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Review Series
T-CELL MALIGNANCIES
Adult T-cell leukemia: molecular basis for clonal expansion and
transformation of HTLV-1–infected T cells
Toshiki Watanabe
Department of Advanced Medical Innovation, St. Marianna University Graduate School of Medicine, Kanagawa, Japan; and Graduate School of Frontier
Sciences, The University of Tokyo, Tokyo, Japan
Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human
T-cell leukemia virus type 1 (HTLV-1) that
develops through a multistep carcinogenesis process involving 5 or more genetic
events. We provide a comprehensive
overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of
genetic abnormalities in ATL that include
alterations highly enriched in genes for
T-cell receptor–NF-kB signaling such as
PLCG1, PRKCB, and CARD11 and gain-of
function mutations in CCR4 and CCR7.
Conversely, the epigenetic landscape of
ATL can be summarized as polycomb
repressive complex 2 hyperactivation
with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27
methyltransferase enhancer of zeste 2
was shown to be induced by HTLV-1 Tax
and NF-kB. Furthermore, provirus integration site analysis with high-throughput
sequencing enabled the analysis of clonal
composition and cell number of each
clone in vivo, whereas multicolor flow
cytometric analysis with CD7 and cell
adhesion molecule 1 enabled the identification of HTLV-1–infected CD41 T cells in
vivo. Sorted immortalized but untransformed cells displayed epigenetic changes
closely overlapping those observed in
terminally transformed ATL cells, suggesting that epigenetic abnormalities are
likely earlier events in leukemogenesis.
These new findings broaden the scope of
conceptualization of the molecular mechanisms of leukemogenesis, dissecting
them into immortalization and clonal progression. These recent findings also open
a new direction of drug development for
ATL prevention and treatment because
epigenetic marks can be reprogrammed.
Mechanisms underlying initial immortalization and progressive accumulation of
these abnormalities remain to be elucidated. (Blood. 2017;129(9):1071-1081)
Introduction
Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused
by human T-cell leukemia virus type 1 (HTLV-1) infection and often
occurs in HTLV-1–endemic areas such as southwestern Japan, the
Caribbean Islands, Central and South America, intertropical Africa, and
the Middle East.1,2 Approximately 1.1 million individuals are carriers
of HTLV-1 in Japan.3,4 The total number of HTLV-1 carriers globally is
estimated to be between 5 000 000 and 10 000 000,5 an apparent
underestimation because of the lack of epidemiological information
in developing countries. Annually, ;1000 people die of ATL in Japan,
based on the national vital statistics data, whereas information on
mortality rates in other countries are limited. Age of onset differs across
geographic areas: the average age at diagnosis in Central and South
America, which is ;40 years, is lower than that in Japan (;60 years).6-8
Estimated lifetime risk of ATL in HTLV-1 carriers is 6% to 7% for
males and 2% to 3% for females in Japan. Additionally, ATL
preferentially develops in individuals infected with HTLV-1 during
childhood and seldom occurs in those infected in adulthood.1 A higher
proviral load (.4 copies/100 peripheral blood mononuclear cells) is an
independent risk factor for progression to ATL.9 Routes of viral
transmission are breast milk, sexual intercourse, and blood transfusion.
However, whether ATL develops after horizontal transmission by
sexual contact or blood transfusion remains unclear, albeit cases of
ATL following organ transplantation were previously reported.10,11
Our recent study revealed .4000 cases of new HTLV-1 infection in
adolescent and old individuals,12 underlying the importance of studies
on HTLV-1–associated diseases among carriers with horizontally
transmitted HTLV-1.
Difference in the age of ATL onset depending on the area in the
world, and evidence for family clustering of ATL, strongly suggests
involvement of environmental and genetic factors in disease
outcome.7,13 As for genetic background, many genes have been
implicated as determinant factors of disease susceptibility such as
HLA class 1, tumor necrosis factor a and killer immunoglobulin-like
receptor.14,15 However, because of the lack of large-scale studies,
clear conclusions have not been reached as to these issues.
Like other retroviruses, the integrated HTLV-1 proviral genome
constitutes of 2 long terminal repeats, gag, pol, and env. The genome
also has an extra sequence designated pX, which has 4 partially
overlapping open reading frames encoding the proteins p12, p13, and
p30, Rex, and Tax. The regulatory proteins, p12, p13, and p30, have
important roles in the establishment and maintenance of HTLV-1
infection in vivo.16 The other regulatory protein, Tax and Rex, act in
combination to regulate HTLV-1 gene expression and replication in
both positive and negative pathways.17 Tax is the viral transcription
activator protein and is also a modulator of cellular gene expression
involved in the proliferation of T lymphocytes mainly via the activation
of the NF-kB and AP-1 pathways. Tax-expressing cells bypass cellcycle checkpoints, affects mechanisms involved in the DNA damage
response and apoptosis pathways, and result in the accumulation of
genetic and epigenetic alterations and RNA stability modifications. Tax
Submitted 6 September 2016; accepted 21 December 2016. Prepublished
online as Blood First Edition paper, 23 January 2017; DOI 10.1182/blood2016-09-692574.
