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LYMPHOID NEOPLASIA
The IKK2/NF-␬B pathway suppresses MYC-induced lymphomagenesis
Kay Klapproth,1 Sandrine Sander,1 Dragan Marinkovic,1 Bernd Baumann,1 and Thomas Wirth1
1Institute
of Physiological Chemistry, University of Ulm, Ulm, Germany
Deregulated c-MYC is found in a variety
of cancers where it promotes proliferation as well as apoptosis. In many hematologic malignancies, enhanced NF-␬B
exerts prosurvival functions. Here we investigated the role of NF-␬B in mouse
and human c-MYC–transformed lymphomas. The NF-␬B pathway is extinguished
in murine lymphoma cells, and extrinsic
stimuli typically inducing NF-␬B activity
fail to activate this pathway. Genetic activation of the NF-␬B pathway induces apo-
ptosis in these cells, whereas inhibition
of NF-␬B by an I␬B␣ superrepressor provides a selective advantage in vivo. Furthermore, in human Burkitt lymphoma
cells we find that NF-␬B activation induces apoptosis. NF-␬B up-regulates Fas
and predisposes to Fas-induced cell
death, which is caspase-8 mediated and
can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-␬B–induced
apoptosis, and persistent inactivity of
NF-␬B signaling is a prerequisite for MYCmediated tumorigenesis. We could also
show that low immunogenicity and Fas
insensitivity of MYC-driven lymphoma
cells are reversed by activation of NF-␬B.
Our observations provide a molecular
explanation for the described absence of
the NF-␬B signaling in Burkitt lymphoma
and question the applicability of NF-␬B
inhibitors as candidates for treatment of
this cancer. (Blood. 2009;114:2448-2458)
Introduction
The c-MYC (MYC) transcription factor has been implicated in
the control of many aspects of tumor cell biology. It promotes
cell proliferation and restrains differentiation. To do so, MYC
controls transcription of multiple genes involved in cell growth
and metabolism, vasculogenesis, cell adhesion, and genomic
stability.1 MYC is a basic helix-loop-helix-leucine zipper transcription factor that dimerizes with the related protein MAX.
MYC/MAX heterodimers bind to specific DNA elements,
designated as E-boxes, located in the promoter regions of target
genes mediating either activation or repression of transcription.2
Complex regulatory networks control MYC activity leading to
up-regulation of its expression in response to mitogens or
suppression upon growth inhibitory signals.3 Increased amounts
of MYC protein are found in many types of human cancer
because control mechanisms keeping MYC in check are inactivated during malignant transformation of cells.1,3 In Burkitt
lymphoma (BL), a highly aggressive non-Hodgkin lymphoma,
overexpression of MYC is invariably connected to chromosomal translocations of the MYC proto-oncogene to immunoglobulin loci. Elevated expression of MYC likewise induces
malignant transformation in mouse models and persistent MYC
expression is required for tumor growth.4
MYC induces proliferation, but at the same time it can induce
cell death. Several MYC-induced pathways, such as activation of
the p53/ARF pathway, changes in expression, and activity of BCL2
proteins or alterations in death receptor signaling have been linked
to apoptosis.3 These most likely serve as safeguard mechanisms
against unrestricted growth and malignant transformation. Inactivation of the MYC-induced apoptotic program is therefore a prerequisite for tumorigenesis.
The NF-␬B transcription factor is also linked to tumor
initiation and progression.5,6 NF-␬B binds to the DNA as homodimer
or heterodimer composed of 2 of 5 related subunits: RelA(p65), RelB,
c-Rel, p50/105 (NF-␬B1), and p52/100 (NF-␬B2).7 Inactive NF-␬B
complexes are sequestered in the cytosol bound to inhibitors, the I␬B
proteins. Activation of NF-␬B involves phosphorylation, ubiquitination,
and proteasomal degradation of the I␬B proteins allowing nuclear
translocation of the NF-␬B dimer. Initiation of transcription relies on
NF-␬B DNA binding that may involve multimerization with additional
proteins such as RPS3.8 The I␬B kinase (IKK) complex consisting of
IKK␣/IKK1, IKK␤/IKK2, and the regulatory subunit IKK␥/NEMO
phosphorylates the I␬B proteins. In the classical pathway predominantly
IKK2 is active. Activation of the IKK complex occurs downstream of
many oncoproteins, and NF-␬B target genes involved in cell proliferation, growth, and survival are induced in many different human tumors.9
Mutations leading to enhanced NF-␬B activity are a recurrent theme in
hematologic malignancies.10 NF-␬B initiates expression of various
antiapoptotic proteins, such as BCL-2, BCL-XL, cIAPs, and CFLAR
(c-FLIP). Thus, apoptosis evasion is an important contribution of
NF-␬B in the pathogenesis of human tumors.9
Surprisingly, in the E␮Myc-mouse model NF-␬B activity was
found to be dispensable for lymphomagenesis.11 Low NF-␬B
DNA-binding activities were also reported for a conditional
MYC-driven human B-cell lymphoma line.12 Moreover, in 2 recent
studies low expression of NF-␬B target genes was reported as
hallmarks of BL.13,14 We therefore investigated the role of NF-␬B
in MYC-driven lymphomas using genetic approaches to either
induce or block the NF-␬B pathway. Both in conditionally MYCtransformed murine lymphomas as well as in human BL we find
that NF-␬B functions as a tumor suppressor.
