|
|
Prepublished online as a Blood First Edition Paper on June 7, 2002; DOI 10.1182/blood-2002-02-0614.
 Previous Article | Table of Contents | Next Article 
Blood, 1 October 2002, Vol. 100, No. 7, pp. 2586-2596
NEOPLASIA
Response to histone deacetylase inhibition of novel PML/RAR
mutants detected in retinoic acid-resistant APL cells
Sylvie Côté,
Angelika Rosenauer,
Andrea Bianchini,
Karen Seiter,
Jonathan Vandewiele,
Clara Nervi, and
Wilson H. Miller Jr
From the Lady Davis Institute for Medical Research, Sir
Mortimer B. Davis Jewish General Hospital and McGill University
Department of Oncology and Medicine, Montreal, Quebec, Canada;
Dipartimento di Istologia ed Embriologia Medica, Università di
Roma "La Sapienza," Rome, Italy; and Department of
Medicine/Neoplastic Diseases, New York Medical College, Valhalla, NY.
 |
Abstract |
Resistance to all-trans retinoic acid (ATRA)
remains a clinical problem in the treatment of acute promyelocytic
leukemia (APL) and provides a model for the development of novel
therapies. Molecular alterations in the ligand-binding domain (LBD) of
the PML/RAR fusion gene that characterizes APL
constitute one mechanism of acquired resistance to ATRA. We identified
missense mutations in PML/RAR from an additional
ATRA-resistant patient at relapse and in a novel ATRA-resistant cell
line, NB4-MRA1. These cause altered binding to ligand and
transcriptional coregulators, leading to a dominant-negative block of
transcription. These mutations are in regions of the LBD that appear to
be mutational hot spots occurring repeatedly in ATRA-resistant APL
patient cells. We evaluated whether histone deacetylase (HDAC)
inhibition could overcome the effects of these mutations on
ATRA-induced gene expression. Cotreatment with ATRA and TSA restored
RAR gene expression in NB4-MRA1 cells, whose PML/RAR
mutation is in helix 12 of the LBD, but not in an APL cell line
harboring the patient-derived PML/RAR mutation, which was between
helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RAR promoter only in NB4-MRA1 cells.
Consistent with these results, the combined treatment induces
differentiation of NB4-MRA1 only. Thus, the ability of an HDAC
inhibitor to restore ATRA sensitivity in resistant cells may depend on
their specific molecular defects. The variety of PML/RAR
mutations arising in ATRA-resistant patients begins to explain how APL
patients in relapse may differ in response to transcription therapy
with HDAC inhibitors.
(Blood. 2002;100:2586-2596)
© 2002 by The American Society of Hematology.
| |
Introduction |
Acute promyelocytic leukemia (APL) is characterized
by a translocation, t(15;17), between the promyelocytic
leukemia (PML) gene on chromosome 15 and the
retinoic acid receptor alpha (RAR) gene on
chromosome 17. This translocation generates a PML/RAR fusion protein
crucial to the pathogenesis of APL.1-8 A dominant-negative effect of the chimeric protein leads to the inhibition of PML and
RAR pathways, a block of myeloid differentiation, and ultimately to
the APL phenotype.9-11
The actions of retinoids are mediated by heterodimers of 2 classes of
ligand-dependent transcription factors: the retinoic acid receptors
(RARs) and the retinoid X receptors (RXRs).12 In the
absence of all-trans retinoic acid (ATRA), RXR/RAR
heterodimers interact with nuclear receptor corepressors (termed SMRT
and NCoR), which recruit histone deacetylases (HDACs) to induce
chromatin modifications and transcriptional
repression.13-15 Binding of the ligand permits release of
the corepressor complex and binding to coactivators, which in turn
recruit histone acetylases that modify chromatin to increase promoter
accessibility, leading to the activation of
transcription.14,16,17
The PML/RAR protein retains most functional domains of RAR
and behaves as an abnormal receptor with altered transactivation functions. In APL, the fusion protein can bind ATRA, but higher concentrations are required to activate
transcription.18,19 This may be due to a more tightly
associated PML/RAR-corepressor complex, in which the corepressor is
not released at physiological ATRA concentrations.20-22
The release of corepressor induced by higher ATRA concentrations allows
recruitment of coactivators and may underlie the cytodifferentiation of
APL cells.20-23
At pharmacologic concentrations, ATRA induces complete remission in a
high percentage of APL patients.24,25 Nevertheless, APL
cells develop resistance to ATRA in vitro and in vivo. Although combined cytotoxic chemotherapy with ATRA cures a high percentage of
patients with APL, relapse with ATRA-resistant cells still occurs.26,27 Different mechanisms have been proposed to
explain this clinical resistance, including altered pharmacokinetics
and genetic changes.28-31
Much has been learned about response and resistance to ATRA by studying
a cell line, NB4, derived from an APL patient.32 We and
others have reported NB4 subclones that are highly resistant to
retinoid-induced cytodifferentiation.33-35 We identified a
point mutation in the LBD of the PML/RAR fusion gene of
the subclone NB4-MR4, which abolishes the ATRA-binding capacity of the
fusion protein and blocks the transcription of ATRA-responsive genes in
a dominant-negative fashion.36 Additional mutations in the LBD of the PML/RAR have been reported in independently established ATRA-resistant NB4 subclones.37-39
A spontaneously ATRA-resistant APL cell line, called UF-1, has been
established directly from an ATRA-resistant APL patient.40 The PML/RAR gene of UF-1 cells harbors a point mutation
in the LBD of its RAR portion replacing the arginine (Arg, R) at
position 276 by a tryptophan (Trp, W).41
Similar mutations of the PML/RAR fusion gene have been
reported in APL cells from a growing number of patients who developed ATRA resistance, demonstrating the clinical relevance of these genetic
alterations as a mechanism of resistance.31,42-45 In a previous study, we analyzed in vitro expression of PML/RAR mutations from ATRA-resistant APL patients. We found that these amino acid changes cause a variety of abnormalities in the ligand-binding transactivation of RAREs and ligand-dependent interactions with the
transcriptional coregulators SMRT and ACTR.46 This
contrasts the nearly complete dominant-negative activity of mutations
in PML/RAR previously characterized in cell lines
developing ATRA resistance in vitro. This variability of binding to
ligand and transcriptional coregulators suggests that strategies to
overcome resistance might work in some cases but not in others.
