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have confirmed that the presence of certain
genetic alterations is associated with overall
prognosis and response to treatment. CLL
is characterized by relatively few recurrent
somatic mutations, of which those in the
TP53 gene are the strongest predictors of
chemoresistance and poor survival.5
In this study, Rossi et al1 have applied
highly sensitive ultra-deep next-generation
sequencing to examine a large cohort of
patients (309) with newly diagnosed CLL for
the presence of very small TP53-mutated
subclones (sensitivity down to 0.3% allele
frequency), which would not have been
detected by Sanger sequencing (which detects
.20% frequency).6 The 5.8% of patients with
such subclones had the same adverse survival
as those 9% in whom TP53 mutations were
detected by conventional methods, and
accounted for a third of all cases with TP53
abnormalities.
The current recommendation is that all
patients should be tested for abnormalities of
TP53 prior to initiating any line of treatment, in
order to select TP53-independent therapy
when appropriate. The prevailing dogma is
that the size of the TP53-deleted clone is
important and that below certain thresholds
(variably reported as 10% or 20% using
FISH), response to treatment is unaffected.
Rossi et al overturn the view that small clones
are clinically unimportant by showing that even
very small subclones (,1%; present below the
threshold of detection using current standard
methods but with no apparent cutoff in the size
of the clone) have an adverse impact on patient
survival. They argue that the effect on outcome
of the presence of these subclones is a yes/no
determinant independent of clonal size.
Importantly, sequential samples showed
that these subclones expanded over time,
particularly under the selective pressure
of chemotherapy, leading ultimately to
chemorefractory disease (see figure). In the
2 patients who did not receive treatment, the
clonal size remained unchanged. This would
be consistent with other studies showing
increasing frequency of TP53 abnormalities
in patients with disease progression and
refractoriness.7
If multiple subclones coexist, what drives
any to become dominant? There are many
reasons, including limited potential for
expansion due to “competition” from other
clones as well as the independent effect of
the surrounding microenvironment. If these
BLOOD, 3 APRIL 2014 x VOLUME 123, NUMBER 14
subclones are vying for space and resources, the
reduction of some clones may unbalance the
status quo. It is therefore unsurprising that
failure of chemotherapy to completely
eradicate CLL cells can result in expansion of
minor, more resistant, and more dangerous
subclones. “Selection” can thus be introduced
artificially by the use of chemotherapeutic
agents. Certain subclones are likely to gain
a competitive advantage due to their “fitness”
in relation to these selection pressures. It is
therefore important to identify low-level
molecular lesions that are known to predict for
chemoresistance so that treatment can be
tailored appropriately. In TP53-mutated CLL,
this may involve use of novel targeted therapies
(eg, Ibrutinib, ABT199)8 which have been
shown to have promising activity in this subset.
What other strategies might be considered
to improve therapeutic efficacy and prevent
emergence of resistance? Cytotoxic drugs are
likely to select for resistant cells by clearing the
ground of more sensitive ones. On the other
hand, cytostatic drugs (some small-molecule
inhibitors) may cause cells to remain in the
tissue space but without either expanding
themselves or allowing expansion of other
subclones. In addition, early intervention with
effective treatment before clonal expansion
may be a more effective way to deal with these
more clinically adverse subclones. It may also
be the case that carefully designed concurrent
or sequential combinations of therapies may
overcome some of the issues related to clonal
diversity. It is important to note, however, that
not all chemoresistant CLL is characterized
by TP53 mutation and it will be crucial to
understand the biology of any other clinically
important subclones which may be present
in order to prevent their dominance. The
underlying principles are likely to be the same
but the therapeutic strategies may be different.
Clearly, CLL is not a static disease, but has
a clonal architecture that changes over time and
is influenced by selection pressures, including
treatment. Certain clinically adverse genomic
changes appear to be present in the CLL
cells from a very early stage of disease.
Understanding how this population becomes
dominant is crucial for the development of new
therapeutic strategies, which will be effective
by rendering them the least fit for survival.
Conflict-of-interest disclosure: The author declares
no competing financial interests. n
REFERENCES
1. Rossi D, Khiabanian H, Spina V, et al. Clinical impact
of small TP53 mutated subclones in chronic lymphocytic
leukemia. Blood. 2014;123(14):2139-2147.
2. Nowell PC. The clonal evolution of tumor cell
populations. Science. 1976;194(4260):23-28.
3. Campbell PJ, Pleasance ED, Stephens PJ, et al.
Subclonal phylogenetic structures in cancer revealed by
ultra-deep sequencing. Proc Natl Acad Sci USA. 2008;
105(35):13081-13086.