© 2017 by The American Society of Hematology
BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
1071
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1072
BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
WATANABE
has transforming activity in rodent fibroblasts and in primary human
lymphocytes, and Tax transgenic mice using a variety of promoters
develop neoplasia including T-cell lymphoma. Therefore, Tax is
considered to be a main viral factor involved in immortalization and
transformation of the infected cells.18-22
HTLV-1 also expresses a minus-strand RNA that can encode a basic
leucine zipper factor, HTLV-1 bZIP factor (HBZ).23,24 HBZ messenger
RNA (mRNA) was shown to promote the proliferation of ATL cells.25
Through the interaction of cellular proteins, HBZ shows a variety of
functions, some of which antagonize those of Tax. HBZ is considered
to play an important role in the oncogenic process because of its ability
to drive infected cell proliferation, to increase hTERT transcription, to inhibit apoptosis, and to disrupt host genomic integrity
depending on several HBZ-induced microRNAs (miRNAs).17,26-28
CD41 T-lymphocyte–specific expression of the HBZ transgene in mice
induces T-cell lymphoma in one-third as well as systemic inflammation.21,29 HBZ mRNA is expressed in all HTLV-1–infected cells,
including ATL cells ex vivo, suggesting its essential roles in transformation, proliferation, and maintenance of HTLV-1–infected T cells.
in a small-scale study,38 and addition of arsenic trioxide was shown
to induce Tax degradation in vitro.39 A large-scale clinical trial will
provide a conclusive evaluation to this new strategy. Other
strategies are up-front allogeneic hematopoietic stem cell transplantation for relatively young patients with aggressive ATL and
defucosylated humanized anti-CC chemokine receptor 4 (CCR4)
monoclonal antibody (mogamulizumab) for relapsed/refractory
CCR4-positive aggressive ATL.36,40
The prognosis of ATL is still very poor. Recently, a Japanese group
investigating treatment and survival outcomes among 1594 ATL
patients reported that the median survival of acute, lymphoma, chronic,
and smoldering ATL subtypes was 8.3, 10.6, 31.5, and 55.0 months,
respectively, and that the 4-year OS rates were 11%, 16%, 36%, and
52%, respectively.41 They also found that ;20% of patients with acute
or lymphoma-type ATL underwent allogeneic hematopoietic stem cell
transplantation, with median survival and 4-year OS rates of 5.9 months
and 26%, respectively. Thus, prognosis of patients with acute and
lymphoma-type ATL was still unsatisfactory despite recent advances in
treatment modalities; however, they also found that the 4-year OS was
better than that reported in a previous survey. Conversely, the prognosis
of smoldering ATL was worse than expected, as only 25% of transplanted patients experienced long survival times.
Clinical features and treatment of ATL
ATL shows a marked diversity in its clinical manifestations: leukemic
manifestations, generalized lymphadenopathy, hepatomegaly, splenomegaly, skin involvement, hypercalcemia, and infiltration of other
organs such as central nervous system and gastrointestinal tract.
Symptoms and signs of ATL include abdominal pain, diarrhea, ascites,
jaundice, pleural effusion, cough, sputum, fever, and unconsciousness
because of hypercalcemia and/or opportunistic infections. ATL is
classified into 4 clinical subtypes (ie, smoldering, chronic, acute, and
lymphoma) based on the diagnostic criteria proposed by Shimoyama
et al30: site of infiltration, presence and degree of leukemic manifestation, lactate dehydrogenase level, and hypercalcemia.
ATL cells in the majority of cases express CD3, CD4, and CD25,
but lack CD7.31,32 In ;10% to 15% cases, ATL cells express both CD4
and CD8. The origin of the ATL cells was suggested to be regulatory
T cells based on the expression of FoxP3 in some tumor cells,33 which,
however, remains a subject under debate.34
International consensus for ATL treatment strategy is based on the
clinical subtype, prognostic factors, and response to initial therapy.35,36 For
patients with aggressive ATL (acute and lymphoma types and chronic
type with poor prognostic factors), intensive chemotherapy regimens
such as VCAP-AMP-VECP (vincristine, cyclophosphamide, doxorubicin, and prednisone; doxorubicin, ranimustine, and prednisone; vindesine,
etoposide, carboplatin, and prednisone) or zidovudine/interferon a (AZT/
IFN-a) combination therapy, except for lymphoma-type patients, is
recommended. For indolent ATL (smoldering type and chronic type
without poor prognostic factors), watchful waiting or AZT/IFN-a
combination therapy is recommended until disease progression. In the
United States and Europe, antiviral therapy using AZT∕ IFN-a is the
standard treatment of leukemic-type ATL. In Europe, chemotherapy is
the first-line therapy for lymphoma-type ATL because overall survival
(OS) with AZT∕ IFN-a alone is very short.37 In Japan, AZT/IFN-a therapy
has not been introduced because the national health insurance system has
not yet approved the use of these 2 drugs in the treatment of ATL patients.