Recently, histone deacetylase inhibitors have been shown to restore
ATRA-induced transcriptional activity and to induce the differentiation
of patient leukemic blasts in vitro and in animal models of
APL.47-49 Indeed, combined treatment with ATRA and an HDAC
inhibitor led to a durable complete remission of one patient with
ATRA-refractory APL.50
Here we report the presence of novel mutations in the LBD of the
PML/RAR fusion gene in a newly established ATRA-resistant NB4 subclone, NB4-MRA1, and in an additional ATRA-resistant APL patient
in relapse. Both PML/RAR mutant cells show impaired ATRA binding and
transcriptional response to ATRA, as well as alterations in their
ability to interact, in a ligand-dependent manner, with the corepressor
SMRT and the coactivator ACTR. We characterized these mutations of
PML/RAR for their response to the combination of ATRA with an
HDAC inhibitor. We investigated whether the induction of histone
acetylation by cotreatment with ATRA and trichostatin A (TSA) could
restore transcriptional activity and expression of an ATRA-target gene,
as well as granulocytic differentiation.
| |
Materials and methods |
Cell culture
NB4-MRA1 was generated by exposing NB4 cells to a stepwise
increase from 1 × 10 8 to 1 × 105 M of
ATRA (Sigma, St Louis, MO) for 3 months. Single clones were isolated
with methylcellulose and expanded. The ATRA-resistant NB4-MRA1 cells
were then propagated for 1 month in ATRA-free media and retested for
resistance in 1 × 105 M ATRA. These cells are
routinely cultured in ATRA-free RPMI 1640 medium (Life Technologies,
[Gibco BRL], Burlington, ON, Canada) supplemented with 10% fetal
calf serum (FCS; Wisent, St Bruno, QC, Canada) and remain ATRA
resistant. In accordance with the proposed nomenclature of Roussel and
Lanotte,51 the previously published NB4-R4 has been
renamed NB4-MR4. The establishment of and conditions for cell culture
of the ATRA-resistant APL cells NB4-MR4 and UF-1 have been published
previously.35,40
Clinical history of the ATRA-resistant APL patient
The patient, a 35-year-old man with bleeding gums and bruises,
received a diagnosis in May 1991, of APL with the presence of a typical
PML/RAR chimeric mRNA of the long type (L, Bcr1). He
achieved complete clinical remission after chemotherapy with mitoxantrone 80 mg/m2, Ara-C 3 g/m2 × 5 and VP-16 150 mg/m2 × 3. He received consolidation therapy with ATRA
(45 mg/m2 per day) for 10 weeks. In August 1993, in early
clinical relapse, he received ATRA (45 mg/m2 per day) for 4 weeks and re-entered clinical remission. In February 1994, he underwent
allogenic bone marrow transplantation without evidence of
graft-versus-host disease. In July 1997, he had another relapse with
progression of APL resistant to ATRA and chemotherapy.
Plasmid constructs
PML/RAR with either R276W or I410T mutations were
cloned into the pSG5 mammalian expression vector harboring wild-type
PML/RAR (L) cDNA using a QuikChange Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA). All the constructs were verified by
sequencing analysis, using the dsDNA Cycle Sequencing System (Life
Technologies). Construction of the PML/RAR (L) harboring the L398P
mutation (PML/RAR-M4) was previously described.36
DNA sequencing analysis
One microgram total RNA was used for reverse
transcription-polymerase chain reaction (RT-PCR) with random primers
and Superscript II reverse transcriptase (Life Technologies). 2 µL of
a 20 µL RT reaction were used for PCR amplification of the
non-rearranged RAR with primers, oligo A: CAG CAC CAG CTT
CCA GTT AG and oligo C: TGT CCG CTC AGA GTG TCC AG and of the RAR
portion of the PML/RAR with oligo B: GTC TCC AAT ACA ACG
ACA GC and oligo C. The PCR products were gel isolated and used as
templates for direct sequencing using the dsDNA cycle sequencing kit
(Life Technologies).
