means of the Gene Editor kit (Promega, Madison, WI) for the
site-directed mutagenesis, whereas the PML/RAR with M413T
mutation was also cloned into the pSG5 expression vector but harboring
the long form (L-form) of the wild-type PML/RAR cDNA. All
the constructs were verified by sequencing analysis, by means of the
dsDNA Cycle Sequencing System (Life Technologies). The construction of
the mammalian expression vector for PML/RAR L-form harboring the
L398P mutation (PML/RAR-m4), from the NB4-R4 resistant cells, was
previously described.31
Assay for ligand-binding activity
The Cos-1 cells were transiently transfected by electroporation
with the expression vectors containing either wild-type or mutant
PML/RAR. Nuclear extracts were prepared from 1 to
5 × 108 cells and incubated for 18 hours at 4°C with
10 nmol/L [3H]-RA (50.7 Ci/mmol; DuPont-NEN, Boston) or
with [3H]-RA in the presence of 200-fold excess of
unlabeled RA, as previously described.11 The extracts were
subsequently fractionated at 4°C by HPLC by means of a
superose 6 HR 10/30 size exclusion column (Pharmacia, Uppsala,
Sweden). The flow rate was 0.4 mL/min; fractions of 0.4 mL
were collected; and radioactivity was determined by means of a liquid
scintillation counter. The HPLC system was calibrated by means of a
series of molecular weight (MW) markers, consisting of the following:
blue dextran, MW 2 000 000; thyroglobulin, MW 669 000;  -amylase,
MW 200 000; bovine serum albumin, MW 66 000; and ovalbumin,
MW 45 000.
Limited proteolytic digestion of translated fusion
proteins
Wild-type and mutant PML/RAR fusion proteins were synthesized
in vitro by means of a coupled transcription and translation reticulocyte lysate system for 90 minutes at 30°C as suggested by the
manufacturer (Promega). The reaction was performed in the presence of
[35S]-methionine (NEN; Streetsville, Ontario, Canada) to
produce radioactive fusion proteins. We incubated 5 µL of in
vitro synthesized wild-type or mutated PML/RAR proteins with 1 µmol/L of all-trans RA (Sigma, St Louis, MO) for up to 30 minutes at room temperature in the dark. After treatment with 0.5 µL
of different concentrations of trypsin (Sigma-Aldrich Canada Ltd;
Oakville, Ontario, Canada) for 10 minutes at room temperature, 20 µL
of denaturing loading dye was added. An equal volume of water
was added for the undigested controls or untreated samples. The samples
were denatured and directly analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (10% wt/vol). The gels
were then dried and exposed for autoradiographic analysis.
Transient transfection experiments for transcriptional
activity
Cos-1 cells were grown in RPMI-1640 with 10% FCS and were
seeded at 120 000 cells per well in 6-well plates 1 day before
transfection. Cells were rinsed with serum-free OPTI-MEM (Life
Technologies) and transfected by the lipofectamine method (Life
Technologies) with 0.7 µg of wild-type or mutant fusion protein
plasmids, 1 µg of the reporter plasmids DR5-tk-CAT41 or
TREpal-tk-CAT,41 and 0.3 µg of pCMV-Galactosidase
(Gal) as an internal control for transfection efficiency. Cells were
transfected for 5 hours, replenished with 2 mL of RPMI-1640 with 10%
FCS, and grown for 24 hours in the absence or presence of different
concentrations of RA. The chloramphenicol acetyltransferase (CAT)
activity was measured by means of a modified protocol of the organic
diffusion method.42 Briefly, 50 µL of cell extract was
incubated at 37°C for 1 to 5 hours with 200 µL of 1.25 mmol/L cold
chloramphenicol (ICN; Costa Mesa, CA) dissolved in 100 mmol/L Tris-Cl,
pH 7.8, and 0.25 µCi [3H]-labeled acetyl coenzyme A
(NEN). The reaction was extracted with Ready Organic Scintillation
Cocktail (Beckman; Mississauga, Ontario, Canada), and 750 µL of the
organic phase was counted on a scintillation counter. The CAT counts
were normalized with Gal activity43 to obtain the