Submitted September 24, 2008; accepted July 8, 2009. Prepublished online as
Blood First Edition paper, July 23, 2009; DOI 10.1182/blood-2008-09-181008.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
Methods
Cell lines and cell culture
Lymphoma cells were cultured in RPMI medium supplemented with 10%
FCS, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 ␮g/
mL), nonessential amino acids, and ␤-mercaptoethanol (50 ␮M). In murine
lymphoma cell lines, MYC expression was abrogated by doxycycline (Dox)
treatment (2 ␮g/mL) for the indicated time periods. For NF-␬B activation,
cells were stimulated with TNF␣ (40 ng/mL), PMA (5 ng/mL) and ionomycin (1 ␮g/mL), LPS (1 ␮g/mL), daunorubicin (5 ␮M), or okadaic acid
(200 nM) for the indicated times.
Western blot analysis and electrophoretic mobility shift assay
Preparation of nuclear and whole-cell extracts was performed as described
earlier.15 For Western blot analysis 40 ␮g whole-cell protein extracts per
lane were separated on 12.5% polyacrylamide gels and transferred onto
polyvinylene difluoride membranes (Millipore). Membranes were blocked
with 7.5% dry milk in TBS containing 0.2% Tween 20. For subsequent
washes, 0.2% Tween 20 in TBS was used. Affinity-purified rabbit antibodies against IKK2 (SC-7607), I␬B␣ (SC-371), RelA/p65 (SC-372) or MYC
(SC-764; Santa Cruz Biotechnology), and horseradish peroxidase–coupled
goat anti–rabbit IgG secondary antibody (Thermo Scientific) were used. For
electrophoretic mobility shift assays (EMSA), 5 ␮g nuclear or whole-cell
extracts was incubated for 20 minutes at room temperature with 3 ␮g
poly(dI/dC), 10 ␮g bovine serum albumin in binding buffer (50 mM NaCl,
1 mM dithiothreitol, 10 mM Tris-HCl, 1 mM EDTA, 5% glycerol), and
radiolabeled oligonucleotides containing specific sites for binding of
NF-␬B (5⬘-GCC TGG GAA AGT CCC CTC AA-3⬘), Oct (5⬘-ACC TGG
GTA ATT TGC ATT TCT AAA AT-3⬘), or SP-1 (5⬘-ATT CGA TCG GGG
CGG GGC GAG C-3⬘). DNA-protein complexes were separated on native
6% polyacrylamide gels.
Retroviral vectors and stable producer cell line
The pCFG-IEGZ retroviral vectors containing TD-I␬B␣ or CA-IKK2
constructs have been described earlier.16 ␾NX producer cells (2 ⫻ 106/10cm plate) were grown in DMEM supplemented with FCS (10%),
L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 ␮g/mL)
and transfected with 10 ␮g plasmid DNA using calcium phosphate.
Forty-eight hours after transfection, efficiency was determined by flow
cytometric analysis of GFP expression (typically 70%-80% positive).
Supernatants were supplemented with 8 ␮g/mL polybrene, and cells
(5 ⫻ 105/well in 24-well plates) were infected with 750 ␮L/well by spin
infection (90 minutes at 1200g and 37°C). Twenty-four hours after
infection, efficiency was determined by flow cytometric GFP analysis.
Infected cells were selected with 100 ␮g/mL Zeocin for 10 to 14 days and
GFP expression was checked by flow cytometry.
IKK2 SUPPRESSES MYC-INDUCED LYMPHOMAGENESIS
2449
catgctctatcagattctcttgaaatctgatagagcatgacccga-3⬘. pcDNA-FLIPL was kindly
provided by P. Krammer.18 Transfections were performed by Nucleofection
(Amaxa). Conditional transgene expression was induced with doxycycline
(0.5 ␮g/mL) and GFP expression was analyzed by flow cytometer.
Cell counts and fluorescence-activated cell sorting analyses
Proliferation of cells was determined by viable cell counts (trypan blue
staining) or by flow cytometer. Cell counts were performed in triplicates.
Apoptosis was determined by annexin-V and 7-AAD stainings (Apoptosis
detection kit; BD Pharmingen) and flow cytometric analysis. Apoptosis was
inhibited by addition of pan-caspase inhibitor z-VAD (20 ␮M). Surface
expression of Fas was determined by staining with APO-I and Alexa-647–
conjugated secondary antibody. GFP⫹ and GFP⫺ fractions were analyzed
separately for Alexa-647 fluorescence.