Cell differentiation
Cell differentiation was evaluated by direct immunofluorescence
staining of CD11b (30455X; PharMingen, Mississauga, ON,
Canada), Coulter Epics XL flow cytometer (Beckman Coulter, Miami,
FL), and nitro blue tetrazolium (NBT) reduction assay as
previously reported.52
Western blot analysis
Nuclear extracts (50 µg) were run on a 10% sodium dodecyl
sulfate acrylamide gel and were transferred to a nitrocellulose membrane (BioRad Laboratories, Mississauga, ON, Canada). The membrane was blocked with 5% skim milk and 0.1% Tween 20 in phosphate-buffered saline (PBS) and was hybridized overnight with a RAR-specific antibody (SC-551; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. After washing in 0.1% Tris-buffered saline, the
membrane was hybridized with a secondary anti-rabbit
antibody and developed with the enhanced
chemiluminescence system (Amersham, Little Chalfont,
Buckinghamshire, United Kingdom).
Assay for ligand-binding activity
Nuclear extracts were prepared and incubated for 18 hours at
4°C with 10 nM [3H]-ATRA (50.7 Ci/mmol; DuPont-NEN,
Boston, MA) or with [3H]-ATRA in the presence of 200-fold
excess of unlabeled ATRA. The extracts were subsequently fractionated
by high-performance liquid chromatography (HPLC) as previously
described.18
Limited proteolytic digestion of translated fusion
proteins
Wild-type and mutant PML/RAR fusion proteins were in vitro
synthesized in the presence of [35S]-methionine (NEN,
Streetsville, ON, Canada), using a coupled transcription and
translation reticulocyte lysate system as suggested by the manufacturer
(Promega, Madison, WI). The radioactive fusion proteins were then
analyzed in a limited proteolytic digestion assay as described
previously.46
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed using
[35S]-labeled in vitro-translated wild-type
and mutant PML/RAR fusion proteins and a direct repeat 5 (DR5)
retinoic acid responsive element, as previously
described.46 Where specified, bacterially expressed and
purified GST fusions containing the receptor interactive domains of
SMRT (GST-SMRT-ID II; amino acid 1073-116820) or ACTR
(GST-ACTR-RID; amino acid 621-82117) were added.
In vitro interaction with GST-DRIP205
Glutathione-S-transferase (GST)-DRIP205 was kindly provided by
Dr Leonard P. Freedman. In vitro interaction of the mutants PML/RAR
with GST-DRIP205 was performed as described previously,53 except that 150 000 cpm of [35S]-labeled in
vitro-translated proteins were used.
Transient transfection experiments for transcriptional
activity
APL cells (5 × 106 cells/transfection) were
transfected by electroporation with 10 µg per transfection of
the reporter plasmid DR5-tk-CAT54 and 10 µg per
transfection of pCMV-Galactosidase (-Gal) as an internal control
for transfection efficiency. Cells were electroporated,
replenished in RPMI 1640 with 10% FCS, and grown for 48 hours in the
absence or presence of different concentrations of drugs.
Chloramphenicol acetyltransferase (CAT) counts were normalized with
-Gal activity to obtain the relative CAT activity.
Ribonuclease protection assay
Fifty micrograms total RNA was used for RNase protection
analysis, as previously described.55 Hybridization of cRNA
probes was performed at 50°C overnight, followed by the addition of
350 µL RNase digestion buffer (10 mM Tris-HCl, pH 7.5, 300 mM
NaCl, 5 mM EDTA [ethylenediaminetetraacetic acid]) containing RNase T1 (Roche Diagnostics, Laval, QC, Canada). RNase digestion was performed at 30°C for 1 hour. RNase-resistant fragments were resolved by electrophoresis on 6% urea-polyacrylamide sequencing gels and visualized by autoradiography.
Chromatin immunoprecipitation
Two million cells were grown the day before treatment with
1 µM ATRA, 200 nM TSA (Sigma), or the combination for 1 hour. To measure histone acetylation levels, formaldehyde-cross-linked and
sonicated chromatin was immunoprecipitated overnight with 5 µL
antibody raised against the acetylated form of histone H4 N-terminal
tail (Upstate Biotechnology, Lake Placid, NY) following the
manufacturer's instructions. For PCR, 1 µL of 20 µL extracted DNA
was used with the FastStart Taq DNA Polymerase kit (Roche Molecular
Biochemicals, Laval, QC, Canada), and 28 to 35 cycles were allowed.
Primers used for PCR detection of the RAR promoter were:
sense, 5'-TCC TGG GAG TTG GTG ATG TCA G-3'; anti-sense, 5'-AAA CCC TGC
TCG GAT CGC TC-3'.
| |
Results |
RA-resistant NB4-MRA1 and relapsed patient APL cells harbor novel
PML/RAR mutations in the LBD
Our laboratory has developed several ATRA-resistant subclones of
NB4 by selection in ATRA-containing media.35 The resistant subclone NB4-MRA1 and the leukemic cells from an ATRA-resistant patient
with relapsed APL were subjected to DNA sequencing analysis of the
PML/RAR gene. Two primers depicted in Figure
1A were used in RT-PCR to specifically
amplify the RAR moiety of the PML/RAR. We identified
missense mutations in the LBD of PML/RAR in both the
ATRA-resistant NB4-MRA1 and patient APL cells. A novel point mutation
was detected in the LBD of the RAR portion of the PML/RAR gene in NB4-MRA1 cells. This mutation resulted in amino acid
substitution of isoleucine (Ile, I) (ATC) for threonine
(Thr, T) (ACC) in the long form (L, Bcr1) of PML/RAR
protein, which corresponds to codon 410 (I410T) in wild-type RAR.