A randomized phase 3 clinical trial is now underway for treating indolenttype ATL with AZT/IFN-a vs watchful waiting. This clinical trial will
provide conclusive information regarding the optimal standard treatment
of indolent-type ATL. Furthermore, efficacy of the combination of AZT/
IFN-a and arsenic trioxide in chronic ATL patients has been reported
Abnormalities in gene expression in ATL
mRNA expression
ATL cells express cytokines, chemokines and chemokine receptors, and
adhesion molecules, most of which are targeted by the viral transactivator protein Tax.33,42-44 Cell adhesion molecule 1 (CADM1), a cell
surface molecule originally isolated as a tumor suppressor in non–small
cell lung cancer,45 was highly expressed in ATL cells and in HTLV1–infected nontransformed T cells in carriers.46 CADM1 is currently
used for the identification of HTLV-1–infected T cells by multicolor
fluorescence-activated cell sorting analysis.47,48 Parathyroid hormonerelated protein is another protein that is highly expressed in ATL cells
and is responsible for humoral hypercalcemia of malignancy observed
during the clinical course of more than half of ATL patients.49 Other
factors that are highly expressed in ATL cells, such as interleukin-1,
transforming growth factor b, tumor necrosis factor b, and receptor
activator of nuclear factor k-B ligand, have been implicated to be
involved in hypercalcemia in ATL patients,50-54 suggesting a complex
mechanism is underlying this characteristic paraneoplastic syndrome.
Overexpression of cell adhesion molecules and chemokine receptors
appear to facilitate organ infiltration of ATL cells.42-44,55-57 One
intriguing aspect of these findings was the expression of genes that were
not normally expressed in T cells,58,59 which was subsequently confirmed by expression array studies. The lineage-independent ectopic
expression of many genes characterized ATL cells and strongly
suggested abnormalities in epigenetic regulation that determined
tissue-specific gene expression.
miRNA expression
Studies on miRNA expression profiles in HTLV-1/ATL cell lines and
primary ATL cells demonstrated both up- and downregulation occurring
in a number of miRNAs. In HTLV-1–transformed cell lines, miR-21,
miR-24, miR-146a, and miR-155 were upregulated, whereas miR-223
was downregulated.60 In primary ATL cells, miR-181a, miR-132, and
miR-125a were downregulated, whereas miR-155 and miR-142-3p were
upregulated by microarray analysis.61 Yet, in another study, among a
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BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
Figure 1. Comparison of molecular abnormalities
between HTLV-1–infected T cells and transformed
ATL cells. Many aspects of the ATL cell phenotype
share common characteristics with that of untransformed HTLV-1–infected T cells expressing viral
proteins, including Tax. Tax was shown to induce the
majority of molecular changes observed in HTLV1–infected cells, most of which are preserved in ATL
cells that do not express Tax. Thus, this phenomenon
is sometimes referred to as the signature of Tax.
CARD11, caspase recruitment domain-containing protein 11; GPR183, G-protein coupled receptor 183;
NRXN3, neurexin-3; PLCG1, phospholipase C, g 1;
PRKCB, protein kinase C b.
CLONAL PROGRESSION OF ATL CELLS
HTLV-1-infected
T cells
Terminally transformed
ATL cells
Genetic
events
Telomere
DNA repair
HTLV-1
Tax
HBZ
TP53
CDKN2A
NRXN3, etc
?
Abnormal transcription
Impairment of DNA repair
Inductionof genome instability
Aberrant signal transduction
Deregulated cell cycle check points
Cell proliferation,
Apoptosis resistance
immortalization
Inflammatroy reaction
total of 6 miRNAs that were upregulated, miR-93 and miR-130b were
shown to target TP53INP1 tumor suppressor.62 Our study on miRNA
expression signature of primary ATL cells demonstrated that the vast
majority of differentially expressed miRNAs were downregulated, with
the most severe suppression observed in miR-31. Intriguingly, we
showed that miR-31 targeted MAP3K14 (NIK), which is overexpressed
in ATL cells, leading to constitutive activation of the NF-kB pathway.63
Signal transduction
A large body of literature has accumulated showing abnormalities in
signal transduction pathways in HTLV-1–infected cell lines and
primary ATL cells. It is widely accepted that HTLV-1 Tax activates
various signaling pathways through direct interactions with signaling
molecules.64 However, only certain aspects of these signaling
pathways were confirmed to be activated in primary ATL cells in
the absence of Tax.64 Thus, Tax-mediated activation of signaling
pathways appears to be involved in the earlier stages of transformation,
and transformed cells retain some of the constitutively activated
pathways that are essential for proliferation and apoptosis resistance
of ATL cells.64,65 Among these, the best-known example is the NFkB pathway, which is constitutively active in ATL cells.66 One of
the mechanisms for constitutive NF-kB activation in ATL cells in the
absence of Tax is NIK overexpression caused by miR-31 silencing.63,67
Inhibition of NF-kB was shown to induce ATL cell apoptosis and
specifically reduce the number of HTLV-1–infected T cells in
asymptomatic carriers.68 Phosphatidylinositol 3-kinase/AKT cascade,
which was also constitutively activated in ATL cells, induced the
formation of multilobulated nuclei characteristic of ATL cells.69
Furthermore, Notch signaling pathway was constitutively activated, as
activating mutations in Notch were found in .30% of ATL patients.
Mutations in F-box and WD repeat domain-containing 7 (FBXW7)
found in 25% of patients, hindered interaction between FBXW7 and
intracellular Notch (NICD), resulting in increased protein stability and
constitutive Notch1 signaling.70 Finally, Jak/STAT pathway, another
signaling pathway activated in ATL cells, was associated with cell
proliferation.71 Although there are a number of other signaling
pathways exhibiting abnormalities in the presence of Tax, they
were not well characterized in primary ATL cells.
