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risk of tumor necrosis and toxicity resulting
from virus reactivation.7 Second, autologous
cytotoxic T cells expanded against EBV
antigens have been tested, but the very
frequent expression of programmed death
ligand 1 (PDL1) and immunosuppressive
cytokines by NK/T lymphoma cells could
inhibit T-cell cytotoxicity.8,9 Indeed, in most
cases, an immune deficiency is not observed in
patients with NK/T-cell lymphoma, strongly
suggesting that tumor cells have developed
strategies to escape the strong immune
response usually developed against EBV
antigen–expressing cells. This expression
of PDL1 by NK/T lymphoma cells is likely
to be involved in this resistance, as seen in
other EBV-associated neoplasia such as
nasopharyngeal lesions, gastric carcinoma, and
Hodgkin disease. This combination of frequent
PDL1 expression and presence of foreign
antigen expression on tumor cells makes the
use of checkpoint inhibitors very attractive.
Kwong and colleagues, on behalf of the Asia
Lymphoma Study Group, demonstrated in
this small series of patients the effectiveness
of pembrolizumab, an anti-PD1 antibody, in
patients with NK/T-cell lymphoma. The 7
patients, previously treated using asparaginasebased regimens, all had advanced disease, with
5 patients having systemic hemophagocytic
syndrome. All patients experienced a rapid
response to pembrolizumab, with 5 complete
responses (median follow-up, 6 months).
Three of 4 patients with strong PDL1
expression achieved complete remission.
Interestingly, residual lesions were biopsied
after pembrolizumab treatment in 3 patients,
and immunohistochemical staining showed
that the infiltrating lymphoid cells were
predominantly CD31 T cells, with a mix of
CD41 and CD81 and only a minority of CD561
EBER1 cells. This finding is consistent with
the hypothesis that pembrolizumab treatment
allows T cells to recognize and kill EBV-infected
NK/T lymphoma cells.
The outcome of NK/T-cell lymphoma
patients with relapsing disseminated diseases
after asparaginase-based regimens is dismal.
The results of anti-inhibitory receptor
programmed death 1 treatment in this very
small series with short follow-up supports the
use of this treatment in compassionate use
programs. Clinical trials should be performed
to extend the indications of checkpoint
inhibitors in NK/T-cell lymphoma. However,
it is still not known if these responses will be
BLOOD, 27 APRIL 2017 x VOLUME 129, NUMBER 17
observed in all patients and what durations will
be. As shown in this small group of patients,
high expression of PDL1 might be a very
strong predictor of response. This report,
however, raises several questions. First, will we
use immune checkpoint inhibitors in first-line
treatment of NK/T-cell lymphoma? Second,
will we be able in the near future to avoid
radiotherapy and its deleterious side effects?
Third, will we use combination therapy (see
figure) to prevent relapses and selection of
resistant cells? L-asparaginase, especially
pegylated forms that are less immunogenic,
will probably remain an important component
to reduce tumor bulk without induction of
a significant immunosuppressive effect.
Combination with other immunotherapies
such as anti-CD38 antibodies may present 2
advantages: that CD38 is expressed on NK
cells and on a subtype of immunosuppressive
regulatory T cells. A recent case report of
a patient relapsing after bone marrow
transplantation argues for this hypothesis.10
Hopefully, we are now approaching the time
when we will see the vast majority of patients
with this terrifying disease cured.
Conflict-of-interest disclosure: A.J. received
honoraria from Jazz Pharmaceuticals and Janssen
and research funding from Janssen. O.H. declares
no competing financial interests. n
REFERENCES
1. Kwong Y-L, Chan TSY, Tan D, et al. PD1 blockade
with pembrolizumab is highly effective in relapsed or
refractory NK/T-cell lymphoma failing L-asparaginase.
Blood. 2017;129(17):2437-2442.
2. Yong W, Zheng W, Zhang Y. Clinical characteristics
and treatment of midline nasal and nasal type NK/T cell
lymphoma [in Chinese]. Zhonghua Yi Xue Za Zhi (Taipei).
2001;81(13):773-775.
3. Jaccard A, Gachard N, Marin B, et al; GELA and
GOELAMS Intergroup. Efficacy of L-asparaginase with
methotrexate and dexamethasone (AspaMetDex regimen)
in patients with refractory or relapsing extranodal NK/
T-cell lymphoma, a phase 2 study. Blood. 2011;117(6):
1834-1839.
4. Yamaguchi M, Kwong YL, Kim WS, et al. Phase II
study of SMILE chemotherapy for newly diagnosed
stage IV, relapsed, or refractory extranodal natural killer
(NK)/T-cell lymphoma, nasal type: the NK-Cell
Tumor Study Group study. J Clin Oncol. 2011;29(33):
4410-4416.
5. Zhang L, Li S, Jia S, et al. The DDGP (cisplatin,
dexamethasone, gemcitabine, and pegaspargase) regimen
for treatment of extranodal natural killer (NK)/T-cell
lymphoma, nasal type. Oncotarget. 2016;7(36):
58396-58404.
