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Blood First Edition Paper, prepublished online November 16, 2015; DOI 10.1182/blood-2015-06-644948
Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
Variegated RHOA mutations in adult T-cell leukemia/lymphoma
Yasunobu Nagata1, Kenji Kontani2, Terukazu Enami3, Keisuke Kataoka1, Ryohei Ishii4,
Yasushi Totoki5, Tatsuki R. Kataoka6, Masahiro Hirata6, Kazuhiro Aoki7, Kazumi
Nakano8, Akira Kitanaka9, Mamiko Sakata-Yanagimoto3, Sachiko Egami2, Yuichi
Shiraishi10, Kenichi Chiba10, Hiroko Tanaka11, Yusuke Shiozawa1, Tetsuichi Yoshizato1,
Hiromichi Suzuki1, Ayana Kon1, Kenichi Yoshida1, Yusuke Sato1, Aiko Sato-Otsubo1,
Masashi Sanada1, Wataru Munakata5, Hiromi Nakamura5, Natsuko Hama5, Satoru
Miyano10,11, Osamu Nureki4, Tatsuhiro Shibata5, Hironori Haga6, Kazuya Shimoda9,
Toshiaki Katada2, Shigeru Chiba3, Toshiki Watanabe7, and Seishi Ogawa1
1
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto
University, Kyoto, Japan; 2Department of Physiological Chemistry, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan; 3Department of
Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 4Department
of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo,
Japan; 5Division of Cancer Genomics, National Cancer Center Research Institute,
Tokyo, Japan; 6Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto,
Japan;
7
Department of Pathology and Biology of Diseases, Graduate School of
Medicine, Kyoto University, Kyoto, Japan; 8Department of Medical Genome Sciences,
Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan;
9
Department of Gastroenterology and Hematology, Faculty of Medicine, University of
Miyazaki, Miyazaki, Japan;
10
Laboratory of DNA Information Analysis, Human Genome
Center, Institute of Medical Science;
11
Laboratory of Sequence Analysis, Human
Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Correspondence should be addressed to:
Seishi Ogawa, M.D., Ph.D.
Department of Pathology and Tumor biology,
Graduate School of Medicine
Kyoto University,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8510, Japan,
Phone: +81-75-753-9285
FAX: +81-75-753-9282
E-mail: [email protected]
Copyright © 2015 American Society of Hematology
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
Key Points:
RHOA mutations are common in adult T-cell leukemia/lymphoma (ATLL) and show
a unique distribution compared to other T-cell lymphomas.
Depending on patients, functionally distinct RHOA mutations are clonally selected
and involved in the pathogenesis of ATLL.
ABSTRACT
Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma
(PTCL) with poor prognosis which is caused by human T-lymphotropic virus type 1
(HTLV-1).
In contrast to the unequivocal importance of HTLV-1 infection in the
pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T-cells has not
been fully elucidated with a handful of genes known to be recurrently mutated.
In this
study, we identified unique RHOA mutations in ATLL through whole genome sequencing
of an index case, followed by deep sequencing of 203 ATLL samples.
RHOA
mutations showed distinct distribution and function from those found in other cancers.
Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across
the entire coding sequence but almost invariably located at the GTP-binding pocket,
with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation
types and positions, these RHOA mutants showed different or even opposite functional
consequences in terms of GTP/GDP-binding kinetics, regulation of actin fibers, and
transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a
dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro)
showed fast GTP/GDP cycling with enhanced transcriptional activation.
These
findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL
leukemogenesis. In summary, our study not only provides a novel insight into the
molecular pathogenesis of ATLL but also highlights a unique role of variegation of
heterologous RHOA mutations in human cancers.
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
Introduction
Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphomas
(PTCLs) caused by human T-lymphotropic virus type 1 (HTLV-1).
T-cells immortalized by HTLV-1 infection during early infancy.
It originates from
Although HTLV-1 can
effectively immortalize T-cells, there is a long latency period of 30~50 years prior to the
onset of ATLL, suggesting that HTLV-1 infection alone may not be sufficient for the
development of ATLL, but that additional acquired genetic hits are thought to be
essential for its pathogenesis.1,2
3
4
5
In this regard, mutations in several genes, such as
6
TP53, FAS, ZEB1, and CCR4, have been implicated in the latter process.
However,
the entire sequence of genetic events that shape an ATLL genome is still unclear. In
this study, we first identified a RHOA mutation (Gly17Val) in combined with a TET2
mutation in a case of ATLL using whole genome sequencing (WGS), which has been
closely implicated in angioimmunoblastic T-cell lymphoma (AITL) and other PTCL not
otherwise specified (PTCL-NOS).
However, a subsequent large-scale analysis
disclosed very different nature of RHOA and TET2 mutations in ATLL cases, in terms of
their distributions and functional consequences.