Abnormal gene expression and signal transduction observed in
ATL cells have much in common with HTLV-1–infected and virusexpressing T cells, although viral genes, except for antisense transcripts,
are not expressed in ATL cells (Figure 1).
1073
TCR signal
NF-κB pathway
PLCG1
PRKCB
CARD11, etc
GPCR
(Cell migration)
CCR4
CCR7
GPR183, etc
Gene
aberration
Genomic abnormalities in ATL
Cytogenetic characteristics
ATL cells exhibit a variety of cytogenetic abnormalities in almost all
patients; however, specific recombination or amplification/deletion
events have not been identified.72 Certain chromosomal aberrations
were shown to correlate with 1 or more of the clinical features as well as
the clinical severity.73 Comparative genomic hybridization analysis
also revealed a correlation of the levels of aberrations with the clinical
course. The most frequent aberrations included gains at chromosomes
14q, 7q, and 3p and losses at chromosomes 6q and 13q. Chromosomal
imbalances, losses, and gains were more frequently observed in aggressive ATL cases than in indolent ATL cases. Analysis of sequential
samples during progression suggested clonal changes at crisis; clonal
diversity was common during progression to ATL, and chromosomal
alterations were associated with the clinical course.74
Abnormalities in specific genes
Inactivation of tumor suppressors by somatic mutations or epigenetic
mechanisms is frequently reported in a wide range of cancers and
is suggested to be involved in tumor initiation and development. In
ATL, main genetic events have been reported to cluster around cyclin
dependent kinase inhibitors such as p15 (INK4A), p16 (INK4B), p18
(INK4C), p19 (INK4D), p21 (WAF1), p27 (KIP1), and p57 (KIP2), as
well as p53 and retinoblastoma (Rb); all these genes play major
regulatory roles during the G1/S transition of cell cycle.75 Frequent
alterations were found in p15 (20%) and p16 (28% to 67%) in
aggressive-type (acute and lymphoma) ATL, whereas abnormalities
were fewer in p15 (0% to 13%) and p16 (5% to 26%) in indolent-type
(chronic and smoldering) ATL. Most of these changes were gene
deletions; mutations occurred less frequently. Patients with deleted p15
and/or p16 had significantly shorter survival than those individuals with
both genes preserved. Genetic alterations in p18, p19, p21, and p27
have rarely been reported.
Conversely, p53 gene was mutated in 10% to 50% of aggressivetype ATL cases, whereas its frequency was lower in indolent-type
ATL.76,77 These data clearly implicate that mutations in these cell
cycle-related genes are more likely to be associated with progression to
more severe stages of ATL than with earlier clinical stages of this
malignancy.
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1074
WATANABE
The leukemic cells of most ATL patients and HTLV-1–transformed
cell lines contain elevated levels of functionally inactive wild-type p53
protein. HTLV-1 Tax oncoprotein alone was shown to be sufficient for
abrogating the transactivating function of p53 and for its stabilization in
the absence of direct binding between Tax and p53.78 In addition, HBZ
was shown to inhibit p53 function through repression of the histone
acetyltransferase activities of p300 and HBO1.79 Given the constant
expression of HBZ in all HTLV-1–infected cells, these data may provide
a clue to explain the underlying mechanisms of p53 inactivation in ATL
cells in the absence of Tax expression in majority of cases.22
The Rb tumor suppressor gene is infrequently altered in
structure77,80; however, ;50% of ATL cases exhibit loss of Rb
protein.81 Additionally, low levels of Rb expression correlated with
poor prognosis and shorter survival.82
Notably, alterations in any one of the cyclin dependent kinase
inhibitors, p53, or Rb appear to obviate the need for inactivation of other
genes in the same pathway. In summary, tumor suppressor genes,
which were shown to be frequently altered in aggressive ATL, are the
likely driving force fueling the clonal progression of tumor cells.
Comprehensive analysis of genomic
abnormalities in ATL
Recently, results of an integrated genomic and transcriptomic analysis
of a cohort of 426 ATL cases were reported.83 Massive genomic,
methylomic, and transcriptomic data, coupled with cell-based
experiments in this study, provided comprehensive and detailed
information to provide insight into ATL pathogenesis and confirmed
the presence of deletions and mutations in the integrated proviral
genome and the lack of expression of the sense strand, including mRNA
encoding Tax, in contrast to the constitutive expression of antisense
transcript HBZ.
Whole-exome sequencing of 81 ATL cases, combined with targeted
resequencing of 370 of the samples, identified 50 genes that were
recurrently and significantly mutated; 13 of these genes were affected in
.10% of the cases. The most frequently mutated genes were PLCG1
(36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), and IRF4 (14%),
all of which are implicated in T-cell receptor (TCR)–NF-kB signaling.