4. Döhner H, Stilgenbauer S, Benner A, et al. Genomic
aberrations and survival in chronic lymphocytic leukemia.
N Engl J Med. 2000;343(26):1910-1916.
5. Gonzalez D, Martinez P, Wade R, et al. Mutational
status of the TP53 gene as a predictor of response and
survival in patients with chronic lymphocytic leukemia:
results from the LRF CLL4 trial. J Clin Oncol. 2011;
29(16):2223-2229.
6. Pospisilova S, Gonzalez D, Malcikova J, et al;
European Research Initiative on CLL (ERIC). ERIC
recommendations on TP53 mutation analysis in chronic
lymphocytic leukemia. Leukemia. 2012;26(7):1458-1461.
7. Zenz T, Häbe S, Denzel T, Winkler D, Döhner H,
Stilgenbauer S. How little is too much? p53 inactivation:
from laboratory cutoff to biological basis of chemotherapy
resistance. Leukemia. 2008;22(12):2257-2258.
8. Byrd JC, Furman RR, Coutre SE, et al. Targeting
BTK with ibrutinib in relapsed chronic lymphocytic
leukemia. N Engl J Med. 2013;369(1):32-42.
© 2014 by The American Society of Hematology
l l l LYMPHOID NEOPLASIA
Comment on Dubois et al, page 2199
An E3 ubiquitin ligase-independent
role
of LUBAC
----------------------------------------------------------------------------------------------------Rudi Beyaert1,2
1
GHENT UNIVERSITY; 2VIB
In this issue of Blood, Dubois et al show a catalytic-independent role of the linear
ubiquitin chain assembly complex (LUBAC) in lymphocyte activation and B-cell
malignancy.1 These data add a new layer of versatility to the recently established
role of LUBAC in nuclear factor-kB (NF-kB) signaling.
2131
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T
and B cells sense antigens via specific
receptors that induce signaling cascades
leading to the activation of multiple
transcription factors such as those of the
NF-kB family.2 NF-kB controls the
expression of multiple genes essential for
the immunogenic response and cell survival.
Hyperactivation of NF-kB is involved in many
autoimmune and inflammatory diseases
and constitutive NF-kB activation is
a characteristic of certain lymphoma types
such as activated B-cell–like diffuse large B-cell
lymphoma (ABC-DLBCL), where NF-kB
signaling drives proliferation and cell survival.3,4
Understanding the early molecular events
leading to NF-kB activation is an active area of
research and much progress has been made
during the last decade. Several molecules that
specifically connect the T-cell receptor (TCR)
and B-cell receptor (BCR) proximal adaptors
Hypothetical model for the role of LUBAC in antigen receptor–induced NF-kB signaling. TCR and BCR stimulation by
antigens triggers the activation of proximal adaptors and kinases, followed by the rapid assembly of a signaling platform
(known as the CBM complex) that contains the scaffold proteins CARD11/BCL10 and the paracaspase MALT1. In ABCDLBCL, the CBM complex is preassembled, which often results from somatic mutations in positive or negative regulatory
signaling proteins. Formation of the CBM complex results in the downstream activation of the IKK complex, consisting of 2
kinases (IKKa and IKKb) and an adaptor protein NEMO. IKK-mediated phosphorylation results in the nuclear translocation
of NF-kB where it induces the expression of several genes that mediate T- and B-cell activation and proliferation. In ABCDLBCL, NF-kB is constitutively activated leading to the expression of anti-apoptotic genes and cell survival. How CBM
connects to the IKK complex is still largely unclear. Dubois et al demonstrate that TCR- and BCR-induced NF-kB signaling
also involves a third multiprotein complex known as LUBAC (consisting of HOIP, HOIL-1, and SHARPIN). LUBAC is best
known for its role in tumor necrosis factor–induced NF-kB signaling, where it modifies specific signaling proteins with headto-tail linked (linear) ubiquitin modules. However, the study of Dubois et al shows a catalytic-independent role of LUBAC in
lymphocytes. Their results are compatible with a model in which antigen receptor stimulation induces the formation of
a large signalosome in which LUBAC functions as an adaptor between the CBM and IKK complexes.
2132
and kinases to the central core of the NF-kB
cascade (the IkB kinase [IKK] complex) have
been identified. More specifically, the antigen
receptor–induced assembly of a signaling
platform containing the scaffold proteins
CARD11 and BCL10 and the paracaspase
MALT1 (known as the CBM complex)
connects proximal antigen receptor signaling to
the IKK complex. In ABC-DLBCL, the CBM
complex is preassembled (which often results
from somatic mutations in key regulatory
proteins), leading to derailed signaling.