Gene expression profiling
Ramos cells were transfected in triplicates with pRTS-GFP or pRTS-CAIKK2, selected with hygromycin, and transgene expression was induced
with doxycycline (0.5 ␮g/mL) for 48 hours. RNA was isolated with RNeasy
mini kit (QIAGEN), and gene expression profiling (GEP) was performed
using Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix). Total
RNA (2 ␮g) was labeled using the GeneChip One-Cycle Target Labeling
assay kit (Affymetrix). After hybridization, arrays were stained and washed
in a FS 450 Fluidics station (Affymetrix) before imaging on an Affymetrix
GeneChip (3000) scanner. Raw data were generated using the GCOS 1.4
software (Affymetrix). Probe level data were obtained using the robust
multichip average (RMA) normalization algorithm and CEL files were
loaded into Genesifter (http://genesifter.net; VizX Laboratories). Genes
were identified as differentially expressed among the 2 classes if a 2-sample
t test revealed a nominal significance level of .05 and the ratio between the
2 classes was at least 2-fold. Calculation of false discovery rate was done
according to the method of Benjamini and Hochberg.19 Biologic significance was determined using Gene Ontology reports.20 Microarray data have
been deposited with Gene Expression Omnibus (GEO) under accession
number GSE17129.21
RNA-isolation and reverse-transcription–PCR
Total RNA was isolated with QIAzol lysis reagent (QIAGEN). Total RNA
(2 ␮g) was reverse transcribed with AMV reverse transcriptase (Roche) and
polymerase chain reaction (PCR) was performed using Taq DNA polymerase (Amersham Pharmacia Biotech). The primers used were hFas_fwd
(5⬘-CAA GTG ACT GAC ATC AAC TCC-3⬘), hFas_rev (5⬘-CCT TGG
TTT TCC TTT CTG TGC-3⬘), hb-actin_fwd (ATC TGG CAC CAC ACC
TTC TAC AAT GAG CTG CG-3⬘), and hb-actin_rev (5⬘-CGT CAT ACT
CCT GCT TGC TGA TCC ACA TCT GC-3⬘). Conditions for Fas PCR were
as follows: 30 cycles at 56°C for 40 seconds, 72°C for 1 minute, and 94°C
for 40 seconds. Conditions for ␤-actin were as follows: 20 cycles at 58°C
for 40 seconds, 72°C for 70 seconds, and 94°C for 40 seconds.
Tumor transplantation
Murine B-lymphoma line 5522 transduced with retroviral IEGZ-empty
vector or IEGZ-TD-I␬B␣ vector were mixed with untransduced parental
cells at a ratio of 1:10. Lymphoma cells (107) of the mixed populations were
injected intraperitoneally into syngeneic mice. After 1 week, recipient mice
were killed and tumor cells were isolated from ascites. Percentages of
GFP⫹ cells in the isolated populations were determined by flow cytometry.
Approval for the use of mice in this study was obtained from the
Regierungspraesidium (TV-709).
Apoptosis induction
After transfection with pRTS-GFP or pRTS-CA-IKK2, Ramos cells were
treated with doxycycline (0.5 ␮g/mL) for 48 hours to induce transgene
expression. APO-I containing supernatant (5% final concentration [f.c.])
was added and apoptosis was determined by trypan blue stainings.
Results
Vectors and transfections
MYC-dependent murine lymphoma cells lack NF-␬B activity
pSFI and pRTS vectors were kindly provided by D. Eick and G. W.
Bornkamm.17 TD-I␬B␣ and CA-IKK2 cDNAs15 were first inserted into
blunt-end EcoRV sites of the pSFI subcloning vector and then recloned into
the pRTS-1 vector. pRETRO-SUPER vectors containing anti–caspase-8
shRNA had the following sequence: forward, 5⬘-gatctcgggtcatgctctatcagatttcaagagaatctgatagagcatgaccctttttggaaa-3⬘ and reverse, 5⬘-agcttttccaaaaagggt-
To evaluate a potential role of the NF-␬B transcription factor for
MYC-driven lymphoma development, we determined its activity in
conditionally MYC-transformed mouse lymphoma cell lines.4,22
Basal NF-␬B activity was measured by EMSA and revealed low to
undetectable NF-␬B in these cells (Figure 1A). We investigated the
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Figure 1. NF-␬B activity is impaired in the MYC-transformed cells. (A) NF-␬B EMSA using nuclear extracts of different murine lymphoma cells. WEHI231 cells show
constitutive NF-␬B activity and were used as control. Oct1 DNA binding served as a control for the integrity of the nuclear extracts. (B) EMSA using protein extracts from
lymphoma cells treated with the standard NF-␬B inducers TNF␣ (40 ng/mL), PMA (5 ng/mL) and ionomycin (1 ␮g/mL), or LPS (1 ␮g/mL) for the indicated time points. Jurkat
cells treated with PMA/ionomycin for 1 hour were used as control for NF-␬B binding. Sp1 DNA was used as a control for the integrity of the cell extracts. (C) NF-␬B EMSA with
nuclear extracts of B-cell lymphoma cell line 5522 treated for 2 hours with daunorubicin (5 ␮M) or okadaic acid (200 nM). Oct1 was used as control for the integrity of the
extracts. (D) Immunoblot with cell extracts of B-cell lymphoma cell lines. Doxycycline treatment abrogates transgenic MYC expression. Cells were treated with doxycycline
(2 ␮g/mL) for 12 hours or were left untreated. Expression of p65 was used as loading control. (E) EMSA using cell extracts of 2 lymphoma cell lines demonstrates induction of
NF-␬B activity in the absence of MYC expression. Immunoblot shows transient loss of MYC expression after doxycycline treatment. Cells were left untreated (lanes 1-2), pulse
treated with doxycycline for 12 hours (lanes 3-4), and again left untreated for the consecutive days (lanes 5-6).