In addition, CCR4 or CCR7 were mutated in 29% and 11% of the cases,
respectively. Furthermore, CCR4 Tyr331 and CCR7 Trp355 were
shown to be sites of gain-of function mutations.83
Single nucleotide polymorphism array–based copy number analysis of 426 ATL cases in the same study revealed 50 copy number
decrease and 26 amplification events. Some of the genes with copy
number abnormalities overlapped with gene mutation sites. To
characterize structural abnormalities, whole-genome sequencing was
performed on 48 paired samples. On average, 60 structural variations
(SVs) per sample were identified, which included accumulated deletions in common fragile sites such as 14q31.1 (NRXN3), 7q31.1
(IMMP2L), 1p21.3 (DPYD), and 3p14.2 (FHIT). NRXN3 deletion was
demonstrated in .60% of ATL cases. These results further reflected
the genomic instability of ATL cells.83
Accumulation of additional mutations affecting the TCR and
NF-kB pathways, together with the inactivation of p53, p16, and other
mutations, eventually transforms T cells into fully malignant cells. Data
also indicate that ATL cells delete, mutate, or hypermethylate genes that
encode components of the class I major histocompatibility complex
(MHC-I), death receptors, and proteins involved in cellular adhesion or
immune checkpoints as a strategy to escape detection by the immune
system83 (Figure 2A-B).
BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
In a study investigating SVs, the 39 region of the programmed cell
death 1 ligand (PD-L1) gene was found to be disrupted by SVs in 27%
of ATL cases.84 These SVs invariably led to marked elevations in
aberrant PD-L1 transcripts that were stabilized by truncation of the 39untranslated region. This is a unique genetic mechanism of immune
escape triggered by SVs.
Deep sequencing of 203 ATL cases by Nagata et al found RHOA
mutations in 30 cases that were widely distributed across the entire
coding sequence but were almost always located at the guanosine
triphosphate–binding pocket.85 Depending on the mutation type and
position, these mutants showed variegated functional consequences,
suggesting the involvement of both loss- and gain-of-RHOA functions
in ATL leukemogenesis.
Another report showed mutations in SUZ12, DNMT1, DNMT3A,
DNMT3B, TET1, TET2, IDH1, IDH2, MLL, MLL2, MLL3, and
MLL4.86 TET2 was the most frequently mutated gene, occurring in 32%
of the samples (10/31). Next-generation sequencing revealed nonsense
mutations accompanied by loss of heterozygosity in TET2 and MLL3,
suggesting important consequences of MLL3 and TET2 inactivation in
the leukemogenesis of HTLV-1–induced ATL.
The same group has reported activating mutations in genes in
Notch-1 signaling, Notch-1 PEST domain, and F-box and WD repeat
domain containing 7 (FBXW7)/hCDC4 in ;20% to 30% of cases, both
of which were shown to contributes to proliferation and tumor
formation.70,87
Epigenetic abnormalities in ATL
DNA methylation abnormalities
The CDKN2 locus encodes proteins such as p16, p14, and p15 and is
involved in cell cycle regulation. It is also considered a hot spot of
genomic and epigenomic abnormalities. In our copy number variation
analysis, genomic deletions at this locus were found in 46 out of 168
cases (27%).63 Additionally, CpG methylation analysis showed
hypermethylation of this locus in 47% and 73% of acute-type and
lymphoma-type ATL cases, respectively, whereas the methylation
levels in both chronic- and smoldering-type ATL cases were 17%.88 In
another report, CpG methylation levels of CDK2B were higher than
those of CDK2A.89 In contrast, our expression profiling of ATL
samples did not show any downregulation in the expression of CDKN2
family members. This underscores the importance of detailed analyses
of expression levels and functional consequences of cell cycle
regulators in ATL cells. Progressive accumulation of CpG methylations
of HCAD, SHP1, DAPK, and other genes with disease progression was
also reported.90 CDKN1A (p21waf/Cip1) was reported to be downregulated by DNA methylation,91 which was in line with our data.63
These results suggest a relationship between abnormal DNA methylation and cell cycle regulation and the possible breakdown of their
functions in ATL.
In addition to cell cycle regulators, DNA hypermethylation of bone
morphogenetic protein-6 (BMP6), adenomatous polyposis coli (APC),
and CD26 was also reported.92-94 A screening study using methylated
CpG island amplification of representational difference analysis
revealed abnormal DNA methylation in 53 genes, including those
involved in apoptosis resistance such as KLF4 and EGR3.95
A comprehensive description of CpG methylation in ATL was also
reported by Kataoka et al.83 ATL genome was characterized by
prominent CpG island DNA hypermethylation; in ATL, a much higher
number of genes were significantly hypermethylated with transcriptional
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BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
TCR
A
CLONAL PROGRESSION OF ATL CELLS
CD28
BCR
B
Intragenic deletions
TP73
CARD11
CD3
PLCγ 1
Fusion 7%
Mutation 2%
Amplification 18%
VAV1
mutation
18%
RHOA
mutation
8%
PLCγ 2
BCL10
CARD11
mutation
24%
IKKβ
Nucleus
IKK γ
IKKα
IRF4
mutation
14%
NF-κ B
IKZF2
mutations
PKCθ
MALT1
1075
PKCβ
CSNK2B
GATA3
CSNK2A1
HNRNPA2B1
CSNK1A1
GPR183
mutation
33%
C
CpG island methylator
phenotype (CIMP)
NOTCH1
mutation
15%
C2H2 zinc finger genes
MHC class I genes
STAT3
> 90%
mutation
21%
Retrovirus expression
Immune evasion
Figure 2. Schematic summary of genetic abnormalities in ATL cells. (A) Accumulation of mutations in TCR signaling and NF-kB pathway. (B) examples of minor
mutations. Intragenic deletions and mutations in genes other than those involved in TCR signaling. (C) possible effects of cytosine guanine dinucleotide (CpG) island
methylator phenotype (CIMP). CSNK2B, casein kinase II subunit b; CSNK2A1, casein kinase 2 a 1; CSNK1A1, casein kinase 1 a 1; HNRNPA2B1, heterogeneous nuclear
ribonucleoproteins A2/B1; IKZF2, zinc finger protein Helios.