We describe unique features of
RHOA mutations in ATLL in comparison with other cancers including AITL and
PTCL-NOS together with their functional implications.
Methods
Tumor specimens.
A total of 203 patients with different ATLL subtypes, including 74 acute, 54 lymphoma,
52 chronic, 6 smoldering, and 17 unknown types, were included in this study after a
written informed consent was obtained.
patients.
Survival data were available for 113 of the 203
This study was approved by the institutional review board at the Kyoto
University and conducted in accordance with the Declaration of Helsinki. Tumor DNA
was extracted from bone marrow, peripheral blood, and lymphoid organs.
Genomic
DNA samples from buccal mucosa were also obtained for 38 patients and used as
germline controls.
Whole-genome sequencing.
DNA samples were prepared for paired-end DNA Sample Prep kit (Illumina) according
to the manufacture's protocol with some modifications.
Sequence data were
generated using the Illumina Hiseq 2000 platform in 2×100 paired-end reads.
Data
processing, variant calling, copy number estimation, and structural alteration detection
were performed as described previously.7
All somatic mutations in protein coding
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
regions were validated by deep sequencing (supplemental Table 1).
analysis were integrated and visualized using Circos.
The results of the
8
Targeted deep sequencing.
Entire coding exons of RHOA, TET2, and DNMT3A and mutational hotspots of IDH1/2
were PCR-amplified using NotI-tagged primers (supplemental Table 2) and subjected to
high-throughput random sequencing using Hiseq 2000.
Mutation calling was
2
performed as previously described, where the data from 95 normal individuals were
used to eliminate SNPs.
All candidate variants were validated by Sanger sequencing
or independent deep sequencing using non-amplified DNA.
SNP array-karyotyping
Genome-wide copy number analysis was performed for 203 samples using Affymetrix
GeneChip Human Mapping 250K NspI Array.
Microarray data were analyzed to
estimate total and allele-specific copy numbers using CNAG/AsCNAR for Affymetrix.9,10
Mutagenesis and vector construction
Plasmids bearing human wild-type, Gly14Val, Gly17Val, and Ala161Glu mutant RHOA
cDNA were described previously.2
Mutagenesis to create constructs encoding the
Cys16Arg, Cys16Phe, Lys118Glu, Ala161Val and Arg161Pro mutants was carried out
with the Primestar Mutagenesis Basal kit (Takara) according to the manufacture’s
protocol. All constructs were verified by Sanger sequencing. These constructs were
subcloned
into
the
tetracycline-inducible
11
CS-TRE-PRE-Ubc-tTA-I2G7,
vector (GE Healthcare).
lentivirus-based
expression
vector
2
the pEF-neo expression vector, and the pGEX-6P-1
In addition, the last four amino acids corresponding to the
CAAX motif were deleted for the pGEX-6P-1 vector.
Biochemical analyses of recombinant RHOA Proteins.
Purification and biochemical characterization of RHOA proteins were performed as
described previously with minor modifications.12
GST-fusion proteins of wild-type and
mutant RHOA were induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside at
20°C for 16 hours in Escherichia coli BL21-CodonPlus DE3 (Stratagene) and purified
with glutathione-Sepharose 4B beads (GE Healthcare Life Sciences).
For measuring
the GTP-binding properties of Gly17Val and Ala161Glu RHOA mutants, the protein
concentrations were estimated based on the total amount of proteins, because most of
them were present in a nucleotide-free form. In other guanine nucleotide exchange
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
assays, the active protein concentrations were estimated based on the amounts of
proteins bound to the guanine nucleotides. For [35S] GTPγS-binding assays, purified
GST-fusion proteins (5 pmol) were incubated with 5 µM of the radiolabeled nucleotide
(4,000 cpm/pmol).
After incubation for the indicated periods, samples were diluted,
and filtered through a nitrocellulose membrane (0.45-µm pore size, Advantech MFS,
Dublin, CA).
The membrane was washed four times and the radioactivity retained on
the membrane was determined by a liquid scintillation counter.
For [35S] GTPγS- and [3H]GDP-dissociation assays, purified proteins (1.5 pmol)
were pre-incubated with 5 µM of the radiolabeled nucleotides (4,000 cpm/pmol for [35S]
GTPγS and 7,000 dpm/pmol for [3H]GDP, respectively) for 2.5 hours.
Dissociation of
the radiolabeled nucleotides from RHOA proteins was then initiated by the addition of
unlabeled GTPγS (final concentration of 50 µM) to the solution.
After incubation for the
indicated periods, the amounts of the radioactivity associated with the proteins were
determined as described above.
F-actin staining.
NIH3T3 cells were transfected with plasmids expressing GFP alone, GFP-fused
wild-type or mutant RHOA.