relative CAT activity.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed with the
use of a direct-repeat 5 (DR5) RARE. The nucleotide sequence of the [32P]-labeled oligonucleotide duplex used was as
follows: 5'-agcttcAGGTCAccaggAGGTCAgagagct-3'. [35S]-labeled wild-type and mutant PML/RAR fusion
proteins were synthesized by means of a coupled transcription and
translation reticulocyte lysate system, according to the
manufacturer's recommended protocol (Promega). Equal counts of in
vitro translated wild-type and mutant fusion proteins, purified
glutathione S-transferase (GST) fusion proteins (1 µg), and
[32P]-labeled DR5 (100 000 cpm) were incubated with or
without RA, as indicated with the following: 0.3 µg poly (dI:dC) for
30 minutes at room temperature in a 24-µL reaction containing 100 mmol/L KCl; 6% glycerol; 10 mmol/L Tris, pH 8.0; 0.05% NP-40; and 1 mmol/L dithiothreitol. Where specified, bacterially expressed and
purified GST fusions containing the receptor interacting domains of
SMRT (GST-SMRT-IDII; amino acid 1073-1168; 1 µg/lane) or
ACTR (GST-ACTR-RID; amino acid 621-821; 1 µg/lane) were
added.13,44 The protein-DNA complexes were
resolved on a 4.5% native polyacrylamide gel electrophoresis in 0.5X
TBE and visualized by autoradiography.
| |
Results |
PML/RAR mutations analyzed in this study
Figure 1 presents the summary of the
LBD PML/RAR mutations associated with RA resistance in
cell lines (numbers 1-5) and in APL patients (numbers 6-11). In this
study, the phenotypes of the first PML/RAR natural
mutations identified in RA-resistant APL patients were characterized.
Transcriptional properties of the mutations, indicated by number 6, 8, 9, 10, and 11 in Figure 1A, were examined to discover the extent of
functional impairment. The locations of the mutations were described
with reference to normal protein of RAR1,32 because
their position in the fusion molecule depends on the isoforms of
PML/RAR messenger RNA. Approximate locations of the
evaluated mutations in the 3-dimensional structure of the RAR LBD are
shown in Figure 1B.
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| Figure 1.
Schematic representations of the PML/RAR mutations.
(A) Schematic of the LBD PML/RAR mutations identified in
RA-resistant APL cell lines (numbers 1-5) and relapsed patients
(numbers 6-11). The alignment of TR LBD and the PML/RAR E-domain
by sequence homology indicates that the mutations in RA-resistant APL
patients and cell lines cluster in accordance with the regions in
resistance to thyroid hormone (RTH) syndrome denoted as I, II, and III.
Numbers 6, 8, 9, 10, and 11 indicate the mutations in the LBD
PML/RAR of RA-resistant APL patients evaluated in this study. The
position of the mutations is described with reference to normal amino
acid sequence of RAR1.32 DBD indicates DNA-binding
domain; LBD, ligand-binding domain; DD, dimerization domain; AD,
activation domain. (B) A 3-dimensional model of the LBD of the
holo-RAR, showing the locations of the evaluated mutants. The
structure of the LBD is based on x-ray crystal structure analysis
of the liganded RAR .45-46
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RA-binding activity of the wild-type and mutant PML/RAR
fusion proteins
To evaluate how these point mutations of PML/RAR alter the
function of the LBD, we examined their RA-binding activity. Binding of
[3H]-RA to nuclear extracts from Cos-1 cells transiently
transfected with the wild-type or the mutant PML/RAR expression
vectors was analyzed. The size exclusion HPLC profile of extracts from
cells expressing the wild-type form of PML/RAR is characterized by 3 main peaks as previously described.11 The 50-kd
peak represents the endogenous RARs in Cos-1 cells. The 110-kd peak
characterizes the binding of PML/RAR monomers, and the approximately
670-kd peak represents macromolecular complexes formed by the
interaction of PML/RAR with itself and/or other nuclear proteins.