effects of typical NF-␬B–inducing stimulators on NF-␬B binding
activity. Most T- and B-cell lymphomas showed either no or very
low induction of NF-␬B activity in response to LPS, TNF␣, or
PMA/ionomycin (Figure 1B). To test whether alternative activation
pathways are functional in these cells, we checked for NF-␬B
activity after stimulation with daunorubicin and okadaic acid. Cells
showed robust increases in NF-␬B binding, indicating the presence
of functional NF-␬B proteins in the cytosol (Figure 1C).
Growth and survival of cell lines established from tumorbearing mice have been shown to be dependent on constant MYC
overexpression. This oncogene addiction is a common attribute of
MYC-induced tumors. To test whether MYC itself has an influence
on NF-␬B activity, we turned off its expression by addition of
doxycycline. This treatment abrogated transgenic MYC expression
in the lymphoma cells (Figure 1D). After washing out doxycycline,
MYC expression was recovered (Figure 1E bottom panel). In cells
lacking MYC, we detected NF-␬B activity by EMSA and nuclear
translocation of p65 (Figure 1Eiii-iv, supplemental Figure 1,
available on the Blood website; see the Supplemental Materials
link at the top of the online article). However, upon re-expression
of MYC, NF-␬B activity was again abolished (Figure 1Ev-vi).
Murine lymphoma cells are sensitive to IKK2 activation
To determine whether absence of NF-␬B signaling is critical for
MYC-induced tumorigenesis, we modulated NF-␬B activity by
retroviral introduction of dominant mutants interfering with the
NF-␬B pathway. Inhibition of NF-␬B activity was achieved by a
transdominant I␬B␣ (S32,36A) superrepressor (IEGZ-TD-I␬B␣),
which cannot be phosphorylated by IKK. A constitutively active
allele of the I␬B kinase 2 (IEGZ-CA-IKK2) was used to activate
the NF-␬B pathway. Internal ribosomal entry sites couple expression of NF-␬B modulators with a GFP-Zeocin fusion protein,
allowing identification and selection of infected cells.23,24
Several MYC-dependent lymphoma B-cell lines were infected
with either the TD-I␬B␣, the CA-IKK2 mutant, or a control vector
(IEGZ-empty) containing only the GFP-Zeocin fusion gene. Twentyfour hours later, frequencies of GFP-expressing cells were evaluated by flow cytometry. Infection efficiencies were similar for the
different viruses (supplemental Figure 2A). Expression of TDI␬B␣ was detectable 24 hours after infection (Figure 2A), accompanied by a reduction of endogenous I␬B␣ levels. The activation of
NF-␬B after MYC down-regulation was strongly reduced in the
presence of the superrepressor (Figure 2B). Expression of CAIKK2 was also detectable 24 hours after infection and resulted in a
strong induction of NF-␬B (Figure 2C).
When we analyzed the infected cells it became apparent that
CA-IKK2–expressing cells displayed a dramatically altered phenotype.
Whereas IEGZ-empty– and IEGZ-TD-I␬B␣–infected cells showed
single-cell distribution, expression of CA-IKK2 induced pronounced
aggregation leading to large clusters of cells (Figure 2D). Selection for
Zeocin resistance was feasible for TD-I␬B␣ or control vector, but failed
for the CA-IKK2 infections. These results implicate that CA-IKK2
expression interferes with proliferation and/or viability of the lymphoma
cells. To investigate this issue we monitored changes in the infected
GFP⫹ compartment in the absence of selective pressure. No changes in
the percentages of GFP⫹ populations were seen in empty vector and
TD-I␬B␣–infected cultures. In contrast, GFP⫹ cells rapidly disappeared
from CA-IKK2–infected cultures (Figure 2E; supplemental Figure 2B).