After pre-cultured for 24 hours, cells were induced for gene
expression with doxycycline, serum-starved for 24 hours, fixed, permeabilized, and
stained by Rhodamine Phalloidin (Cytoskeleton).
Nuclei were stained with
4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Dojindo).
Images were
obtained by confocal-laser scanning microscopy (Olympus).
Reporter assay.
For luciferase reporter assay, 1.2 × 105 /well 293T cells were seeded in 12-well plates
and co-transfected with wild-type or mutant RHOA in pEF-neo together with
pSRα/β-galactosidase and SRF-RE /pGL4.34 [luc2P/SRF-RE/Hygro] Vector (Promega),
by using XtremeGENE 9 DNA transfection reagents (Roche) according to the
manufacture’s protocol. Activity of firefly luciferase in cell lysates was measured as
previously described.2
Flow cytometry.
Peripheral blood- and bone marrow-derived cells were stained with fluorescent-labeled
antibodies and analyzed by flow cytometry using an LSRFortessa (BD Biosciences)
with standard filter sets.
Antibodies used were fluorescein isothiocyanate-labeled
anti-CD4 (RPA-T4, BD Biosciences), phycoerythrin (PE) Cy7-labeled anti-CD25 (2A3,
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
BD Biosciences), and PE-labeled anti-PD-1 (MIH4, BD Biosciences). For intracellular
staining, Alexa Fluor 647 Mouse anti-Human FOXP3 and human Foxp3 Buffer set
(259D/C7BD Biosciences) was used after surface staining.
All flow cytometry data
were analyzed with FlowJo software (TreeStar).
Immunohistochemistry
For immunostaining, 3 µm sections were cut, deparaffinized, subjected to a
heat-induced antigen retrieval with Target Retrieval Solution (Dako), and pretreated with
0.3% hydrogen peroxide for blocking. They incubated with primary antibodies against
CD25 (clone 4C9, dilution 1:1; Nichirei Bioscience) and CD45RO (clone UCLH-1,
dilution 1:100; DAKO).13,14
The bound antibodies were detected using the Histofine
Simple Stain MAX PO reagent with diaminobenzidine (DAB) as a chromogen, and the
sections were counterstained with Mayer's hematoxylin.
Immunohistochemistry
analysis with anti-CD3 (clone 2GV6, dilution 1:1; Roche) and anti CD4 (clone SP35,
dilution 1:1; Roche) antibodies was performed with the Ventana Benchmark automated
staining system (Ventana Medical Systems) according to the manufacturer’s
instruction.13
For double staining of FOXP3 and PD-1, the sections were first
incubated with anti-FOXP3 antibody (clone 236A/E7, dilution 1:100; Abcam) and
visualized using ImmPRESS HRP-conjugated anti-mouse polymeric system (Vector
Laboratories) together with DAB.
antibody
(clone
NAT105,
Then, the sections were stained with anti-PD-1
dilution
1:50;
Abcam),
detected
with
anti-rabbit
ImmPRESS-AP polymer reagent and Vector Blue (Vector Laboratories), and followed
by the Nuclear Fast Red counterstaining.15
The tissue section images were captured
using the NanoZoomer 2.0-HT (Hamamatsu Photonics).
Statistical analysis
Overall survivals were assessed using Kaplan-Meier curves and two-sided log-rank
tests. Hazard ratios (HRs, given in numbers) and their 95% confidential intervals were
calculated by Cox regression.
Analyses were performed using SPSS version 19.0.0
(IBM Corporation, Armonk, NY).
Results
Identification of RHOA mutations in ATLL
To obtain an insight into the ATLL genome, we performed WGS of paired tumor/normal
DNA from a single case with ATLL.
The mean coverage of WGS for paired
tumor/normal DNA was 60.4× and 33.1×, with which 96% and 88% of the entire
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
genomes were analyzed at more than 20 independent reads on average, respectively.
WGS revealed a high complexity of this particular ATLL genome, in which 15,848 single
nucleotide variants (SNVs), 1,012 insertions/deletions, 91 structural variations, and
numerous copy number variations and allelic imbalances were detected (Figure 1).
The SNVs included a number of potential drivers that has been previously implicated in
ATLL and other T-cell malignancies as well as solid cancers, such as ZEB1, POT1,
TET2, SETD2, TGFB1, FYN, and RHOA genes (supplemental Table 1). Among these,
especially intriguing were mutations of RHOA (p.Gly17Val) and TET2 (p.Gln232*),
because this combination of mutations were characteristically found in other distinct
types of PTCLs, i.e. AITL and PTCL-NOS with follicular helper T-cell (TFH-cell)
phenotype2,16,17; being mutated in 83% (TET2) and 70% (RHOA) of the cases, both
were among the most frequently mutated genes in the latter tumors, together with
DNMT3A and IDH2.