APL cells from the ATRA-resistant patient in relapse expressed the long
form (Bcr1) of PML/RAR, with a C-to-T substitution
changing the codon specificity from arginine (Arg, R) to tryptophan
(Trp, W) at position 276 (R276W) (Figure 1A). Locations of the
mutations were described with reference to the normal amino acid
sequence of RAR1.56 Sequencing both strands of NB4-MRA1
and patient cDNA confirmed the point mutations. No other mutations were
found in the PML/RAR or the coexpressed wild-type
RAR of NB4-MRA1 or the resistant patient cells. Figure 1B
and Table 1 present a summary of reported
LBD PML/RAR mutations associated with ATRA resistance in
cell lines (numbers 1-6) and in APL patients (numbers 7-17). Of note,
the R276W mutation in our patient-resistant APL cells is identical to
the previously reported PML/RAR mutation of the
patient-derived APL cell line, UF-1.41 This allowed us to
use the UF-1 cell line as an in vivo model to study the effects of the
patient PML/RAR mutation on ATRA response.
View larger version (11K):
[in this window]
[in a new window]
| Figure 1.
Identification of novel point mutations in the LBD of
the RAR moiety of PML/RAR in RA-resistant APL.
(A) Schematic representation of PML/RAR, describing point
mutations identified in ATRA-resistant APL cells from a patient in
relapse and from a newly established cell line, NB4-MRA1, showing the
approximate positions of 2 primers used in RT-PCR. (B) Summary of the
LBD PML/RAR mutations identified in ATRA-resistant APL cell lines
(numbers 1-6) and patients in relapse (numbers 7-17). Numbers 5 and 9 indicate the novel mutations in the LBD PML/RAR of ATRA-resistant
APL cells identified and characterized in the current study and
correspond to NB4-MRA1 and the patient, respectively. Numbers 1 to 17 in Figure 1B correspond to numbers 1 to 17 in Table 1. The lengths of
the arrows reflect the frequency of each mutation. The position of the
mutations is described with reference to normal amino acid sequence of
RAR1.56 The alignment of the PML/RAR E-domain and
TR ligand-binding domain by sequence homology indicates that the
mutations in ATRA-resistant patients with APL and cell lines cluster in
accordance with the regions in RTH syndrome denoted as I, II, and III.
DBD indicates DNA-binding domain; LBD, ligand-binding domain; DD,
dimerization domain; AF-2, ligand-dependent activation
function.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Summary of the PML/RAR mutations identified in
ATRA-resistant APL cell lines (numbers 1-6) and patients in relapse
(numbers 7-17)
|
|
Characterization of cell lines expressing novel PML/RAR mutants
and their ATRA-binding activity
NB4-MRA1 cells were cultured in ATRA-free media for more than 6 months and remained resistant to ATRA-induced growth inhibition (data
not shown) or cell differentiation (see "Results" and Figure 8). The long-term maintenance of resistance in ATRA-free
culture and the presence of a genetic change mediating resistance argue that this cell line is truly ATRA resistant, not transiently
insensitive.57 Figure 2A
confirms the expression of PML/RAR fusion protein in NB4-MRA1 cells
and shows the ATRA-induced PML/RAR protein degradation in the
ATRA-sensitive NB4 but not in the ATRA-resistant NB4-MRA1 APL cells.
The lack of response of UF-1 cells to ATRA has previously been
described.41
View larger version (38K):
[in this window]
[in a new window]
| Figure 2.
Characterization of cell lines expressing novel
PML/RAR mutants and their ATRA-binding activity.
(A) ATRA induces PML/RAR protein degradation in the ATRA-sensitive
NB4 cells, but not in the ATRA-resistant NB4-MRA1 APL cells. Cells were
treated with 1 µM ATRA for 24 hours, and 50 µg of nuclear extracts
were used in Western blot analysis for the expression of PML/RAR
protein. NB4 and NB4-MRA1 expressed a long PML/RAR (L) isoform (110 kDa). The lower panel shows laminin B expression to confirm protein
loading. (B) Specific HPLC ATRA-binding profiles of nuclear extracts
from NB4 cells ( ) compared with those from the ATRA-resistant
NB4-MRA1 ( ) and UF-1 ( ) APL cells. Nuclear extracts were
incubated with 10 nM [3H]-ATRA (, , ) or with
[3H]-ATRA in the presence of 200-fold excess of unlabeled
ATRA ( ). Extracts were subjected to HPLC analysis using a 6 HR 10/30
size exclusion column.
|
|
To evaluate how these point mutations of PML/RAR alter
the function of the LBD, we examined their ATRA-binding activity
(Figure 2B). The size exclusion HPLC profile of extracts from NB4 cells expressing the wild-type form of PML/RAR is characterized by the
50-kDa peak representing the binding of ATRA to the endogenous RARs and
the approximately 670-kDa peak representing macromolecular complexes
formed by the interaction of PML/RAR with itself or other nuclear
proteins.18 The HPLC profiles of both NB4-MRA1 and UF-1
cells show an altered pattern of high-molecular-weight binding
complexes. Nuclear extracts from NB4-MRA1 cells expressing the
PML/RAR mutation I410T showed an HPLC profile consistent with ATRA
binding to the mutated chimeric protein but at a much decreased level.