6. Perrine SP, Hermine O, Small T, et al. A phase 1/2
trial of arginine butyrate and ganciclovir in patients with
Epstein-Barr virus-associated lymphoid malignancies.
Blood. 2007;109(6):2571-2578.
7. Kim SJ, Kim JH, Ki CS, Ko YH, Kim JS, Kim WS.
Epstein-Barr virus reactivation in extranodal natural killer/
T-cell lymphoma patients: a previously unrecognized
serious adverse event in a pilot study with romidepsin.
Ann Oncol. 2016;27(3):508-513.
8. Bollard CM, Gottschalk S, Torrano V, et al. Sustained
complete responses in patients with lymphoma receiving
autologous cytotoxic T lymphocytes targeting Epstein-Barr
virus latent membrane proteins. J Clin Oncol. 2014;32(8):
798-808.
9. Bi X-W, Wang H, Zhang W-W, et al. PD-L1 is
upregulated by EBV-driven LMP1 through NF- B
pathway and correlates with poor prognosis in natural
killer/T-cell lymphoma. J Hematol Oncol. 2016;9(1):109.
10. Hari P, Raj RV, Olteanu H. Targeting CD38 in
refractory extranodal natural killer cell-T-cell lymphoma.
N Engl J Med. 2016;375(15):1501-1502.
DOI 10.1182/blood-2017-03-769075
© 2017 by The American Society of Hematology
l l l PLATELETS AND THROMBOPOIESIS
Comment on Mills et al, page e38
Ribosomes in platelets
protect
the messenger
----------------------------------------------------------------------------------------------------Jesse W. Rowley and Andrew S. Weyrich
UNIVERSITY OF UTAH SCHOOL OF MEDICINE
In this issue of Blood, Mills et al comprehensively examine ribosome-bound
messenger RNAs (mRNAs) in resting and activated platelets and describe a role
for the ribosome rescue factor Pelota (PELO) in regulating mRNA decay in
platelets (see figure).1
P
rior studies have demonstrated that
nucleated megakaryocytes invest their
anucleate platelet progeny with thousands
of capped and poly-adenylated mRNAs2 and
ribosomes.3 Platelets use this translational
machinery to synthesize a subset of proteins,
2343
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Ribosome profiling:
• Ribosomes are bound to
numerous mRNAs in
unactivated platelets
• Thrombin increases ribosomal
occupancy on mRNAs
Loss of PELO
Ex vivo culture:
• Ribosomes remain
attached to some mRNAs
• Ribosomes bound to
3´ UTRs prevent mRNA
degradation
Nascent proteins
3´ UTR
5´
mRNA
Ribosome
Experimentally increased
PELO
Platelet
Meg01
PLPs
mRNA decay
ex vivo
(half-life = 4-6 h)
Accelerated
mRNA decay
in vitro
• Ribosomes are recycled
from mRNA 3´ UTRs
• mRNA is degraded
Ribosome profiling of platelets and platelet-like particles (PLPs) demonstrates a role for PELO in mRNA decay.
(Top) Megakaryocytes (left) produce platelets that are naturally devoid of PELO protein. Ribosome profiling
measurements on platelets demonstrate that thousands of mRNAs are translationally active and that thrombin
increases translation of many of these transcripts. Because platelets are anucleate, new mRNAs cannot be made,
and their mRNA is degraded over time thus limiting translation. However, because the ribosome rescue factor
PELO is naturally absent from platelets, ribosomes remain attached to the 39 untranslated region (UTR) of platelets
and slow their degradation thus potentially prolonging their availability for translation into protein. (Bottom) When
PELO is transgenically increased in PLPs from a megakaryocyte cell line (Meg01), ribosomes are removed from the
39UTR, and mRNA degradation is accelerated, potentially decreasing protein synthesis.
a function that is potentiated by platelet
activation.4 Translation is higher in reticulated
(ie, young) platelets, and mRNA levels and
translation seem to decline as platelets age
in the circulation.5 Although several targets
have been identified and functionally defined,2
the repertoire of mRNAs that are translated
into protein in resting and activated platelets
is generally unknown. It is also unclear how
mRNAs are stably expressed in circulating
platelets.
To address these knowledge gaps, Mills
et al screened platelets by using a sophisticated
RNA sequencing (RNA-Seq)–based technique
called genome-wide ribosome profiling that
Ingolia et al developed in 2009.6 This technique
relies on the concept that ribosomes are tightly
bound to translating mRNAs and that bound
sequences elude RNAse degradation whereas
ribosome-free mRNAs are degraded. RNA-Seq
of the ribosome-protected fragments (ie,
ribosomal footprints) yields a genome-wide,
single nucleotide resolution survey of translating
mRNAs. In a recent report, Mills et al found that
ribosomes occupy more than 6000 mRNAs in
platelets.7 In their study, they found that
ribosomal footprint levels generally correlate
with protein levels in platelets.8 They also found
that thrombin increases ribosomal occupancy on
2344
mRNAs, consistent with previous work
demonstrating that bulk translation increases
in activated platelets.9 Whether all or a subset
of ribosomal-occupied mRNAs are actually
translated into full proteins is not known
and requires further validation. There is a
possibility that ribosomes are stalled during
translation of mRNAs, which would reveal a
new mode of translational control for platelets.