Posttranslational modification of several
components of the CBM and IKK complexes
by phosphorylation and different types of
polyubiquitination are believed to contribute to
CBM-mediated NF-kB signaling, but the
exact molecular mechanisms remain obscure.5
In this issue, Dubois et al provide new
insights into the molecular basis of the link
between CBM and NF-kB signaling.1 They
performed a mass spectrometry proteomic
screen in stimulated T cells after
immunoprecipitation of casein kinase a,
a kinase that binds to CARD11 and governs
antigen receptor–induced NF-kB signaling.
By this approach, they identified HOIP (also
known as RNF31) as a new component of
the CBM complex after antigen receptor
stimulation. HOIP is 1 of the 3 proteins of the
recently identified LUBAC that mediates
NF-kB signaling in response to cytokines,
bacteria, and genotoxic stress.6 Furthermore,
coimmunoprecipitation experiments revealed
that all 3 LUBAC components enter the CBM
and IKK signalosome after antigen receptor
stimulation. The authors further explored
whether LUBAC is required for TCR-induced
NF-kB signaling by assessing the outcome of
small interfering RNA–mediated silencing of
each of the 3 LUBAC components. Deficiency
of HOIP and SHARPIN, but not HOIL-1,
significantly reduced NF-kB activation and
the binding of BCL10/MALT1 to the IKK
adaptor protein NEMO. These data indicate
that LUBAC contributes to optimal NF-kB
activation in response to antigen receptor
stimulation by enabling the interaction
between CBM and IKK complexes.
HOIP is known as an E3 ubiquitin ligase
whose catalytic activity is necessary for the
LUBAC-mediated modification of specific
NF-kB signaling proteins with multiple
ubiquitin modules that are linked to each other
via their N and C termini.6 This type of
polyubiquitination is known as linear or
BLOOD, 3 APRIL 2014 x VOLUME 123, NUMBER 14
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
M1-linked polyubiquitination and is the focus
of intense research. Surprisingly, Dubois
and colleagues found that expression of
a catalytically inactive HOIP mutant is able
to restore reduced NF-kB signaling in
HOIP-deficient cells to normal levels, indicating
that HOIP mediates TCR signaling independent
of its catalytic activity. This is further supported
by the observation that silencing of OTULIN,
a negative regulator of linear polyubiquitination,
did not change TCR-induced NF-kB signaling.
It should be mentioned that some modest
linear polyubiquitination is detected in
TCR-stimulated cells, indicating a possible role
for linear ubiquitination in other TCR signaling
pathways than NF-kB signaling.
Dubois et al show that LUBAC is also
part of the preassembled CBM complex in
ABC-DLBCL cell lines and that combined
silencing of all 3 LUBAC components inhibits
constitutive NF-kB activation in these cells.
Consistent with these findings and the known
anti-apoptotic function of NF-kB, they show
that LUBAC silencing also reduces cell
survival. Together, these data indicate that
LUBAC guarantees cell proliferation and
survival of ABC-DLBCL by maintaining
constitutive NF-kB activity.
The results of Dubois et al suggest a novel
catalytic-independent role of LUBAC
in lymphocytes and B-cell lymphoma. The
underlying molecular mechanism is still
unclear but the finding that HOIP is necessary
for the association between CBM and IKK
complexes is indicative for an adaptor function.
The exact mechanism could, however, be more
complex as many ill-defined components
compose the CBM complex. The data
of Dubois et al complement the recent
demonstration that BCR-mediated NF-kB
activation does not require LUBAC catalytic
activity in splenocytes.7 In addition, another
parallel study also reports that LUBAC
associates with the CBM complex in
ABC-DLBCL and is required for cell
viability.8 However, the latter study shows
that LUBAC mediates constitutive linear
polyubiquitination of the IKK adaptor protein
NEMO in ABC-DLBCL, and describes 2
rare HOIP germline mutations that promote
LUBAC E3 ubiquitin ligase activity and
activate NF-kB in ABC-DLBCL. At first
look, these findings do not fit the catalyticindependent role of LUBAC that is proposed
by Dubois et al in this issue. However, it should
be mentioned that they only analyzed the
BLOOD, 3 APRIL 2014 x VOLUME 123, NUMBER 14
dependency on HOIP catalytic activity in
T cells and not in ABC-DLBCL cell lines.