Specific ATRA-binding activity was not detectable in nuclear extracts
prepared from UF-1 cells harboring the PML/RAR mutation R276W,
suggesting that this mutation completely prevents binding of the fusion
protein to labeled ATRA.
Conformational change analysis of PML/RAR mutants
We predicted that modifications in the ligand binding of fusion
proteins 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 mutated fusion proteins in the absence and in the
presence of ATRA. Distinct fragment patterns were observed for the
wild-type and for mutant fusion proteins, especially after ATRA
treatment (Figure 3). The distinction
centered around a fragment at 32-kDa (lower asterisk in Figure 3) and a fragment at 37-kDa (upper asterisk in Figure 3). In the absence of
ATRA, a 32-kDa fragment was more resistant to trypsin digestion. After
1 µM ATRA treatment, the 32-kDa fragment completely disappeared, whereas a fragment of 37-kDa was more resistant to protease treatment of the wild-type PML/RAR. The digestion pattern of the mutant R276W
is different from that of the wild-type PML/RAR after ATRA treatment. The identical digestion patterns in the absence and in the
presence of ATRA suggest that the mutant R276W fusion protein does not
bind the ligand, which is consistent with our ligand-binding analysis
(Figure 2B). Analysis of the mutation I410T showed that the trypsin
digestion pattern is similar to that of the wild-type PML/RAR. This
result indicates that ATRA can still bind to and alter the conformation
of the mutated fusion protein LBD, even though binding is shown to be
reduced by the HPLC studies (Figure 2B).
View larger version (52K):
[in this window]
[in a new window]
| Figure 3.
ATRA-induced conformational changes in wild-type and
I410T mutant PML/RAR fusion proteins but not in R276W mutant.
Limited trypsin digestion analysis of wild-type and mutant PML/RAR
proteins. In vitro [35S]-methionine synthesized
PML/RAR proteins were incubated without ( ) or with (+) 1 µM
ATRA, and were subsequently treated with increasing trypsin
concentrations (0-25 µg/mL). Digestion products were analyzed by
denaturing electrophoresis. The arrows indicate the intact PML/RAR
proteins. Lower and upper asterisks indicate resistant fragments at 32 kDa and 37 kDa, respectively.
|
|
Loss of ligand-dependent association with transcriptional
coregulators by mutant PML/RAR fusion proteins
Ligand-dependent activation of nuclear receptors is associated
with displacement of corepressors and recruitment of coactivating proteins. Interactions of the PML/RAR 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 (ID
II) of the corepressor SMRT (Figure 4A).
In vitro-translated wild-type and mutants PML/RAR bound a
radiolabeled 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 4A. Binding of GST-SMRT-IDII to each of the mutants was
similar to the wild-type PML/RAR in the absence of ATRA. As
previously reported, the wild-type PML/RAR fusion protein completely
dissociated SMRT-IDII at 1 µM ATRA. In contrast, the fusion proteins
harboring the mutations R276W and I410T could not be dissociated from
the corepressor SMRT-IDII, even at 10 µM ATRA.
View larger version (49K):
[in this window]
[in a new window]
| Figure 4.
Alteration of the ligand-dependent association with
transcriptional coregulators by mutant PML/RAR fusion proteins.
(A) Interaction of SMRT with wild-type and mutant PML/RAR proteins
on a DR5 RARE in gel mobility shift assay. 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 ATRA. The
position of the complex shifted by SMRT is indicated. (B) Interaction
of ACTR with wild-type and mutant PML/RAR proteins on a DR5 RARE in
gel mobility shift assay. 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 ATRA. The position of the complex shifted
by ACTR is indicated. (C) Interaction of mutant PML/RAR fusion
proteins with the coactivator DRIP205 by GST pull-down analysis. In
vitro-translated 35S-labeled wild-type and mutant
PML/RAR fusion proteins were incubated with bacterially expressed
and purified GST-DRIP205 in the absence and in the presence of
increasing concentrations of ATRA as indicated. GST alone was included
as the negative control.
|
|
To test whether these mutants of PML/RAR could recruit the
coactivator ACTR, the central receptor-interacting domain of ACTR (ACTR-RID) fused to the GST protein was used in a gel-shift study (Figure 4B). In the absence of ATRA, 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 µM and was
maximal at 0.1 µM ATRA. The mutation R276W is fully associated with
ACTR-RID only at 10 µM ATRA, which is 100-fold greater than for the
wild-type association. PML/RAR harboring the mutation I410T
showed only minimal recruitment of ACTR at 10 µM ATRA,
indicating that the coactivator needs more than 100-fold ATRA
concentrations for its association with this mutated fusion protein.