The HPLC profiles of extracts from cells expressing the PML/RAR
mutations R394W and M413T are similar to the pattern observed with the
wild-type form, indicating that these mutations in the LBD of
PML/RAR do not substantially impair the binding of ligand (Figure
2). Nuclear extract from cells expressing
the PML/RAR mutations M297L and R272Q showed HPLC profiles
consistent with the elution of PML/RAR macromolecular nuclear
complexes in fractions corresponding to MWs of about 400 to 200 kd. In
addition, the mutation R272Q of PML/RAR decreased RA binding to the
fusion protein. Specific RA-binding activity was not detectable in
nuclear extracts prepared from cells transiently transfected with the
PML/RAR containing the mutation L290V (Figure 2A).
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| Figure 2.
Specific nuclear RA-binding activity in Cos-1 cells
transfected with wild-type in comparison with those transfected with
mutant PML/RAR fusion proteins.
HPLC RA-binding profiles of wild-type (top panels) and mutant (bottom
panels) PML/RAR S-form (A) and L-form (B). Nuclear extracts were
incubated with 10 nmol/L [3H]-RA or with
[3H]-RA in the presence of 200-fold excess of unlabeled
RA. Extracts were subjected to HPLC analysis using a 6 HR 10/30 size
exclusion column.
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Proteolytic analysis of the wild-type and mutant
PML/RAR
We predicted that changes in the ligand binding of mutated
receptors would correlate with altered conformations of the receptors. Because proteolytic analysis is a powerful method for analyzing conformational changes within proteins, we performed a limited trypsin
digestion of the wild-type and mutated fusion proteins in the absence
and presence of RA (Figure 3). Distinct
fragment patterns were observed for the wild-type and for certain
mutant fusion proteins, mainly after RA treatment. The distinction
centered around a fragment at 32 kd and 2 closely migrating fragments
at 36 to 37 kd. The S- and L-forms of wild-type PML/RAR did not differ in digestion pattern (Figure 3). In the absence of RA, a 32-kd
fragment was more resistant to trypsin digestion. After 1 µmol/L RA
treatment, the 32-kd fragment disappeared, whereas 2 fragments of 36 to
37 kd were more resistant to protease treatment. As with human
RAR,47 human progesterone
receptor,47 and human estrogen
receptor,48 ligand binding causes a
conformational change of PML/RAR, resulting in the modification of
accessibility of trypsin cleavage sites. Analysis of the mutations
M297L and M413T showed that their trypsin digestion patterns are
identical to that of the wild-type. The digestion patterns for the
mutated R272Q and R394W PML/RAR differ from that of the wild-type by the presence of an intense 32-kd resistant fragment after RA treatment. These results indicate that RA can still bind to the mutated fusion protein LBD, but induces a different conformational change than in the
wild-type LBD. In the case of the mutation L290V, the trypsin digestion
pattern after RA treatment is completely different from that of the
wild-type: the 32-kd fragment is still present and the 36- to 37-kd
fragments are absent. The identical digestion patterns in the absence
and presence of RA indicate that the mutated fusion protein does not
bind the ligand, which is consistent with our ligand-binding analysis
(Figure 2A). The same results were obtained with the mutation found in
NB4-R4 cells, PML/RAR (m4), which we have previously shown is not
able to bind RA.31
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| Figure 3.
RA-induced conformational changes in wild-type and
mutant PML/RAR fusion proteins.
Limited trypsin digestion analysis of wild-type and mutant PML/RAR
S-form (A) and L-form (B). In vitro [35S]-methionine
synthesized PML/RAR proteins were incubated without ( ) or with (+)
1 µmol/L RA, and subsequently treated with increasing trypsin
concentrations (0 to 25 µg/mL). Digestion products were analyzed by
denaturing electrophoresis. The arrows indicate the intact PML/RAR
proteins. Asterisks indicate resistant fragments.