silencing. Approximately 40% of the samples showed more extensive hypermethylation, known as the CpG island methylator
phenotype (CIMP). CIMP status was associated with aggressive
phenotypes but not with the mutation status of epigenetic regulators
known to be associated with CIMP in other cancers (TET2, IDH2,
and DNMT3A). Additionally, C2H2-type zinc finger genes implicated
in the suppression of both endogenous and exogenous retroviruses
were hypermethylated and silenced. Furthermore, MHC-I genes were
hypermethylated and silenced in many ATL cases, and loss of MHC-I
expression was observed in ;90% of ATL cases, contributing to
immune evasion by ATL cells (Figure 2C).
Although these data provide a basic understanding of the DNA
methylation status in ATL cells, mechanisms underlying the abnormal
DNA methylation and its biological significance remain to be determined.
in ATL cells and HTLV-1–infected cell lines.96,97 Although several
clinical trials assessing several HDACis in ATL are ongoing, future
studies are paramount to determine the mechanisms of histone
deacetylase abnormalities and the efficacy of HDACis.
Trimethylation of H3K27 (H3K27m3) is well known for its role in
survival, proliferation, de-differentiation, invasion, and metastasis of
many human cancers such as breast and prostate cancer, and B-cell
lymphoma.98 H3K27m3 is a marker for the suppression of gene
expression in euchromatin region. Polycomb group (PcG) proteins
form multiprotein complexes that function as transcriptional repressors
of several thousand genes controlling differentiation pathways during
development. PcG proteins work as transcriptional repressors through
ATL cells
Abnormal histone modification patterns
Various chemical histone modifications are involved in the modulation
of chromatin structure and gene expression. Gene expression profile of
ATL cells is characterized by marked deviations from that of normal
CD41 T cells, with upregulated expression of genes encoding various
receptors and cytokines as well as lineage-independent aberrant expression of various differentiation-associated genes63 (Figure 1). These
data suggest the presence of complex aberrations in histone modifications in ATL cell and provide a rationale for aberrant gene expression
profiling. However, in contrast to DNA methylation studies, significant
progress in characterization of histone modifications in ATL cells has
not been made until recently, mainly because of technical difficulties.
Although histone acetylation abnormalities in ATL cells have not
been well characterized, apoptosis induction and inhibition of NF-kB
activity by histone deacetylase inhibitors (HDACis) have been reported
EZH1/2
dual inhibitor
PRC2
EZH2
HTLV-1
H3K27me3
PRC2
EZH2
PRC2
EZH2
EZH1
Tax
EZH1
ON
Normal
CD4+ T cell
H3K27me3
OFF
EZH1
OFF
Tumor suppressor
Transcription factors
miRNAs, epigenetic
modifiers, etc
HTLV-1 infected
HTLV-1 infected
transformed T cell
immortalized T cell
Accumulation of epigenetic abnormalities
Figure 3. Epigenetic landscape of ATL cells. High levels of EZH2 expression is
observed in HTLV-1–infected cells as well as in ATL cells. Tax and NF-kB can induce
EZH2 expression. ATL cells are characterized by PRC2 overexpression and H3K27 m3
accumulation, the level of which appears to progress with clonal progression.
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NF-κB
?
ATL-specific gene expression
HTLV-1
Tax
Tumor
suppressors
PRC2
secondary
effects
on
gene
expression
EZH1/2
EED
SUZ12
microRNAs
Epigenetic
modifiers
H3K27me3
Transcription
factors
Non-canonical
function?
Cellular and Clinical Characteristics
Figure 4. Accumulation of H3K27m3 as the basis of ATL cell phenotype. PRC2mediated accumulation of H3K27 me3 suppresses important genes such as tumor
suppressors, miRNAs, epigenetic modifiers, and transcription factors, culminating in
abnormalities in regulation of downstream genes that determine the phenotype of
ATL cells.
chemical modifications of histones by 2 major PcG protein complexes:
polycomb repressive complex 1 (PRC1) and PRC2. PRC2 contains
enhancer of zeste 1 (EZH1) and EZH2 that function as methylases for
H3K27. PRC1 recognizes H3K27 m3 and induces ubiquitination of
H2AK119 (H2K119Ub), stabilizing the chromatin structure.
Our recent comprehensive study on the polycomb-dependent
epigenetic landscape of ATL cells provided data to decipher the ATLspecific epigenetic code critical for ATL pathogenesis.99 Integrative
analyses of the epigenome (n 5 3) and transcriptome (n 5 58) of
primary ATL cells revealed that PRC2 components, including EZH2,
were highly expressed and that H3K27m3 was significantly and
frequently reprogrammed in over half of the genes (53.8%). Furthermore, H3K27me3 pattern in ATL was distinct from that in other cancer
types and PcG-dependent cell lineages. No EZH2 mutation was found
among 50 ATL patients included in our study. Progressive downregulation of gene expression was demonstrated with disease progression from indolent to aggressive ATL. Genes that were downregulated
included key genes such as miR-31, BCL2L11, EVC1/2, CDKN1A, and
NDRG2. Another finding was the presence of diverse outcomes
resulting from the remote regulation of a broad spectrum of gene
regulators, including various transcription factors, miRNAs, epigenetic
modifiers, and developmental genes. Increased EZH1 expression,
which compensated for the functions of EZH2, was also confirmed in
peripheral T cells. Collectively, PRC2 hyperactivation with genomewide H3K27m3 accumulation characterized the ATL epigenome.