Annexin-V and 7-AAD staining revealed increased levels of apoptosis
after expression of CA-IKK2 (Figure 2F; supplemental Figure 2C). We
therefore conclude that the reduction of CA-IKK2–infected cells is at
least partially the result of apoptosis induction.
To test whether blocking of NF-␬B might have a selective advantage
for lymphoma cells, we performed in vivo competition assays. MYCdriven lymphoma cells transduced with retroviral IEGZ-empty or
IEGZ-TD-I␬B␣ were combined with parental cells at a ratio of 1:10.
Flow cytometric analysis confirmed that approximately 10% of these
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
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IKK2 activation induces growth inhibition and apoptosis in
human Burkitt lymphoma cells
NF-␬B activity has been described in human MYC-expressing
lymphomas,25,26 arguing against a general incompatibility of these
transcription factors in human cancer cells. We therefore analyzed
consequences of NF-␬B modulation in human MYC-driven lympho-
30
25
20
15
10
5
0
GFP
mixed populations were GFP positive (Figure 2G). Murine lymphoma
cells (107) of the mixed populations were injected intraperitoneally into
syngeneic mice. Recipient mice developed ascites and lymphocytes,
isolated from ascites, were analyzed for GFP (Figure 2G). The fraction
of GFP-positive cells within the group carrying the TD-I␬B␣–
transduced cells was increased to 23.8 plus or minus 2.0, whereas it
stayed at roughly 10% when empty virus–infected cells were mixed
(Figure 2H). This indicates a selective advantage for lymphoma cells
with blocked NF-␬B activation.
H
GFP positive cells in ascites (%)
E
relative changes GFP+ cells (%)
Figure 2. Modulation of NF-␬B signaling pathway in
murine lymphoma cells. (A) Immunoblot analysis of
TD-I␬B␣ expression in transduced murine lymphoma
cell line. Cells were spin-infected with retroviral vectors
IEGZ-empty or IEGZ-TD-I␬B␣ and protein extracts
were isolated 24 hours after infection. (B) EMSA of
NF-␬B binding activity in transduced lymphoma cell
lines. NF-␬B activation after doxycycline-mediated MYC
inactivation was detectable only in IEGZ-empty– but
not in IEGZ-TD-I␬B␣–transduced cells. Doxycycline
(2 ␮g/mL) was added for 12 or 24 hours, respectively.
(C) Immunoblot analysis of CA-IKK2 expression in
transduced lymphoma lines. Protein extracts of transduced cells were isolated 24 hours after the infection.
EMSA of the extracts displays induction of NF-␬B
binding in CA-IKK2–expressing cells. (D) Morphologic
examination of transduced cell line 5522. Bright field
and fluorescence microscopy of transduced cells
2 days after infection. Images were taken with a Leica
DMIRBE microscope 10⫻ NA ⫽ 0.3 PH1 acquired with
a Hamamatsu digital camera C4742-95 (Hamamatsu
Photonics) and Open Lab software Version 4.0.4 (Improvision). (E) Flow cytometric determination of changes
in the GFP-positive fractions in transduced cultures.
Murine lymphoma cells were transduced with IEGZempty (〫), IEGZ-TD-I␬B␣ (䡺), or IEGZ-CA-IKK2 (Œ),
and percentage of GFP⫹ cells was measured by flow
cytometer at the indicated time points after retroviral
infection. Percentage of GFP⫹ cells at day 1 after
infection was set to 100% to facilitate comparisons.
(F) Apoptosis detection of murine lymphoma cell line
transduced with IEGZ-empty, IEGZ-TD-I␬B␣, or IEGZCA-IKK2. At day 3 after the infection the cells were
stained and analyzed for annexin-V–APC and 7-AAD
binding. (G) In vivo tumor competition assay. Murine
B-lymphoma cell line 5522 was transduced with either
IEGZ-empty or TD-I␬B␣. Transduced GFP⫹ cells were
then mixed with parental nontransduced cells at a ratio
of 1:10. Cells of mixed populations (107) were injected
intraperitoneally into syngeneic recipient mice and
lymphocytes were isolated from ascites 1 week later.
Flow cytometric determination of the GFP⫹ fractions is
shown for the cells before transplantation (top panels)
and after isolation of ascites of recipient mice (bottom
panels). (H) Statistical analyses of competitive tumor
transplantation. Mean values of the percentage of the
GFP⫹ fraction of cells isolated from recipient mice
(n ⫽ 6 for each group) detected by flow cytometry.
IKK2 SUPPRESSES MYC-INDUCED LYMPHOMAGENESIS
IEGZ-empty IEGZ-TD-IκBα
(n=6)
(n=6)
mas. The best model is the highly aggressive human Burkitt
lymphoma (BL), where transcriptional deregulation of MYC due to
chromosomal translocation is the pivotal event in lymphomagenesis.