The Mills et al study group previously
found that ribosomes accumulate on the
39UTR of certain transcripts in platelets and in
PLPs, which have naturally low expression of
the ribosomal rescue and surveillance factor
PELO.7 Conversely, overexpression of PELO
decreases ribosomal tracking into the 39UTR.7
Because PELO mediates mRNA degradation
when ribosomes enter the 39UTR,10 the
authors examined roles for PELO in mRNA
decay in PLPs harvested from a megakaryocytic cell line. By using RNA-Seq, they
demonstrated that the half-life of mRNAs
in cultured PLPs is 5.7 hours, a result that
coincides with previous measurements of ex
vivo mRNA decay in platelets.5 Transgenic
overexpression of PELO in PLPs significantly
decreased the half-life of the majority of PLP
mRNAs. Given that PELO expression is
low in platelets,7 these data suggest that
megakaryocytes invest low levels of PELO into
platelets as a mechanism for preserving mRNA
levels in anucleate platelets that are incapable
of transcribing new mRNA. In addition to
promoting mRNA decay, the release of stalled
ribosomes by PELO frees the ribosomes for
additional rounds of translation. This raises the
possibility that decreased expression of PELO
may also be a mechanism for suppressing
translation in circulating platelets, especially in
resting (ie, unactivated) cells. Consistent with
this possibility, reduced PELO expression in
erythrocytes, another anucleate cytoplast, is
associated with decreased protein synthesis.7
Because suppressed translation may accompany
increased mRNA stability, the net effect of
reduced PELO on individual or total protein
production in platelets remains to be
determined.
The fascinating articles by Mills and
colleagues7 provide the first demonstration that
ribosomes bind mRNAs in platelets and that
ribosomal footprints are fluid, as evidenced
by differential mRNA/ribosome occupancy
patterns in activated platelets. Their studies
also raise the possibility that different soluble
agonists, adhesion to extracellular matrices,
or aggregation (homotypic and heterotypic)
alter platelet ribosomal occupancy patterns
in a trigger-specific fashion. Other questions
require further inquiry. One of these is
whether results obtained in megakaryocytederived PLPs mimic what is observed in
bona fide platelets. Studies will also be required
to determine how changes in platelet protein
synthesis, including targets altered by
PELO, control platelet function and in vivo
physiology/pathology. As has been done
with mRNA profiling to identify differentially
expressed mRNAs in platelets in health and
disease, we anticipate that the ribosomal
profiling approach pioneered by Mills and
colleagues will be useful to identify the in
vivo conditions and diseases that alter platelet
translation and the functionally relevant
differentially translated proteins.
Conflict-of-interest disclosure: The authors declare no competing financial interests. n
REFERENCES
1. Mills EW, Green R, Ingolia NT. Slowed decay of
mRNAs enhances platelet specific translation. Blood. 2017;
129(17):e38-e48.
2. Schubert S, Weyrich AS, Rowley JW. A tour through the
transcriptional landscape of platelets. Blood. 2014;124(4):493-502.
BLOOD, 27 APRIL 2017 x VOLUME 129, NUMBER 17
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3. Weyrich AS, Schwertz H, Kraiss LW, Zimmerman
GA. Protein synthesis by platelets: historical and new
perspectives. J Thromb Haemost. 2009;7(2):241-246.
4. Warshaw AL, Laster L, Shulman NR. Protein
synthesis by human platelets. J Biol Chem. 1967;242(9):
2094-2097.
5. Angénieux C, Maı̂tre B, Eckly A, Lanza F, Gachet
C, de la Salle H. Time-dependent decay of mRNA and
ribosomal RNA during platelet aging and its correlation
with translation activity. PLoS One. 2016;11(1):
e0148064.
BLOOD, 27 APRIL 2017 x VOLUME 129, NUMBER 17
6. Ingolia NT, Ghaemmaghami S, Newman JR,
Weissman JS. Genome-wide analysis in vivo of translation
with nucleotide resolution using ribosome profiling.
Science. 2009;324(5924):218-223.
9. Weyrich AS, Dixon DA, Pabla R, et al. Signaldependent translation of a regulatory protein, Bcl-3, in
activated human platelets. Proc Natl Acad Sci U S A. 1998;
95(10):5556-5561.
7. Mills EW, Wangen J, Green R, Ingolia NT.
Dynamic regulation of a ribosome rescue pathway in
erythroid cells and platelets. Cell Reports. 2016;17(1):
1-10.
10. Doma MK, Parker R. Endonucleolytic cleavage of
eukaryotic mRNAs with stalls in translation elongation.
Nature. 2006;440(7083):561-564.
8. Rowley JW, Weyrich AS. Coordinate expression of
transcripts and proteins in platelets. Blood. 2013;121(26):
5255-5256.
DOI 10.1182/blood-2017-03-770180
© 2017 by The American Society of Hematology
2345
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2017 129: 2343-2345
doi:10.1182/blood-2017-03-770180
Ribosomes in platelets protect the messenger
Jesse W. Rowley and Andrew S. Weyrich
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