Consequently, simultaneous depletion of EZH1 and EZH2 by shRNAs
was demonstrated to significantly reduce cellular H3K27m3 levels and
Lymphoma
Smoldering
Carriers
Chronic
Acute
High Risk Carrier
Characteristics of infected cells in vivo
Indolent Type
Aggressive Type
Clonality based on integration sites
mir - 31
P
CD7
EZH2
D
IKZF2:
splicing
abn
N
mir - 31
EZH2
CADM1
IKZF2:
abn splicing
Polyclonal
Oligoclonal
Monoclonal
Figure 5. Schematic description of clonal progression and phenotypic changes. HTLV-1–infected T cells and ATL cells in vivo are now available for molecular analyses.
Accumulating data indicate that epigenetic abnormalities occur early during leukemogenesis, as untransformed HTLV-1–infected cells show evidence of epigenetic
abnormalities that are observed in ATL cells as well. The extent of clonality during HTLV-infection and progression to ATL have been characterized in detail by recent studies
that provide information on clonal progression based on integration sites.
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BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
Infancy
HTLV-1
Infection
CLONAL PROGRESSION OF ATL CELLS
Old age
Young and middle age
Immortalization
clonalization
1077
Clonal
expansion
Malignant
transformation
Clone=a
b’
B’
b’
Clone=b
TN
TEM
b”
TSCM
TCM
Clone=c
b”
x’
Clone=x
x”
Epigenetic change
Genetic change
Figure 6. Schematic presentation of the clonal progression and hierarchical structure of HTLV-1–infected T cells. A clone that is defined by the integration site
contains subclones with different genetic abnormalities. Among these subclones, there is a hierarchical structure where TSCM cells behave as ATL-initiating stem cells. TCM,
CD45RO1 central memory T cells; TEM, CD45RO1 effector memory T cells; TN, CD45RA1 naive T cells.
to dramatically inhibit ATL cell growth compared with single depletion
of either EZH, suggesting that the compensatory actions of EZH1/2
were critical for ATL (Figures 3 and 4).
Clonal growth and identification of
HTLV-1–infected T cells in vivo
Clonality of ATL cells and HTLV-1–infected T cells in carriers
Clonal growth of ATL cells was first demonstrated by Southern blot
analysis using HTLV-1 provirus DNA as a probe.8 ATL cells were
found to be monoclonally expanded HTLV-1–infected cells except for
rare cases with 2 or more provirus copies in ATL cells. Subsequent
studies demonstrated that some ATL patients had multiple simultaneously occurring ATL clones at certain time points during the clinical
course of disease.100-103 Clonal exchange during the clinical course was
also reported.104,105 Array comparative genomic hybridization analysis
of paired samples from the peripheral blood and lymph nodes of
patients with acute-type ATL revealed multiple subclones of tumor
cells with distinct genomic aberrations.106 The data suggested that
clonal progression takes place in lymph nodes and a selected subclone
among the lymph node subclones appears in the peripheral blood.
Collectively, these reports strongly suggested the presence of clones
concurrently undergoing transformation in vivo.
Inverse polymerase chain reaction (PCR) detected multiple
amplified bands consisting of HTLV-1 proviral and flanking cellular
DNA in HTLV-1–infected T cells in asymptomatic carriers, demonstrating the polyclonal nature of HTLV-1–infected cells in
asymptomatic carriers.107 This technique has been widely used to
characterize the clonality in various clinical settings.108-110 Difference
in the stability of clonality among patients was shown to depend on the
mode of virus transmission. The clonality of adults who acquired
HTLV-1 horizontally from their spouses was more heterogeneous and
less stable than that of long-term carriers who appeared to be infected
through breastfeeding in infancy.111,112 Linker-mediated PCR was
more sensitive, allowing detection of a greater number of integrated
proviruses.113 HTLV-1 carriers doubly infected with Strongyloidiasis
stercoralis showed oligoclonal expansion of HTLV-1–infected cells
and high proviral loads.114 These techniques provided information on
the clonality of HTLV-1–infected cells in various clinical conditions,
although the findings were descriptive but not quantitative.
Subsequently, Bangham and colleagues developed a novel, highthroughput protocol using next-generation sequencing to map and
quantify provirus integration sites.115 They found that there were
between 10 000 and 100 000 clones in the peripheral blood of HTLV1–infected individuals.115 They also claimed that HTLV-1 integration
into the host genome was not random but showed associations with
specific transcription factor binding sites116 and that specific sites
upstream of certain proto-oncogenes were associated with ATL.117
Although these new findings significantly advanced the understanding
of the clonality of HTLV-1–infected T cells in vivo, this technique
unfortunately had limited utility in determining the number of infected
cells belonging to a specific clone. To successfully measure the number
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BLOOD, 2 MARCH 2017 x VOLUME 129, NUMBER 9
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of cells in individual clones, Bangham et al depended on the size
variation of sheared genomic DNA fragments that showed the maximum variety of 400, because the size of recovered DNA fragments
were distributed between 300 and 700 base pairs after shearing. To
overcome this limitation, Firouzi et al introduced oligomer tags of 8
nucleotides that marked sheared DNA before amplification, which
enabled the discrimination of .60 000 cells, dramatically expanding
the ability to quantify the number of cells in individual clones.118 The
results of these analyses provided firm evidence for the hypothesis that
HTLV-1 carriers harbored a large number of HTLV-1–infected and
immortalized clones and that each clone originated from a single infected
T cell (Figure 5). The size of cell populations belonging to specific clones
(ie, clone sizes) also varied greatly, and some clones had populations
large enough to be detected as clonal expansion by conventional
techniques, such as Southern blot hybridization and inverse PCR.