Frequent mutations of RHOA in ATLL
Prompted by this finding, we first interrogated mutations of RHOA and TET2, as well as
DNMT3A and IDH2, in a cohort of 203 ATLL cases using targeted deep sequencing, in
which their entire coding exons were individually amplified and subjected to deep
sequencing (mean coverage:18,346×) (supplemental Table 2).
All patients had
documented HTLV-1 infection, where HTLV-1 integration had been confirmed in 201
cases (data not shown).
RHOA and TET2 mutations were detected and validated in
30 (15%) and 20 (10%) cases of our cohort, respectively, whereas DNMT3A and IDH2
were rarely mutated and found only in different few cases (Figure 2A; supplemental
Tables 3 and 4).
Somatic origin of mutations was confirmed for nine RHOA and three
TET2 variants and all mutated cases had viral integration (Figure 2A and 3A;
supplemental Figure 1).
None of the RHOA variants detected in our ATLL series were
observed in 95 normal Japanese samples (data not shown) or in other single nucleotide
polymorphism databases, including dbSNP Build 131, and NHLBI GO Exome
Sequencing Project (ESP) [Feb, 2012 release], as well as our in-house data base from
127 germline controls from cancer-borne patients, except for two variants recorded in
Catalogue of Somatic Mutations in Cancer (COSMIC) v 71 [Nov 2014 release].
In AITL
and related lymphomas, RHOA mutations are always accompanied by TET2 mutations,
2
whereas the latter mutations were less common in ATLL, accounting for only 17% of
RHOA-mutated cases (Figure 2B).
Nevertheless, TET2 mutations, if present, had a
higher valiant allele frequency (VAF) than RHOA mutations, suggesting that TET2
mutations predated the RHOA mutations (Figure 2C).
There were no significant
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
correlations of RHOA and TET2 mutations between disease subtypes or clinical
outcomes (supplemental Figure 2). To examine the association of RHOA mutations
and copy number variations (CNVs), SNP array-karyotyping was performed for the
entire sample set (N = 203).
Overall, high frequency of copy number gains (36%) and
copy neutral loss of heterozygosities (LOH, 2%) was observed at the RHOA locus,
although most CNVs affected large regions of 3p chromosome. CNVs at the RHOA
locus were more frequently detected in RHOA-mutated patients (63%) than in RHOA
wild-type (WT) patients (35%) (Figure 2D).
In addition, patients with CNVs at the
RHOA locus had significantly higher VAFs of RHOA mutations than those without CNVs
(Figure 2E), suggesting the preferential amplification of the mutant allele.
These
results suggest that RHOA is one of the potential targets of copy number change
involving 3p, emphasizing the relevance of RHOA mutations in the pathogenesis of
ATLL.
Unique distribution of RHOA mutations in ATLL
Unexpectedly and conspicuously, RHOA mutations in ATLL exhibited a very different
pattern of distribution from those reported in AITL, even though both mature T-cell
neoplasms are very similar and often indistinguishable from each other on
histopathology except for HTLV-1 infection (Figure 3A).
RHOA mutations in AITL
almost invariably involved the Gly17 residue that participates in the GTP-binding pocket,
causing an identical amino acid change (Gly17Val).
In contrast, mutations in ATLL
were widely distributed but clearly targeted to the GTP-binding pocket, showing discrete
mutational hotspots at the Cys16, Gly17, and Ala161 residues with Cys16Arg mutations
being most prevalent (n = 9).
The Gly17Val mutation was also found in three ATLL
cases but other Gly17-involving mutations had never been reported in AITL, including
Gly17Glu (n = 3) and Gly17Arg (n = 1).
Being commonly involving the same amino
acid within the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively
found in ATLL and AITL, respectively.
detected in 3 cases.
Two independent RHOA mutations were
In ATLL-C-05, Gly17Arg and Gly14Val mutations were on the
same allele in the majority of tumor cells (Figure 4A), whereas a minority carried a
Gly14Val mutation alone, indicating that a Gly14Val-carrying cell that acquired the next
Gly17Arg mutation had outgrown and dominated the tumor population (Figure 4A).
In
the remaining cases harboring Cys16Arg/Thr19Ile and Cys16Leu/Lys118Gln mutations,
a low allelic burden of the second RHOA mutation precluded accurate determination of
the clonal structure (supplemental Table 3).
Almost all mutations found in ATLL were
distributed across all the highly conserved amino acids within or in the vicinity of the
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
GTP-binding pocket (Figure 3A and 4B), most likely affecting GTP-binding capacity
(Figure 4C).