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Ligand-dependent transcriptional activity
The conformational changes induced by certain mutations might also
be expected to alter protein-protein interactions that are required for
RARE binding and activation. Thus, we compared the transcriptional
activity of PML/RAR mutants with that of the wild-type fusion
protein on 2 retinoid-responsive elements. Fusion protein plasmids were
cotransfected into Cos-1 cells with tk-CAT reporters driven by either a
DR5 RARE (Figure 4A) or a palindromic
thyroid response element TREpal (Figure 4B). Figure 4 showed that RA
slightly induced transcription of both DR5-tk-CAT and TREpal-tk-CAT
reporters cotransfected with the empty vectors (pSG5 and pCMX),
consistent with the known endogenous RAR present in Cos-1 cells. The
wild-type PML/RAR S-form and L-form acted as dominant negative
inhibitors of the control transcriptional activity for both DR5-tk-CAT
and TREpal-tk-CAT reporters (Figure 4), reducing the baseline CAT
activity as compared with the empty vectors. This dominant negative
activity of the fusion protein is maintained in all PML/RAR mutants,
as exhibited by a smaller baseline CAT activity than was seen with the
empty vectors. As shown in Figure 4, ligand-dependent transcriptional
activity of both S-form and L-form of the wild-type PML/RAR chimeric
proteins was increased in a manner dependent on RA concentrations. The PML/RAR L-form stimulated the RA-induced transcription more
efficiently than the S-form (Figure 4). The PML/RAR chimeric
proteins harboring either M297L or R394W mutations showed a
ligand-dependent transcriptional activity on both tk-CAT reporters that
was similar to the wild-type S-form fusion protein (Figure 4). In
contrast, the mutant R272Q and L290V fusion proteins had an altered
transcriptional activity on DR5 and TREpal reporters in the presence of
RA. The mutant R272Q showed no increase in CAT activity in the presence
of 0.01 µmol/L RA on the DR5 (Figure 4A) and on the TREpal
(Figure 4B). In the presence of 0.1 µmol/L RA, both reporters
produced increased transcriptional activity that remained less than
that of the wild-type PML/RAR. The mutated form, PML/RAR L290V,
has significantly lost ligand-dependent transcriptional activity on
both reporters (Figure 4). As previously reported, PML/RAR (m4) does
not activate transcription in response to even 1 µmol/L RA, on all
reporters tested (Figure 4 and data not shown). The PML/RAR fusion
protein harboring M413T mutation showed a loss of approximately half of the CAT activities on RARE reporters, as compared with the wild-type L-form PML/RAR, in the presence of RA (Figure 4). Similar results were obtained with the use of a DR5-tk-LUC reporter (data not shown).
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| Figure 4.
Transcriptional activity of wild-type and mutant
PML/RAR fusion proteins.
DR5-tk-CAT (A) or TREpal-tk-CAT (B) reporters were cotransfected with
the indicated PML/RAR. The pCMX and the pSG5 represent the vectors
alone. Relative CAT activity without (control) or with indicated
concentrations of RA is shown, with the calculated corresponding fold
induction below. Each data point represents results from at least 3 independent transfections. Control,  ; RA 0.01 µmol/L,  ; RA
0.1 µmol/L,  ; RA 1 µmol/L,  .
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Ligand-dependent corepressor SMRT release by wild-type and mutant
PML/RAR fusion proteins
Ligand-dependent activation of nuclear receptors is
associated with displacement of corepressors and recruitment of
coactivating proteins. To determine the molecular mechanism of RA
resistance caused by these mutants, the interactions of wild-type and
mutants with the corepressor, SMRT, and the coactivator, ACTR, were
tested in gel shift assays. We first evaluated the binding of mutants to the receptor interacting domain (IDII) of the corepressor
SMRT, using an electromobility shift assay. In vitro translated
wild-type and mutant PML/RAR bound a radio-labeled DR5 element in
the absence of SMRT-IDII. Addition of purified GST-SMRT-IDII shifted
the bound complex, as indicated by an arrow in Figure
5. Binding of GST-SMRT-IDII to each of
the mutants was similar to the wild-type PML/RAR in the absence of
RA. In contrast, ligand-induced dissociation of SMRT-IDII from certain
mutants was very different from the wild-type. As previously reported,
the wild-type PML/RAR fusion proteins completely dissociated
SMRT-IDII at RA concentrations between 10 6 and
105 mol/L (Figure 5). PML/RAR harboring the mutations
M297L or R394W required about 10-fold higher concentrations of RA to
dissociate the corepressor SMRT than did the wild-type (Figure 5A). The
fusion proteins harboring the mutations R272Q, L290V, M413T, or
PML/RAR (m4) could not be dissociated from the corepressor SMRT-IDII
even at 10 µmol/L RA concentration (Figure 5), indicating that these mutants required more than 100-fold higher concentrations of RA to
dissociate SMRT than did the wild-type.