A distinct coactivator complex has been identified by 2 separate
groups for its ligand-dependent interaction to the vitamin D3 receptor
(DRIP) or to the thyroid hormone receptor (TRAP).58,59 These studies suggest that the DRIP/TRAP complex acts as a
ligand-dependent positive transcriptional regulator of various nuclear
receptors. We previously showed that RAR and the wild-type
PML/RAR chimeric protein interact directly with the DRIP
complex.53 To assess whether PML/RAR mutants modify the
interaction with the DRIP complex, in vitro-translated
[35S]-labeled mutant PML/RAR proteins were allowed to
interact with the purified GST-DRIP205 (Figure 4C). Wild-type
PML/RAR showed a ligand-dependent interaction with the DRIP205
protein. As we previously reported, the M4 mutant PML/RAR of NB4-MR4
failed to bind DRIP205. In contrast, both mutants R276W and I410T
showed an interaction with DRIP205 similar to that of the wild-type
PML/RAR, though PML/RAR mutant I410T interacted less with DRIP205
in presence of 0.1 µM ATRA.
TSA cooperates with ATRA to induce transcriptional activity of a
RARE in ATRA-resistant APL cell lines
Because a histone deacetylase-dependent transcriptional
repression of the ATRA-signaling pathway may underlie the
differentiation block of APL, we evaluated the capacity of the HDAC
inhibitor, TSA, to modulate the ATRA response of PML/RAR mutants in
ATRA-resistant APL cell lines on a retinoid-responsive element.
ATRA-sensitive NB4 and ATRA-resistant NB4-MRA1, NB4-MR4, and UF-1 APL
cell lines were cotransfected with a tk-CAT reporter driven by a DR5
RARE and a -Gal expression vector as a control for the efficiency of
transfection. As shown in Figure 5,
ligand-dependent transcriptional activity of the wild-type PML/RAR
chimeric protein present in ATRA-sensitive NB4 cells was increased by
concentrations of ATRA from 0.01 to 1 µM, and treatment with
ATRA + TSA further increased transactivation. Figure 5 shows that
ATRA-resistant NB4-MRA1, UF-1, and NB4-MR4 APL cells have significantly
impaired ligand-dependent transcriptional activity. However, TSA
cooperates with ATRA to increase transcriptional activity on a
DR5-tk-CAT in ATRA-resistant APL cells. The combination of 200 nM TSA
with 1 µM ATRA increased the DR5-tk-CAT activity in NB4-MRA1 and UF-1
cells to 160- and 67-fold, respectively, compared with a 160-fold
induction in NB4 cells.
View larger version (25K):
[in this window]
[in a new window]
| Figure 5.
TSA cooperates with ATRA to induce transcriptional
activity of a RARE in ATRA-resistant cell lines harboring point
mutations in the LBD of PML/RAR.
Relative CAT activity of ATRA-sensitive NB4 and ATRA-resistant
NB4-MRA1, NB4-MR4, and UF-1 APL cells without (control) or with
indicated concentrations of ATRA or 200 nM TSA is shown. A DR5-tk-CAT
reporter was cotransfected with a -galactosidase expression vector
into the cells for normalization of the transfection. Each data point
represents the mean of at least 3 independent transfections, and bars
denote SD.
|
|
RA synergizes with the HDAC inhibitor TSA to induce
RAR gene expression only in NB4-MRA1 ATRA-resistant
cells
Given that these in vitro transcriptional activity data show that
the PML/RAR mutants can respond to HDAC inhibitors, we next
evaluated the capacity of ATRA and TSA to activate transcription of an
endogenous ATRA target gene. RAR is a direct ATRA target whose induction has been implicated in several tumor cell models in
which retinoids inhibit growth and induce
differentiation.12 We investigated the effects of ATRA
alone and in combination with TSA on the expression of
RAR in ATRA-sensitive NB4 and ATRA-resistant NB4-MRA1,
NB4-MR4, and UF-1 cells by ribonuclease protection analysis (Figure
6). No constitutive expression of
RAR transcript in ATRA-sensitive or -resistant APL cell
lines was observed. RAR mRNA levels were markedly
increased by the induction of differentiation of NB4 cells by ATRA.
However, ATRA was unable to induce RAR expression in
ATRA-resistant NB4-MR4 and UF-1 cell lines, though a slight induction
was detected in NB4-MRA1 cells. We found no modification of the levels
of RAR mRNA transcript by TSA alone in ATRA-sensitive NB4
and ATRA-resistant NB4-MRA1 and UF-1 APL cells and a very weak
induction independent of ATRA treatment in NB4-MR4 cells. Treatment
with ATRA and TSA produced a significant up-regulation of the levels of
RAR mRNA in NB4 and, interestingly, in the ATRA-resistant NB4-MRA1 APL cells, but it failed to stimulate RAR in
UF-1 and NB4-MR4 cells.
View larger version (64K):
[in this window]
[in a new window]
| Figure 6.
HDAC inhibitor TSA potentiates ATRA induction of
RAR mRNA expression in the ATRA-resistant NB4-MRA1 APL
cells harboring the mutant I410T PML/RAR.
Ribonuclease protection analysis for RAR expression of 50 µg total RNA isolated from ATRA-sensitive NB4 and ATRA-resistant
NB4-MRA1, NB4-MR4, and UF-1 APL cells untreated or treated with 1 µM
ATRA or 200 nM TSA and the combination of ATRA with TSA for 28 hours.
The RNase-protected band corresponding to RAR is identified, and
GAPDH expression was used as a quantitative loading control.