Kinetics of different RHOA mutants for GDP/GTP-binding
RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch
in a wide variety of biological processes through cycling between active (GTP-bound)
and
inactive
(GDP-bound)
states.18
It
has
been
demonstrated
that
the
AITL-associated Gly17Val mutant does not bind GTP and inhibit wild-type RHOA
function in a dominant-negative manner.2,16,17
To evaluate the functional impact of
different RHOA mutations, we first examined the affinity of wild-type and mutant RHOA
proteins to GTP and GDP by measuring kinetics of their binding to a radio-labeled
nonhydrolyzable GTP analog such as [35S]GTPγS and [3H]GDP in vitro.
The Gly17Val
mutant hardly bound [ S]GTPγS and another AITL-associated RHOA mutant
35
(Ala161Glu) also showed little binding to [35S]GTPγS (Figure 5A).
On the other hand,
two mutants newly identified in ATLL (Cys16Arg and Ala161Pro) bound [35S]GTPγS
more rapidly than wild-type RHOA (Figure 5B).
Moreover, dissociation of [35S]GTPγS
and [3H]GDP were prominently accelerated for Ala161Pro and to a lesser extent for
Cys16Arg mutants (Figure 5C-D).
Given a much higher intracellular concentration of
GTP compared to GDP, these data indicated that Cys16Arg and Ala161Pro RHOA
mutants function as a fast-cycling mutant having an increased intrinsic GDP/GTP
exchange rate, favoring the active GTP-bound form.
Biological activity of different RHOA mutants
Since RHOA is one of the master regulators of actin cytoskeleton dynamics in various
cell types and regulates gene transcription through activation of the serum response
factor (SRF) signaling pathway,2 we next assessed the impact of RHOA mutations on
F-actin formation and transcriptional activation of SRF.
immuno-staining,
Gly17Val-
and
Ala161Glu-transduced
When examined by
NIH3T3
cells
showed
attenuated actin stress fiber formation, whereas similar to wild-type RHOA, the
Cys16Arg and Ala161Pro mutants exhibited enhanced actin fiber formation
(supplemental Figure 3).
We further examined transcriptional activity of these
mutations from an SRF-responsive element (SRF-RE) by luciferase assay using
mutant-transduced 293T cells.
The Gly17Val and Ala161Glu RHOA mutants
down-regulated transcription from the SRF-RE, supporting that both mutants can act as
a dominant-negative molecule (Figure 5E).2
In contrast, the Cys16Arg and Ala161Pro
mutants significantly enhanced transcriptional activity, similar to a known constitutively
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VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
active mutant (Gly14Val),19,20 suggesting that these fast-cycling Cys16Arg and
Ala161Pro mutants represented gain-of-function molecules that augment intrinsic
RHOA functions and modulate the downstream transcriptional activity.21
In addition,
Cys16Phe, Lys118Glu and Ala161Val mutants, which were specifically observed in
ATLL cases, also significantly enhanced SRF-RE luciferase activity, suggesting that
these mutations possess a gain-of-function effect similar to Cys16Arg and Ala161Pro
mutations.
Surface and intracellular markers of primary ATLL cells with unique RHOA
mutations
As for the cell-of-origin of ATLL, it has been suggested that ATLL may arise from
different subsets of CD4+ T-cells, i.e. naïve helper, activated or regulatory T-cells,1
whereas TFH-cells are assumed to be the cell-of-origin of AITL and a subset of
PTCL-NOS.2
Thus, a possible explanation of unique variegation of RHOA mutations in
ATLL would be that different mutations reflect different origin of ATLL cells.
To address
this hypothesis, we performed flow cytometric analysis of 12 patient-derived ATLL cells
carrying different RHOA mutations (Table 1 and Figure 6A).
Most of the ATLL samples
carrying wild-type RHOA, Cys16Arg, Cys16Gly and Ala161Pro mutations showed
CD4+CD25+FOXP3+PD-1− or CD4+CD25+FOXP3−PD-1+ phenotypes, suggesting their
origin of regulatory T-cells or effector T-cells.22,23
+
In contrast, Gly17Val positive ATLL
−
cells had CD4 CD25 phenotype, supporting that memory T-cells are likely to be their
cell-of-origin.22-26
Moreover, formalin-fixed paraffin-embedded (FFPE) specimens
obtained from the lymph nodes from two RHOA-mutated ATLL cases were examined by
immunohistochemistry.
The neoplastic cells of the patient with Cys16Arg mutation
(ATLL_A-02) showed CD4+CD25+FOXP3+PD-1−CD3+CD45RO+ phenotype, which was
consistent with the flow cytometry results (Figure 6B).
with
Gly17Glu
+
−
−
mutation
−
+
On the other hand, tumor cells
(ATLL_L-03)
exhibited
+
CD4 CD25 FOXP3 PD-1 CD3 CD45RO phenotype, suggesting that they are derived
from memory T-cells.24-26
These results support the hypothesis that differential RHOA
mutations are linked to distinct cell-of-origins.