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| Figure 5.
Ligand-dependent dissociation of the corepressor SMRT
from wild-type and mutant PML/RAR fusion proteins on a DR5 RARE in
gel mobility shift assay.
Interaction of SMRT with wild-type and mutant PML/RAR S-form (A) and
L-form (B). In vitro translated PML/RAR fusion proteins were
coincubated with the [32P]-labeled DR5 RARE, along with
bacterially expressed GST-SMRT-IDII in the presence of increasing
concentrations of RA. The position of the complex shifted by SMRT is
indicated.
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Recruitment of the coactivator ACTR by mutants
PML/RAR
In the presence of ligand, proteins termed coactivators are
recruited to mediate the ligand-dependent response. To test whether mutants of PML/RAR could normally recruit the coactivator ACTR, the
central receptor-interacting domain of ACTR (ACTR-RID) fused to the GST
protein was expressed in bacteria. Purified GST-ACTR-RID and in vitro
translated fusion proteins were then employed in a gel-shift study of
wild-type and mutated PML/RAR to a DR5 probe (Figure
6). In the absence of RA, ACTR-RID did
not form a complex with the wild-type or mutated fusion proteins. The
wild-type PML/RAR-ACTR-RID complex was first noted at 0.01 µmol/L
and was maximal at 0.05 µmol/L RA. The M297L mutant appeared to be
similar to the wild-type in its ability to recruit ACTR-RID over the RA
concentrations tested (Figure 6A). The mutant M413T required about
10-fold greater RA concentrations, relative to the wild-type, to begin
the recruitment of ACTR-RID (Figure 6B). The protein harboring the
mutation R394W started recruiting the coactivator at 0.01 µmol/L as
did the wild-type, but its association is maximal only at 1 µmol/L
RA, as compared with 0.05 µmol/L for the wild-type. The mutant R272Q
and L290V fusion proteins were least able to associate with the
coactivator. The mutation R272Q is fully associated with ACTR-RID only
at 1 µmol/L RA, which is 20-fold greater than for the wild-type
association. PML/RAR harboring the mutation L290V showed only
minimal recruitment of ACTR at 1 µmol/L RA, which indicates that the
coactivator needs more than 100-fold RA concentrations for its
association with this mutated fusion protein. The mutation PML/RAR
(m4) showed that even with 1 µmol/L RA, ACTR-RID was not able to bind
the fusion protein (Figure 6B).
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| Figure 6.
Differential recruitment of the coactivator ACTR by
wild-type and mutant PML/RAR fusion proteins on a DR5 RARE in gel
mobility shift assay.
Interaction of ACTR with wild-type and mutant PML/RAR S-form (A) and
L-form (B). In vitro translated PML/RAR fusion proteins were
coincubated with the [32P]-labeled DR5 RARE, along with
bacterially expressed GST-ACTR-RID in the presence of increasing
concentrations of RA. The position of the complex shifted by ACTR
indicated.
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Discussion |
APL has the unique characteristic of responding in vitro and in
vivo to differentiation therapy with RA. The chimeric protein PML/RAR retains the majority of the functional domains of the RAR gene, in particular the DNA- and the ligand-binding
domains, maintaining the capacity to bind the ligand and activate
transcription. RA used alone in APL treatment induces clinical
remission in a range of 72% to 100%.15 Unfortunately,
acquired clinical RA resistance appears within a few months of
treatment, and relapse occurs in almost all patients who do not receive
additional cytotoxic therapy. Therefore, the development of resistance
to RA in patients with acute promyelocytic leukemia is one of the major
problems that limit the effectiveness of therapy with this agent.