We modulated NF-␬B activity in the Ramos BL cell line using
an episomal conditional expression system (pRTS) allowing high
expression of the transgene upon doxycycline addition without any
background activity in the absence of inducer.17 Ramos cells were
transfected with a control plasmid containing only GFP (pRTSGFP), the I␬B␣ superrepressor (pRTS-TD-I␬B␣), or the constitutive active IKK2 (pRTS-CA-IKK2). Selection for hygromycin
resistance and induction with doxycycline resulted in more than
90% GFP-positive cells (supplemental Figure 3) and transgene
expression (Figure 3A). Ramos cells show low basal NF-␬B levels
(Figure 3B) consisting of RelB-containing heterodimers (supplemental Figure 4),15,27 which were reduced in TD-I␬B␣–expressing
cells (Figure 3B). CA-IKK2 mediated an induction of NF-␬B
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Figure 3. Transfection of Ramos cells with inducible NF-␬B modulators. (A) Immunoblot analysis of transfected Ramos cells untreated or
treated with doxycycline (0.5 ␮g/m) for 48 hours. Cell extracts were
analyzed for CA-IKK2, for TD-I␬B␣, and as a control for actin expression. (B) EMSA of cell extracts shows induction of NF-␬B activity after
doxycycline addition. Oct1 DNA was used as control for the integrity of
NF-κB
the extracts. (C) Morphologic examination of transfected Ramos. Bright
field and fluorescence microscopy of cells treated with doxycycline for
Oct
5 days. Images were taken with a Leica DMIRBE microscope 10⫻
NA ⫽ 0.3 PH1 acquired with a Hamamatsu digital camera C4742-95
(Hamamatsu Photonics) and Open Lab software Version 4.0.4 (Improvision). (D) Proliferation assay of Ramos expressing pRTS-GFP (〫),
pRTS-TD-I␬B␣ (䡺), or pRTS-CA-IKK2 (Œ). GFP-positive cells were
counted by flow cytometer at the indicated time points after doxycycline
addition. Error bars represent SDs from triplicate experiments.
D
70
pRTS-GFP
pRTS-TD-IκBα
pRTS-CA-IKK2
cell number (x104)
60
50
40
30
20
10
0
0
2
5
7
days after DOX addition
activity (Figure 3B), which consists of p50 and p65/RelA subunits
(supplemental Figure 4).
Concerning morphologic appearance of the infected cultures,
no differences between TD-I␬B␣– and control-transfected cells or
nontransfected cells were seen (Figure 3C and data not shown).
However, upon CA-IKK2 induction Ramos cells showed rapid
clumping, highly reminiscent of the phenotype induced in murine
lymphoma lines (Figure 3C; Figure 2E). This phenotype was also
observed in the transfected BL cell lines Raji, Namalwa, and BL-30
upon transgene induction (supplemental Figure 5). We analyzed the
proliferation by counting GFP-positive cells over a time course of
1 week. Whereas no difference was seen between TD-I␬B␣–
expressing cells and controls, the CA-IKK2–expressing cells
showed reduced proliferation (Figure 3D). CA-IKK2–transfected
cultures showed a higher percentage of annexin-V–positive cells
(Figure 4A). In addition, we found increased percentages of dead
cells in Ramos-expressing CA-IKK2 (Figure 4B). Also in Raji,
Namalwa, and BL-30 cells expressing CA-IKK2 we observed 2- to
4-fold increases in dead cell numbers compared with pRTS-GFP–
or pRTS-TD-I␬B␣–transfected cells (supplemental Figure 6).
Increased cell death after CA-IKK2 expression was prevented by
addition of the pan-caspase inhibitor zVAD, suggesting that NF-␬B
induces apoptosis (Figure 4B).
An important antiapoptotic target gene of NF-␬B is CFLAR
(cFLIP), which prevents recruitment of caspase-8 to the deathinducing signaling complex. Interestingly, a direct repression of
CFLAR by MYC has been reported, which renders cells more
susceptible to extrinsic stimuli.28 CFLAR expression was hardly
detectable by Western blot in Ramos pRTS-GFP control cells and
expression increased in cells expressing CA-IKK2 (Figure 4C).
However, levels were low compared with other cell lines with
constitutive NF-␬B activity (eg, Hodgkin cell line L428; data not
shown). We tested whether ectopic CFLAR protects Ramos cells
from CA-IKK2–induced apoptosis and found that transfection with
a CFLAR expression vector blocked the apoptosis induced by
CA-IKK2 (Figure 4D).
As CFLAR prevents caspase-8 activation, we asked whether
knockdown of this protein could protect Ramos cells from CAIKK2–induced apoptosis. Ramos cell lines stably expressing a
specific shRNA showed reduced caspase-8 protein levels (Figure
4E). In these cells, CA-IKK2–induced cell death was almost
completely abolished (Figure 4F). These results imply that the
extrinsic death signaling pathway is responsible for the IKK2induced apoptosis.