Discussion
We have described novel recurrent RHOA mutations in ATLL, accounting for 15% of the
current cohort.
RHOA mutations have been most frequently reported in AITL and
PTCL-NOS with TFH phenotype, where the mutations almost invariably affect the Gly17
residue and accompanied by coexisting TET2 mutations.2,16,17
In contrast, RHOA
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VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
mutations in ATLL are more widely distributed near or within the GTP binding pocket,
suggesting the distinct pathogenesis of ATLL compared other PTCLs.
Also, the RHOA
gene is a plausible target of 3p gain, which is one of the most frequent CNVs in ATLL.27
In this context, a notable finding in the current study is the variegated nature of
RHOA mutations in ATLL.
We identified two types of RHOA mutations in ATLL;
loss-of-function (Gly17Val and Ala161Glu) and gain-of-function (Cys16Arg, Cys16Phe,
Lys118Glu, Ala161Val and Ala161Pro) mutations.
Currently, there is no clear
explanation to the underlying molecular mechanism for this variegation of RHOA
mutations within the same tumor type that shows unique distribution compared to other
PTCLs histologically closely related to ATLL.
A correlation between cellular
phenotypes and mutation types might suggest the different cell-of-origin of ATLL
depending on mutation types.
ATLL cells with gain-of-function mutations had cellular
phenotypes similar to effector or regulatory T-cells, whereas those in cells with
loss-of-function mutations were consistent with memory T-cells. These data suggest
that some HTLV-1 infected T-cells subsets could favor gain-of-function RHOA mutations,
whereas others do loss-of-function type mutations, both being selected for neoplastic
proliferation to develop ATLL.
Alternatively, it is also possible that altered downstream
signaling by RHOA mutations may result in a change of immunophenotype in ATLL cells.
Indeed, RHOA is a key regulator in T-cell receptor signaling, which has been known to
affect the expression of several T-cell subset markers, such as PD-1 or FOXP3.28
Another interesting aspect of the variegated nature of RHOA mutation in ATLL is
two functionally different mutations detected in the same tumors. Particularly, in one
case (ATLL_C-05), Gly14Val and Gly17Arg mutations were found to be present on the
The Gly14Val is a previously known gain-of-function mutant,29 as it
same allele.
prevents intrinsic and GTPase-activating proteins (GAP)-induced GTP hydrolysis, thus
keeping the protein in its active state.18 While Gly17 is located in P-loop, which is a
highly conserved consensus sequence essential for GDP/GTP-binding and GTPase
activities.30
As several kinds of mutations at Gly17 (i.e., Gly17Val and Gly17Ala) have
been shown to act in a dominant-negative manner by disrupting the binding of
GTP/GDP,2,16,17,31 the Gly17Arg mutant is predicted to represent a dominant-negative
RHOA molecule, although the exact functional consequence of this mutant has not been
elucidated. The mechanism for the clonal selection of these double mutants having
apparently opposite functional consequences is totally unknown as well and requires
further studies.
Recently, different RHOA mutations have been reported in a wide variety of
human cancers other than PTCLs.
These mutations exhibit different distributions
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
depending on tumor types; in Burkitt’s lymphoma32 and gastric adenocarcinoma,33-35
Arg5 and Tyr42 are predominantly mutated, whereas head and neck cancers have a
different mutational hotspot at Glu40.36
The strong correlation between phenotype
(tumor type) and mutational distribution indicates that the functional consequence of a
RHOA mutation during tumor development could be conditional on their derived tissues
and cells.
Several genes are affected by mutations having apparently opposite
functional consequences such as both gain-of-function mutations and loss-of-function
mutations. For example, NOTCH1 mutations found in acute T-lymphoblastic leukemia
exclusively reside in the C-terminal PEST and HD domains, leading to constitutive
activation of NOTCH1 signaling,37 whereas those found in many solid cancers show
mutational hotspots within the EGF-like domain and are thought to result in
loss-of-function. Another example of tumor type-specific mutations of the same gene
is gain-of-function mutations of EZH2 at Tyr641 associated with diffuse large B-cell
lymphoma and loss-of-function mutations found in myeloid neoplasms.38
In contrast to
the functionally dichotomous nature of these mutations, a unique and more complex
feature of RHOA mutation is a higher order of functional heterogeneity among different
mutations that are differentially selected in specific tumor types.
More comprehensive
and detailed functional characterization of different RHOA mutants in proper cell
contexts should be warranted to understand the oncogenic role of different RHOA
mutation in ATLL and other RHOA-mutated human cancers and to better target this
molecule for therapy.