Different mechanisms have been proposed to explain this clinical
resistance. The decrease in RA plasma concentrations, as a result of
enhanced activity of P450,21,22 as well as up-regulation
of the CRABP20 and MDR22 gene
products, could be responsible for the decrease in RA sensitivity
observed in some resistant APL patients.
However, several groups have identified specific genetic lesions
associated with RA resistance, both in cell lines and in patients.
Several missense mutations in the LBD of the RAR moiety of the
fusion protein PML/RAR have now been reported, as shown in Figure 1.
The mutation that we found in the NB4-R4 resistant cell line, L398P,
abrogates the binding of the fusion protein PML/RAR to its ligand
and blocks the transcription of RA-responsive genes in a dominant
negative manner.31 In these cells, we found that RA could
not release the corepressor SMRT from the mutant PML/RAR (m4), even
with suprapharmacological concentrations,13 suggesting
that the phenotypic RA resistance may couple directly to the inability
to dissociate the corepressor complex and activate retinoid target
genes. Here, we report that the fusion protein is able to interact with
the coactivator ACTR only in the presence of the ligand, whereas the
mutant PML/RAR (m4) is unable to recruit the coactivator, even at the
highest tested concentrations of RA (Figure 6). This is consistent with
the total inhibition of transcriptional activity by PML/RAR (m4) on
different RARE reporters (Figure 4).
Recently, 2 independent groups identified the presence of the first
missense mutations in the LBD of the RAR moiety of the PML/RAR chimeric gene in 5 RA-resistant APL patients who
had received prolonged or intermittent administration of RA before relapse.37,38 In this study, we found that these
PML/RAR mutations caused a variety of alterations in the
known functions of the RAR LBD.
The L290V mutation is located in the -turn between H5 and H6, and
nearby residues (F286, S287) in this -turn are also involved in the
binding pocket for RA.45 Although this amino acid change from leucine to valine is the most conservative substitution that we
analyzed, the mutated PML/RAR fusion protein dramatically lost its
RA-binding capacity (Figure 2) and exhibited no change in the pattern
of resistant fragments in limited proteolytic digestion in the presence
of RA (Figure 3). Consistent with these results, the L290V mutation in
PML/RAR protein completely impaired the release of the corepressor
SMRT and abolished the ability of the mutant protein to recruit the
coactivator ACTR, even at high doses of RA (Figures 5 and 6). These
ligand-binding and protein-interaction studies are consistent with the
transient transfection analyses, which show that of the tested patient
mutations, this is the least responsive to transcriptional activation
by RA (Figure 4). We propose that amino acid L290, in the
-turn between H5 and H6, is probably directly implicated in the
formation of the RA binding pocket, and so even a conservative mutation
of this amino acid could disrupt the binding of the ligand. This L290V
mutation of PML/RAR identified in a RA-resistant patient is the most
similar, phenotypically, to the L398P mutation observed in NB4-R4 APL
resistant cells.
In contrast, other mutations observed in cells from resistant patients
had quite varying effects on RAR LBD functions. The mutation of the
amino acid 272, located in H5, changed a positively charged arginine to
a polar uncharged glutamine. This arginine is one of the 24 amino acids
directly implicated in the formation of the ligand binding
pocket, specifically by making van der Waals contact with the
carbon molecule of the acyl chain of RA.45 The replacement
of this arginine at position 272 by a glutamine considerably decreased
the capacity of the fusion protein PML/RAR to bind RA (Figure 2). In
accord with our results, the site-directed mutagenesis of R272 of the
nonrearranged RAR has been shown to impair the binding of
RA.49 The R272Q mutant also exhibited an impaired
responsiveness to low concentrations of ligand in transient
transfection assays (Figure 4). Nevertheless, the R272Q mutant
PML/RAR was able to activate transcription at a higher RA
concentration (107 mol/L), although still not as
efficiently as did the wild-type fusion protein. These aberrant
transcription properties of the R272Q mutant were associated with an
impaired ability to release the corepressor SMRT and to recruit the
coactivator ACTR: 10 µmol/L RA is not sufficient to release SMRT from
the fusion protein, while its association with ACTR does occur at high
levels of ligand.