Yeast tRNA (y-tRNA) was included as a negative control to verify the
specificity of the probes.
|
|
HDAC inhibition with TSA potentiates ATRA-induced histone
hyperacetylation on chromatin of RAR promoter in
ATRA-resistant NB4-MRA1 cells
To directly assess whether the effects of TSA and ATRA induction
on RAR gene expression correlate with modifications of
histone acetylation, we analyzed histone H4 acetylation at the receptor target gene RAR by the chromatin immunoprecipitation
(ChIP) assay. As shown in Figure 7, ChIP
analysis with antibodies to acetylated H4 revealed that ATRA treatment
of ATRA-sensitive NB4 cells induces the acetylation level on H4. The
addition of TSA increases acetylation of histone H4 in the absence and
in the presence of ATRA. Treatment of ATRA-resistant NB4-MR4 cells with
TSA showed a very weak increase in the acetylation of H4 on
RAR, which was not changed by the presence of ATRA.
Treatment of ATRA-resistant UF-1 APL cells with ATRA and TSA used as
single or combined agents demonstrated no modification of the level of
acetylation of histone H4 on RAR. However, in NB4-MRA1
ATRA-resistant cells, consistent with our data on RAR RNA
expression, the combination of ATRA and TSA strongly induced the
acetylation of H4 on the RAR promoter.
View larger version (45K):
[in this window]
[in a new window]
| Figure 7.
HDAC inhibition by TSA intensifies ATRA-induced histone
H4 hyperacetylation on chromatin of RAR promoter in
ATRA-resistant NB4-MRA1 APL cells.
Cross-linked chromatin preparations of ATRA-sensitive NB4 and
ATRA-resistant NB4-MRA1, NB4-MR4, and UF-1 APL cells untreated or
treated with 1 µM ATRA or 200 nM TSA, and the combination of ATRA
with TSA were immunoprecipitated with an antibody against acetylated
tails of histone H4. Immunoprecipitated and input material was analyzed
by PCR using primers corresponding to the RARE region of the
RAR promoter.
|
|
Combined effect of ATRA and TSA on differentiation of the
ATRA-resistant NB4-MRA1 APL cells
To determine whether the observed effects of ATRA and TSA on
transcription and gene expression translate into induction of differentiation, we investigated the extent of differentiation response
of the cell lines by CD11b staining and NBT reduction (Figure
8). Although ATRA alone induces a
moderate increase of the early differentiation marker, CD11b, in
NB4-MRA1 (Figure 8A), there is no effect of ATRA alone on the terminal
differentiation marker, NBT, in either NB4-MRA1 or UF-1 APL cells
(Figure 8B). However, the combination of ATRA with TSA causes
substantial differentiation in NB4-MRA1, but not in UF-1 APL cells,
consistent with our data on transcription and gene expression.
View larger version (25K):
[in this window]
[in a new window]
| Figure 8.
Combined effect of ATRA and TSA on differentiation of the
ATRA-resistant NB4-MRA1 APL cells.
(A) Expression of the differentiation marker CD11b by flow cytometry
analysis. NB4, NB4-MRA1, and UF-1 APL cells were treated with 25 nM
TSA, 1 µM ATRA, and the combination for 3 days. Numbers in
parentheses indicate mean channel fluorescence. This experiment is
representative of 3 that gave comparable results. (B) NBT
reduction analysis of NB4, NB4-MRA1, and UF-1 APL cells treated with
the indicated concentrations of agents for 5 days. Each data point
represents the mean of 3 independent experiments, and bars denote SD.
|
|
| |
Discussion |
Different groups have identified specific genetic lesions
associated with ATRA resistance, in cell lines and in patients. As
shown in Figure 1B, several missense mutations in the LBD of the RAR
moiety of the fusion gene PML/RAR have now been reported in APL cells, highlighting the role of the fusion protein in mediating the sensitivity and response to ATRA. Rather than being randomly distributed, the mutations cluster mainly in 3 areas, denoted by I, II,
and III, corresponding to the amino-terminus (between H1 and H3), the
central region (within or between H5 and H6), and the carboxy-terminus
(within H11 and H12) of the LBD, respectively. Interestingly, these
PML/RAR mutations can be compared to clusters of mutations in
thyroid receptor beta (TR) associated with the syndrome of
resistance to thyroid hormone (RTH).60-62
In this report, we characterize 2 novel missense point mutations in the
E-domain of PML/RAR, associated with the development of resistance
to ATRA, that cluster in regions II and III described above (Figure 1).
The growing list of mutations identified in the LBD of PML/RAR is an
indication of the importance of this mechanism for the development of
ATRA resistance in APL cells in vitro and in vivo.
The R276W PML/RAR mutation was identified from leukemic cells of a
patient in relapse. A spontaneously ATRA-resistant APL cell line, UF-1,
established directly from an ATRA-resistant patient with APL, harbors
exactly the same point mutation, R276W, in its PML/RAR
gene41 as in the APL cells of our ATRA-resistant
patient with APL. Importantly, this point mutation has been
independently observed in a third ATRA-resistant patient with relapsed
APL.43 Very recently, a different mutation at the same
position, Arg276Gly, has been identified in 2 different
ATRA-resistant patients.44 The identification of
mutations of the arginine at position 276 by independent groups in
cells of 5 different ATRA-resistant patients suggests that this amino
acid is probably very important in the mechanism of ATRA response and,
therefore, is a mutational "hot spot" involved in the mechanism of
ATRA resistance.