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
Acknowledgements
We thank K. Ishiyama and S. Miyawaki for providing specimens. This work was
supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare of Japan and
Grants-in-Aid for Scientific Research on Innovative Areas and by the Japan Society for
the Promotion of Science (JSPS) through the Funding Program for World-Leading
Innovative Research and Development on Science and Technology (FIRST Program)
and by the Medical Research Support Center, Graduate School of Medicine, Kyoto
University. We thank Maki Nakamura, Hitomi Higashi and Miki Sago for their technical
assistance.
Genome sequence data are available at the European Genome-phenome
Archive under accession EGAS00001001210.
Authorships
Y.N., Y.Shiozawa, T.Y., H.S., A.Kon, K.Y., Y.Sato, A.S. and M.S. performed experiments
and data analysis.
Y.Shiraishi, K.C., H.T., Y.T., S.M. and T.S. were committed to
bioinformatics analysis of the sequencing data.
K.Kontani, T.E., T.K., M.H.,
M.Sakata-Yanagimoto., S.E., K.A., H.H., T.K. and S.C. performed the functional
analyses of RHOA mutants.
A.Kitanaka, K.N., K.S. and T.W. collected specimens and
were also involved in planning the project. Y.N., K.Kataoka., R.I. and S.O. generated
figures and tables and wrote the manuscript. S.O. leads the entire project. All authors
participated in discussions and interpretation of the data and results.
Conflict-of-interest disclosure: All authors declare that there are no competing
financial interests.
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
Table 1. Flow cytometry data of patient-derived primary ATLL cells
ID
Subtype
RHOA
mutations
VAFs
CD25
FOXP3
PD1
(% of
(% of
(% of
positive)
positive)
positive)
ATLL_A-02
Acute
C16R
0.12
43
26
0
ATLL_A-04
Acute
C16R
0.31
15
26
1
ATLL_A-03
Acute
C16G
0.14
44
64
21
ATLL_C-08
Chronic
A161V
0.42
42
36
16
ATLL_A-06
Acute
C16F
0.18
80
3
41
ATLL_C-06
Chronic
K118E
0.29
60
27
47
ATLL_A-13
Acute
A161P
0.04
63
8
73
ATLL_A-08
Acute
G17V
0.24
8
4
1
ATLL_A-01*
Acute
G17V
0.31
25
11
6
ATLL_A-20
Acute
WT
-
81
3
77
ATLL_A-66
Acute
WT
-
34
75
42
ATLL_C-42
Chrnoic
WT
-
23
21
6
Control
Carrier
WT
-
9
9
5
* A case was analyzed by whole genome sequencing
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
FIGURE LEGENDS
Figure 1.
Identification of RHOA mutations in ATLL.
Circos plot of genomic alterations obtained from WGS for a single ATLL case.
Locations of somatic non-silent mutations (n = 96) are indicated as orange circle.
Abnormalities in genomic copy number are shown in red for gains, blue for losses, and
green
for
copy
number
neutral
loss
of
heterozygosities.
Chromosomal
rearrangements are indicated in the inner circle (black lines).
Figure 2.
Spectrum and correlation of RHOA mutations in ATLL.
(A) Mutational status of RHOA, TET2, IDH2 and DNMT3A genes in 203 ATLL cases.
Subtype of each case is shown by indicated colors.
(B) Comparison of distributions of
RHOA and TET2 mutations between AITL and ATLL.
Coexistence of RHOA and TET2
mutations are shown in AITL and ATLL.
Red and blue circles indicated cases with
RHOA and TET2 mutations, respectively.
(C) Comparison of variant allele frequencies
(VAFs) between RHOA and TET2 mutations. ATLL cases with both TET2 and RHOA
mutations (n = 5) were analyzed.
Each axis shows the VAFs of mutations. VAFs with
genomic copy number abnormalities were corrected according to the usual methods.
P-value was calculated by Mann-Whitney's U test.
(D) Frequency of CNVs at the
RHOA locus in ATLL cases with or without RHOA mutations.
P-value was calculated
by Fisher’s exact test. (E) VAFs of RHOA mutations in cases with or without CNVs at
the RHOA locus.
Only patients with a single RHOA mutation were considered.
P-value was determined using Student’s t-test.
Figure 3.
Distribution of RHOA and TET2 mutations in ATLL.
(A) Distribution of RHOA mutations in ATLL and other cancers such as AITL,2 Burkitt
lymphoma,32 gastric carcinoma,33-35 and head and neck cancers.36
TET2 mutations identified in ATLL is shown with triangle.
mutations are marked by red triangles.
Figure 4.
(B) Distribution of
Confirmed somatic
Asterisk shows citations of references.
Feature and structural modeling of RHOA mutations in ATLL.
(A) Representative two independent co-occurring RHOA mutations in ATLL cases.
ATLL_C-05 and ATLL_A-07 cases have Gly17Arg/Gly14Val and Thr19Ile/Cys16Arg
mutations, respectively.