Our results with the mutation R272Q of PML/RAR provide a link
between RA resistance in APL and mutations in thyroid receptor beta
(TR) associated with the syndrome of resistance to thyroid hormone
(RTH). RTH is a dominant negative syndrome characterized by reduced
tissue responsiveness to thyroid hormones caused by mutations in TR
that abolish the hormone binding. In a recent study, Privalsky and
Yoh50 showed that the V264D mutation of TR, located in
the omega loop between H1 and H3, exhibits a phenotype very similar to
the mutation R272Q in PML/RAR. The V264D TR mutant shows impaired
hormone binding and corepressor release, but retains the ability to
recruit coactivator and activate transcription at high hormone
concentrations. Privalsky and Yoh suggested that this substitution may
be able to recruit both corepressor and coactivator simultaneously and
that the regulatory properties of this receptor may therefore be a
combined manifestation of both corepressor and coactivator functions.
They further proposed that the V264D mutated protein serves as a bridge
to simultaneously tether the corepressor and the coactivator. These
data suggest the possibility that the R272Q mutant of PML/RAR might
be able to bind simultaneously to both SMRT and ACTR. Multiple sites
for coactivator and corepressor interaction have been mapped within the
RAR protein, particularly in H5 where the R272Q mutation is located,
so it may be possible, under certain circumstances, for SMRT and ACTR
to occupy the same receptor molecule. In this case, the predicted
result would be a composite transcriptional activity reflecting the
counteracting contributions of the corepressor and the coactivator, as
in the results we obtained. Additional analyses of these
mutations will be necessary to confirm these hypotheses.
An additional case, the substitution of the methionine at position 413 by threonine (M413T), suggests that loss of ligand-inducible coactivator binding does not always accompany loss of corepressor release. This methionine, located in H12 of the LBD is believed to
contribute directly to stabilization of the RA binding pocket through
the formation of hydrogen bonds or van der Waals contacts with the
bound RA molecule.45 Although this substitution is not
conservative, our analysis of ligand binding, as well as our limited
proteolytic-digestion assay, indicated that this mutation has only a
minor effect on the binding of RA for PML/RAR (Figures 2 and 3).
This effect on binding may be limited because contact of the H12 of the
receptor with RA occurs only after the ligand-induced conformational
flip of the H12 over the binding pocket. This is consistent with
studies showing that ligand is still able to bind the RAR receptor
in presence of mutations in this particular region of the molecule,
even though the mutations cause constitutive repression of gene
expression.45,51 Rather, the major dysfunction of this
mutation may be due to its central location in the AF-2 activation
domain, which mediates essential interactions with coregulators of
transcription.45-46,52-53 With this M413T mutation, ligand
binding does not induce dissociation of the corepressor, whereas the
ligand dependence of coactivator recruitment is minimally altered. As
with the R272Q mutation, even in the continued presence of significant
corepressor binding, the association with the coactivator correlated
with the considerable transcriptional activity (Figure 4). These
data suggest that an intact AF-2 domain is required for
corepressor release, but transactivation in vitro may better correlate
with coactivator recruitment than with corepressor release.
We also characterized 2 patient mutations in PML/RAR that exhibit
additional alterations associated with RA resistance. R394W substitutes
arginine for tryptophan in a region of H11 contributing to
stabilization of the RA binding pocket.45 Surprisingly,
our analysis of ligand binding showed that this nonconservative
substitution did not impair the RA binding to the fusion molecule
(Figure 2). However, our analysis of limited trypsin digestion of R394W
suggested that the conformation of the mutated PML/RAR after RA
treatment is different from that of the wild-type fusion protein
(Figure 3). This may explain the impairment of the ligand-dependent
release of SMRT and the recruitment of ACTR by the mutated protein
(Figures 5 and 6), since the final configuration of the H12 of the
holo-receptor determines the association of the
coregulators.45-46,52-53
Finally, we analyzed the substitution of the methionine at position 297 for a leucine (M297L), within H6, which presented almost the same
phenotype as that of the wild-type chimeric protein. These results
suggest that the methionine at 297 does not play a major role in the
ligand binding and in the interaction with transcriptional
coregulators. The nature of the conservative substitution may explain
the very minor effect of this mutation on the tested functional
properties of the fusion protein PML/RAR. Alternatively, the RA
resistance of this patient may be due to other mechanisms that our in
vitro assays of LBD function did not detect.