IKK2 effects are NF-␬B dependent
To determine whether CA-IKK2–induced cell death is also seen in other
lymphoma entities, we analyzed a human classical Hodgkin lymphoma
cell line (KM-H2) and a cell line representing primary mediastinal
B-cell lymphoma (MedB1). These cells already show considerable
NF-␬B activity. Conditional CA-IKK2 expression again exceeded the
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
A
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20
35
CONTROL
zVAD
30
16
dead cells (%)
Annexin positive cells (%)
25
20
15
10
12
8
4
5
0
0
GFP
CA-IKK2
pRTS-GFP
C
pRTS-CA-IKK2
dead cells (%)
D
CA-IKK2
Ramos-pRTS
GFP
Figure 4. IKK2 expression induces the extrinsic apoptotic pathway. (A) Apoptosis detection in Ramos cells transfected with empty
vector or CA-IKK2. Doxycycline (0.5 ␮g/mL) was added to the culture
medium for 5 days before cells were analyzed for annexin-V binding.
(B) Percentages of dead cells after induction of transgene expression
) or in the presence of caspase inhibitor zVAD (
). Viable
without (
and dead cell numbers were determined by trypan blue exclusion. Cells
were treated with doxycycline and in the presence or absence of zVAD
(20 ␮M) for 4 days. (C) Expression of CFLAR in Ramos-pRTS-GFP and
Ramos-pRTS-CA-IKK2. Immunoblot analysis of Ramos cells transfected with pRTS-GFP or pRTS-CA-IKK2 and treated with doxycycline
(0.5 ␮g/m) for 48 hours. Cell extracts were analyzed for CFLAR and as
a control for actin expression. (D) Percentages of dead cells after
transfection of Ramos-pRTS-GFP and Ramos-pRTS-CA-IKK2 with
) or a CFLAR expression vector (
). Cells
either pcDNA (control,
were treated with doxycycline for 7 days to induce the pRTS-system.
(E) Immunoblot analysis of Ramos-pRTS-CA-IKK2 cells stably transduced with shRNA expression vectors encoding either a scrambled
shRNA (sh-co) or shRNA against caspase 8 (sh-C8). (F) Percentages
) or the
of dead Ramos cells expressing the nonsense shRNA (
) after CA-IKK2 transgene expression.
shRNA against caspase-8 (
Cells were treated with doxycycline (0.5 ␮g/m) for 6 days. All error bars
represent SDs from triplicate experiments.
IKK2 SUPPRESSES MYC-INDUCED LYMPHOMAGENESIS
14
pcDNA
12
CFLAR
10
8
6
4
CFLAR
2
0
Actin
pRTS-GFP
E
pRTS-CA-IKK2
F
25
Ramos
pRTS-CA-IKK2
shRNA-C8
20
sh-C8
Caspase 8
Actin
dead cells (%)
sh-co
sh-control
15
10
5
0
endogenous levels of IKK2 (Figure 5A and supplemental Figure 7A).
When apoptosis in CA-IKK2–expressing KM-H2 and MedB1 cells was
analyzed, no significant increase was seen (Figure 5B). As the level of
NF-␬B is limited by the amount of available inhibited NF-␬B complexes, all observed NF-␬B inductions were well within the physiologic
range (supplemental Figure 7B).
Although phosphorylation of the I␬B proteins is the best-established
function of IKK2, other targets of this kinase have been described.29 To
identify the relevant pathway, we transduced Ramos cells with a
retroviral vector expressing TD-I␬B␣ (IEGZ-TD-I␬B␣). Infected cells
(more than 95% positive for GFP; Figure 5C bottom panels; supplemental Figure 8) were transfected with pRTS-CA-IKK2 or a control
construct. EMSA revealed that TD-I␬B␣ completely blocks CA-IKK2–
induced NF-␬B activity (Figure 5C). The CA-IKK2–induced massive
clumping phenotype was largely attenuated (Figure 5D). Furthermore,
TD-I␬B␣ expression blocks CA-IKK2–mediated effects on proliferation (Figure 5E) and cell death, indicating that apoptosis induction is
entirely NF-␬B dependent (Figure 5F).
To rule out the possibility that CA-IKK2 expression leads to
supraphysiologic levels of NF-␬B in the transfected BL, we
induced physiologic levels of endogenous NF-␬B by stimulation
of CD40. Treatment with anti-CD40 antibody (G28.5) induced
NF-␬B binding in Ramos but not in Namalwa cells (supplemen-
Ramos
pRTS-CA-IKK2
tal Figure 9A). Treated Ramos cells showed rapid clumping,
whereas no alterations were seen for Namalwa cells (supplemental Figure 9B). Furthermore, Ramos cells showed reduced
proliferation and viability after anti-CD40 treatment (supplemental Figure 9C-D).