(B) Amino acid alignment of RHOA proteins from different
species. Evolutionally conserved amino acids among species are shown in blue, while
the amino acid positions of mutation in ATLL are highlighted by arrowhead.
functional domains are also indicated.
Conserved
(C) Mutated amino acid positions identified in
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Nagata et al
VARIEGATED RHOA MUTATIONS IN ADULT T-CELL LEUKEMIA
ATLL are mapped to the 3D structure of the GTP-biding pocket of human RHOA protein
(Protein Data Bank identification: 1A2B).
GTPγS and the magnesium ion are shown
as white sticks and a green sphere.
Figure 5.
Functional impact of RHOA mutants.
(A-D) Kinetics of GDP/GTP-binding for different RHOA mutants in the presence of
0.8mM Mg2+. WT, Wild-Type (A, B) 5 µM of radio-labeled GTP analogue ([35S]GTPγS)
was incubated with indicated RHOA mutants, and the amounts of RHOA-bound
[35S]GTPγS at indicated time points are plotted.
(C, D) RHOA-bound [35S]GTPγS and
[3H]GDP were measured at indicated time points after their dissociation was initiated by
the addition of unlabeled GTPγS.
Data represent mean ± s.d. (n = 3 for each group).
The significance of difference was determined by two-tailed unpaired t-test.
*P < 0.05,
**P < 0.005, ***P < 0.0005; 3 independent experiments (E) Activity of the SRF-RE
reporter in 293T cells transduced with indicated RHOA mutants in luciferase assay.
The differences between mutants and the wild-type RHOA were all statistically
significant (*P < 0.05, **P < 0.005).
Data represent mean ± s.d. (n = 4). P-value was
determined using Student’s t-test. 3 independent experiments were performed.
Figure 6.
Analysis of cell surface and intracellular markers in RHOA mutated
cases.
(A) Analysis of cell surface marker in RHOA mutated cases. Expression of PD-1 and
FOXP3 (lower) in the CD4+CD25+ tumor cell fraction (upper) in primary ATLL samples.
Result of representative 3 cases is shown.
Case ID, type of RHOA mutations and their
VAFs are also indicated. Numbers in boxes show percentage of cells in the indicated
fractions. (B) Immunohistochemical analysis of FFPE specimens obtained from the
lymph nodes of two RHOA-mutated ATLL cases. Representative images of double
staining with FOXP3 (intra-nuclei, brown) and PD-1 (cytosol, blue) and single staining
with CD25, CD3, CD4, CD45RO (brown) are shown.
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A
ATLL_A-66
RHOA : Wild-type
104
33.9%
103
CD25
ATLL_A-08
RHOA : G17V
VAF : 24%
105
105
Unfractionated cells
ATLL_A-03
RHOA : C16G
VAF : 14%
105
104
104
43.6%
103
102
101
102
101
7.9%
103
102
101
101102 103 104 105
101102 103 104 105
101102 103 104 105
CD4
105
104
Gated cells
FOXP3
103
105
105
39.1%
35.9%
102
101 6.8%
6.1%
101102 103 104 105
104
50.5%
104
13.5%
4.3%
0.0%
103
103
102
101 19.2%
7.9%
101102 103 104 105
102
101 92.3%
0.7%
1 2
3
10 10 10 104 105
PD-1
B
FOXP3
PD-1
CD3
CD4
CD45RO
ATLL_A-02
RHOA : C16R
VAF : 12%
CD25
100μm
100μm
100μm
100μm
100μm
100μm
100μm
100μm
100μm
ATLL_L-03
RHOA : G17E
VAF : 6%
100μm
Figure 6. Analysis of cell surface and intracellular markers in RHOA-mutated cases.
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Prepublished online November 16, 2015;
doi:10.1182/blood-2015-06-644948
Variegated RHOA mutations in adult T-cell leukemia/lymphoma
Yasunobu Nagata, Kenji Kontani, Terukazu Enami, Keisuke Kataoka, Ryohei Ishii, Yasushi Totoki,
Tatsuki R. Kataoka, Masahiro Hirata, Kazuhiro Aoki, Kazumi Nakano, Akira Kitanaka, Mamiko
Sakata-Yanagimoto, Sachiko Egami, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Yusuke Shiozawa,
Tetsuichi Yoshizato, Hiromichi Suzuki, Ayana Kon, Kenichi Yoshida, Yusuke Sato, Aiko Sato-Otsubo,
Masashi Sanada, Wataru Munakata, Hiromi Nakamura, Natsuko Hama, Satoru Miyano, Osamu Nureki,
Tatsuhiro Shibata, Hironori Haga, Kazuya Shimoda, Toshiaki Katada, Shigeru Chiba, Toshiki Watanabe
and Seishi Ogawa
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