The relationship of different mutations to the severity of the clinical
phenotype of RA resistance in APL patients is difficult to establish,
in part because it was not possible to define differences in the degree
of clinical resistance among patients. A given mutation of the
PML/RAR may also have different phenotypic consequences in different
leukemic clones. The complexity of characterizing the role of mutations
in RA resistance is also due to the location of these genetic lesions
in regions of the receptor that play multiple roles, including ligand
binding, receptor dimerization, and interaction with the
transcriptional machinery. Indeed, the clustering of mutations within
these "hot spot" regions in the RA resistance disorder highlights
their functional importance.
The comparison of RA-resistant APL with the RTH syndrome may help us
understand the functional roles of these mutations. More than 70 different natural TR mutations have been identified in the RTH
syndrome. Rather than being randomly distributed, the mutations cluster
mainly in 3 areas, denoted by I, II, and III in Figure 1; these
correspond to the amino-terminus, the central region, and the
carboxy-terminus of the LBD, respectively.54-56 These
mutations in TR LBD inactivate the ligand binding of the receptor,
but retain the receptor-dimerization and DNA-binding functions and act
as dominant negative inhibitors on the wild-type receptor. More
importantly, a comparison of all PML/RAR mutations identified in APL
RA-resistant patients and cell lines highlights the clusters of
mutations identified as hot spots in the RTH syndrome, as shown in
Figure 1.
Since the discovery of these mutations, new mutations have been found
in the above hot spots of the LBD of the fusion protein PML/RAR in
additional RA-resistant APL relapsed patients.39,40 These
mutations are located between H1 and H3, within or between H5 and H6,
and within H11 and H12. The growing list of new mutations identified in
the LBD of the fusion protein is an indication of the importance of
this mechanism for the development of RA resistance in patients with
APL. The findings of naturally occurring point mutations in PML/RAR
of RA-resistant relapsed APL patients confirmed the results obtained in
the cellular models, indicating that stable genetic alterations in APL
cells could mediate RA resistance.
It is now clear that the acquisition of mutations in PML/RAR plays a
critical role for the development of RA resistance in patients with
APL. In this report, we demonstrated how mutations of PML/RAR could
impair the functions of the RAR/E-domain at a molecular level. We
showed that specific mutations of the LBD of PML/RAR led to altered
interactions with transcriptional coregulators that may be directly
involved in the molecular mechanism of RA resistance. We previously
reported that retinoid resistance of the NB4-R4 subclone was associated
with transcriptional repression by increased association of the mutated
PML/RAR with histone deacetylase (HDAC). In these cells,
pharmacologic inhibition of HDAC function could partially induce
RA-mediated transcription and phenotypic
differentiation.13 One patient with RA-refractory APL
received combined treatment with RA and HDAC inhibitor, leading to a
durable, complete remission.57 However, no other such
patients have been reported to date, nor have other acute leukemias
whose fusion oncoproteins may repress transcription by recruitment of HDACs yet responded to this therapy. The diversity of alterations observed here in patient-derived mutations suggests the hypothesis that
specific mutations of PML/RAR may vary in response to manipulation of HDAC activity. Further studies of additional mutations of PML/RAR from RA-resistant patients will provide a clearer picture of how structural features of the PML/RAR protein determine interactions with transcriptional coregulators and modulators of histone
acetylation. This molecular understanding may allow novel treatment
strategies for retinoid-resistant patients with APL, and possibly other leukemias.
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Acknowledgments |
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Top
Abstract
Introduction