The R276W mutation is centrally located in the LBD of PML/RAR at the
end of H5. It has been shown in x-ray structure analysis that this
arginine is conserved in the steroid nuclear receptor family and
participates in a network of hydrogen bonds and salt bridges anchoring
the carboxylate group of the ligand. This positively charged arginine
may also serve as an electrostatic field guide for the
ligand.63,64 This amino acid substitution, involving a
radical shift from the most hydrophilic positively charged Arg to the
nonpolar hydrophobic Trp, is likely to be responsible for the
disruption of these bonds and the electrostatic field. The mutated
PML/RAR chimeric protein of UF-1 cells completely lost its
ATRA-binding capacity (Figure 2B). In vitro expression of this mutant
PML/RAR exhibited no change in the pattern of resistant fragments in
limited proteolytic digestion in the presence of ATRA (Figure 3).
Consistent with these results, this R276W mutation severely impaired
the ability of the PML/RAR protein to interact in a ligand-dependent
manner with coregulators (Figure 4). These ligand-binding,
conformational, and protein-interaction analyses are consistent with
transient transfection studies in the ATRA-resistant UF-1 cells showing
that this R276W mutant PML/RAR is minimally responsive to
transcriptional activation by ATRA (Figure 5).
The importance of this amino acid as a target for mediating nuclear
hormone resistance is further supported by the analysis of a natural
TR mutant R320H from a patient with RTH.65 This amino
acid corresponds in TR to the R276 in RAR. The R320H in TR has
a reduced ligand-binding affinity and a decreased
T3-dependent release of NCoR, and it mediates a weaker
transactivation, but these effects are of a lesser degree than those we
observed with R276W. Indeed, this difference may be explained by the
more conservative amino acid substitution in the TR, where a polar
positively charged arginine is replaced by an amino acid of the same
group. This observation suggests that the type of amino acid
substitution may influence interactions with ligands and nuclear coregulators.
NB4-MRA1 is a newly established ATRA-resistant NB4 subclone harboring a
novel mutation, I410T, in the LBD of the RAR moiety of PML/RAR.
This amino acid is one of 8 residues constituting the H12, which
includes the AF-2 domain. H12 appears to be crucial for ATRA binding
and transcriptional activity. In the presence of the ligand, the H12 of
PML/RAR is repositioned, stabilizing the binding pocket and
providing a surface for coactivators to bind and activate
transcription.63,64 The isoleucine at position 410 produces an almost entirely apolar interaction by making Van der Waals
contact with the beta-ionone ring of the ligand.66 The
replacement of the nonpolar isoleucine at position 410 by a polar
uncharged threonine considerably decreases the capacity of the fusion
protein PML/RAR to bind ATRA (Figure 2B), probably by disrupting
this apolar interaction. However, tryptic digestion analyses showed
that ATRA can still bind to the mutated PML/RAR and induce
conformational changes similar to those observed in the wild-type
receptor (Figure 3). Although 2 assays show that the mutant I410T
can bind ATRA, it also exhibited a decreased transcriptional response
to ATRA (Figure 5), and ATRA regulation of binding to coregulators is
completely lost (Figure 4). This suggests that a mutation in the AF-2
domain can have more prominent effects on binding to coregulators than
to ligand. Further, observed differences between assays comparing
PML/RAR mutations45,46 (Figures 3-5) suggest that no one
in vitro assay will fully describe the altered biology of mutated
PML/RAR.
Again, there is an interesting correlation with studies of TR
mutations. Consistent with our data, the L454S TR mutant in RTH,
which corresponds to the amino acid I410 in RAR, has been shown to
bind ligand, whereas ligand-dependent release of the corepressor NCoR
and the ability of the ligand to activate transcription are
impaired.67 The natural occurrence of mutations in the
amino acids of TR corresponding to each of the mutated residues of PML/RAR described here and the observed similarities in phenotype support their potential importance in mediating thyroid and ATRA resistance. Further comparison of mutations identified in
ATRA-resistant APL with those found in TR with RTH syndrome may
increase our understanding of the functional roles of these mutants.
Mutants R276W and I410T showed an interaction with DRIP205 similar to
that of the wild-type PML/RAR protein, in contrast to the M4 mutant
of NB4-MR4, which failed to interact with this coactivator (Figure 4C).
Dilworth and Chambon68 propose a model for ATRA-induced
initiation of transcription where DRIP/TRAP is directly involved in the
activation pathway by RAR/RXR heterodimers, at a step that occurs after
displacement of p160 proteins from AF-2. Consistent with this model,
the interaction of R276W and I410T mutants with DRIP205 may explain the
higher ATRA-induced transcriptional activity of UF-1 and NB4-MRA1 than
of NB4-MR4 cells (Figure 5). Thus, our data suggest that the
interaction of retinoid receptors with DRIP/TRAP may provide an
additional regulatory step that can be involved in ATRA resistance. The
data further support our hypothesis that ATRA resistance may be
mediated by mutations that target different elements of the complex
mechanism of ligand-dependent transcriptional activation by nuclear receptors.
The increased association that we found between mutants PML/RAR and
the corepressor SMRT suggests an abnormal interaction with HDACs. An
addit |
Top
Abstract
Introduction