Nevertheless, the finding that LUBAC is
part of the CBM complex and mediates
NF-kB signaling and cell survival is of high
importance for our understanding of the
regulation of physiological and pathological
signaling in adaptive immunity. The CBM
complex is an attractive therapeutic target for
diseases associated with aberrant lymphocyte
activation and B-cell lymphomas, and
recent developments using MALT1 protease
inhibitors are very promising.9 A better
knowledge of the function and regulation of
LUBAC in the CBM complex may provide
additional ways for therapeutic targeting.
Conflict-of-interest disclosure: The author declares
no competing financial interests. n
REFERENCES
1. Dubois SM, Alexia C, Wu Y, et al. A catalytic-independent
role for the LUBAC in NF-kB activation upon antigen
receptor engagement and in lymphoma cells. Blood. 2014;
123(14):2199-2203.
2. Hayden MS, Ghosh SNF. NF-kB in immunobiology.
Cell Res. 2011;21(2):223-244.
3. Sun SC, Chang JH, Jin J. Regulation of nuclear factorkB in autoimmunity. Trends Immunol. 2013;34(6):282-289.
4. Shaffer AL III, Young RM, Staudt LM. Pathogenesis of
human B cell lymphomas. Annu Rev Immunol. 2012;30:565-610.
5. Thome M, Charton JE, Pelzer C, Hailfinger S. Antigen
receptor signaling to NF-kappaB via CARMA1, BCL10,
and MALT1. Cold Spring Harb Perspect Biol. 2010;2(9):
a003004.
6. Iwai K. Diverse roles of the ubiquitin system in NF-kB
activation. Biochim Biophys Acta. 2014;1843(1):129-136.
7. Sasaki Y, Sano S, Nakahara M, et al. Defective immune
responses in mice lacking LUBAC-mediated linear
ubiquitination in B cells. EMBO J. 2013;32(18):2463-2476.
8. Yang Y, Schmitz R, Mitala JJ Jr, et al. Essential role of
the linear ubiquitin chain assembly complex in lymphoma
revealed by rare germline polymorphisms. Cancer Discov.
[published online ahead of print February 3, 2014]. doi:
10.1158/2159-8290.CD-13-0915.
9. Yang C, David L, Qiao Q, Damko E, Wu H. The CBM
signalosome: potential therapeutic target for aggressive
lymphoma? Cytokine Growth Factor Rev. [Published
online ahead of print December 24, 2013]. doi:10.1016/
j.cytogfr.2013.12.008.
© 2014 by The American Society of Hematology
l l l MYELOID NEOPLASIA
Comment on Lundberg et al, page 2220
Many
roads lead to MPN
----------------------------------------------------------------------------------------------------Heike L. Pahl1
1
UNIVERSITY MEDICAL CENTER FREIBURG
In this issue of Blood, Lundberg et al correlate the presence of known mutations
in patients with myeloproliferative neoplasms (MPNs) with clinical outcome,
thereby proposing a molecular risk stratification.1
T
he clinical presentation of patients
with MPNs is heterogeneous, and the
individual disease course is difficult to predict
at diagnosis. Although in some patients the
disorder remains indolent for many years,
others experience multiple complications and
rapid disease progression. It is therefore gratifying
to read that Lundberg et al can corroborate this
clinical heterogeneity at the molecular level.1 The
authors investigated the “clonal architecture”
of MPNs, that is the nature of different mutations
detected in individual patients and the order in
which they appear.
Because the authors selected known cancer
genes for analysis, many of which have been
previously shown to be affected in MPNs, the
message of this study is less in the nature of
the mutations found but rather in the variable
pattern of their acquisition, which this study
demonstrates. However, it is noteworthy that
in this cohort, mutations in some novel genes,
such as p53 and NF-E2, appear to be more
frequent than others, such as c-Cbl or c-Mpl,
which have been known for several years.2-4
A model presented in Figure 5 of the
Lundberg et al paper depicts the many different
constellations observed and uses them to stratify
patients by risk of leukemic transformation.
As is the case in other myeloid neoplasias,
such as acute myeloid leukemia (AML) and
myelodysplastic syndrome (MDS),5,6 a higher
number of mutations is associated with poorer
outcome. In MDS, the number of mutations
is likewise correlated with the time to leukemic
transformation.6 The question remains,
however, whether the acquisition of additional
2133
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2014 123: 2131-2133
doi:10.1182/blood-2014-02-556076
An E3 ubiquitin ligase-independent role of LUBAC
Rudi Beyaert
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