Furthermore, newly developed techniques to characterize the
integration sites of HTLV-1 provirus provided a new set of evidence
that encourages reappraisal of the classic promoter insertion model of
retrovirus-mediated tumor genesis.119
Identification of HTLV-1–infected cells in vivo
For the identification of HTLV-1–infected T cells in vivo, a technique
using multicolor flow cytometry and fluorescence-activated cell sorting
was developed. The CD4 cells expressing CADM1 with reduction of
CD7 expression represent the transformation status of infected cells and
allow for the discrimination of immortalized but not untransformed
cells from transformed cells.47,48 This method enabled molecular
characterization of HTLV-1–infected T cells in vivo during both immortalization and transformation. Expression array analysis of fractionated CD41 cells revealed that H3K27m3 target genes were
suppressed in immortalized HTLV-1–infected cells as well as in
terminally transformed ATL cells; suppression was more extensive in
the latter cells47 (Figure 5). These data provided evidence that epigenetic abnormalities were one of the earlier leukemogenic events
of HTLV-1–infected T cells. Currently, studies utilizing mutation
analyses of cells derived from each fraction are underway to determine the
timing of genetic mutations during the development of ATL. Ultimately,
these data will provide in-depth information on the chronological
landscape of genetic and epigenetic changes accumulated during
immortalization and transformation of HTLV-1–infected CD4 T cells.
Recently, a hierarchical structure of ATL cell clones has been
reported. CD45RA1 T memory stem (TSCM) cells were identified as the
hierarchical apex that is capable of reconstituting identical ATL clones.
TSCM cells may be a venue for clonal evolution and reservoir for ATL
cells that can be exploited for the development of future molecular
targeted therapies.120
Summary and perspectives
Significant progress has been made in the field of ATL research, with
a growing body of information on the cellular and molecular
characteristics of ATL cells available for better understanding of
disease progression. In addition, “microenvironment” of the ATL cells
is supposed to be involved in ATL cell survival and resistance to
chemotherapy. Several reports suggested importance of interaction
with normal epithelial cells and mesenchymal stem cells.121-123
Furthermore, involvement of angiogenesis and fibroblast-derived
osteopontin has been suggested in proliferation and survival, as well
as possible roles of exosomes produced by ATL cells.124-126 In this
regard, a possible role of mutations of Notch1 signaling70,87 appears to
deserve further examination. Further studies are expected to understand
the mechanisms underlying resistance and survival of ATL cells in vivo.
Xenograft models transplanted with ATL-derived cell lines or primary
ATL cells using SCID mice have been reported, which is usually used
for testing the efficacy of drugs.127 However, several questions remain
unanswered. First, information on the mechanisms underlying immortalization after HTLV-1 infection of T cells is limited because of
technical difficulties for in vitro infection experiments and the lack of
appropriate animal models of HTLV-1 infection. Second, molecular
events during clonal progression have not yet been fully elucidated. The
reported landscape of genetic and epigenetic abnormalities is mostly
that of terminally transformed ATL cells; thus, studies on HTLV1–infected clones at various stages during transformation will provide
crucial data needed to understand the mechanisms of immortalization
and clonal expansion (Figure 6). Third, analysis of genetic and
epigenetic abnormalities accumulating in individual HTLV-1–infected
clones is necessary to understand the mechanism of clonal progression
and malignant transformation. Finally, elucidation of factors that
determine the provirus load is anticipated, given that proviral load is one
of the major risk factors that appear to be determined by host factors.
Taken together, clarification of molecular processes involved in the
natural history of HTLV-1 infection is paramount for the prevention and
treatment of HTLV-1-associated diseases, including ATL.
Acknowledgments
This work was supported by grants from the Japan Society for the
Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI)
(JP26293226) and the Japan Agency for Medical Research and
Development (AMED), Practical Research for Innovative Cancer
Control (16ck0106133h0003 and 16ck0106136h0003) (T.W.).
Authorship
Contribution: T.W. collected all the information and prepared the
manuscript and figures.
Conflict-of-interest disclosure: Part of the research by T.W. was
supported by a research fund from Daiichi Sankyo Company.
ORCID profiles: T.W., 0000-0002-1198-3773.
Correspondence: Toshiki Watanabe, St. Marianna University
Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki,
Kanagawa 216 8512, Japan; e-mail: [email protected].
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2017 129: 1071-1081
doi:10.1182/blood-2016-09-692574 originally published
online January 23, 2017
Adult T-cell leukemia: molecular basis for clonal expansion and
transformation of HTLV-1−infected T cells
Toshiki Watanabe
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