Physiologic NF-␬B stimulation had similar effects in responsive
murine MYC-driven lymphoma cells. Whereas most of the tested cell
lines did not induce NF-␬B in response to CD40 stimulation, we
identified 1 responsive cell line. In this cell line, anti-CD40–induced
NF-␬B was accompanied by cell clumping, reduced proliferation, and
reduced viability (supplemental Figure 10A-D).
IKK2 induces proapoptotic and antiapoptotic genes
To identify genes regulated by CA-IKK2, we performed expression
profiling using Affymetrix chips. By applying a threshold of 2, we
identified a total of 378 genes that were differentially expressed in
CA-IKK2 versus control cells. Three-hundred eight genes were upregulated and 70 genes were down-regulated in Ramos-CA-IKK2 cells
(supplemental Table 1). We were particularly interested in genes that
could mediate the observed phenotypes in cell adhesion, proliferation,
and apoptosis (Table 1). Several genes coding for cell adhesion
molecules were up-regulated including ICAM-1, a known target
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KLAPPROTH et al
B
A
30
KM-H2-pRTS-
GFP CA-IKK2
GFP CA-IKK2
pRTS-GFP
pRTS-CA-IKK2
25
IKK2
actin
dead cells (%)
MedB1-pRTS-
20
15
10
5
0
Ramos
D
C
TD-IκBα
empty
CACApRTS- GFP
GFP
IKK2
IKK2
Namalwa
pRTS-GFP
MedB1
KM-H2
pRTS-CA-IKK2
pCFG-IEGZ-empty
IEGZ
NF-κB
pCFG-IEGZ-TD-IκBα
Oct
IKK2
IκBα 32,36
IκBα
E
F
120
35
dead cells (%)
cell number (x105)
100
pRTS-GFP
pRTS-CA-IKK2
40
pRTS-GFP
pRTS-CA-IKK2
IκBα-pRTS-GFP
IκBα-pRTS-CA-IKK2
80
60
40
20
30
25
20
15
10
5
0
2
4
6
8
0
IEGZ empty
IEGZ-TD-IκBα
days after DOX addition
Figure 5. Morphologic alterations and induced cell death after CA-IKK2 expression are NF-␬B dependent. (A) Immunoblot of MedB1 and KM-H2 cell transfected with
either pRTS-GFP or pRTS-CA-IKK2. Cells were treated with doxycycline (0.5 ␮g/mL) for 48 hours to induce transgene expression. Cell extracts were analyzed for CA-IKK2
) or pRTS-CA-IKK2 (
) after induction of transgene expression. Viable
and, as a control, for actin expression. (B) Percentages of dead cells transfected with pRTS-GFP (
and dead cell numbers were determined by trypan blue exclusion. Cells were treated with doxycycline (0.5 ␮g/m) for 6 days. (C) Immunoblot analysis of Ramos cells stably
transduced with IEGZ-empty or IEGZ-TD-I␬B␣ and transfected with inducible pRTS-GFP or pRTS-CA-IKK2. Doxycycline was added to cultures (0.5 ␮g/mL) for 48 hours to
induce transgene expression. (D) Morphologic examination of transfected Ramos cells. Fluorescence microscopy of cells treated with doxycycline for 5 days. Images were
taken with a Leica DMIRBE microscope 10⫻ NA ⫽ 0.3 PH1 acquired with a Hamamatsu digital camera C4742-95 (Hamamatsu Photonics) and Open Lab software Version
4.0.4 (Improvision). (E) Proliferation assay of Ramos-expressing NF-␬B modulators. Ramos cells were stably transduced with IEGZ-empty and transfected with either
pRTS-GFP (f) or pRTS-CA-IKK2 (E), or transduced with IEGZ-TD-I␬B␣ transfected with either pRTS-GFP (F) or pRTS-CA-IKK2 (‚). Viable cell numbers were determined by
) or pRTS-CA-IKK2 (
). Increased
trypan blue exclusion at the indicated time points. (F) Viable cell counts of transduced Ramos cells transfected with either pRTS-GFP (
percentages of dead cells after CA-IKK2 expression were reduced in the presence of TD-I␬B␣. Viable and dead cell numbers were determined by trypan blue exclusion. Cells
were treated with doxycycline (0.5 ␮g/mL) for 6 days. All error bars represent SDs from triplicate experiments.
of NF-␬B.30 In addition, several integrins (CD11b/ITGAM,
CD18/ITGB2-LFA-1, and CD58/LFA-3) and genes regulating
antigen processing and presentation (CIITA, TAP, IFI30), and
several chains of MHC class I and class II complexes (supple-
mental Table 1) were induced. We found several genes involved
in cell-cycle regulation. Most prominently, we detected enhanced expression of RASSF4, a gene that has been described as
a potential tumor suppressor due to its antiproliferative effects.31
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
IKK2 SUPPRESSES MYC-INDUCED LYMPHOMAGENESIS
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Table 1. Genes regulated following CA-IKK2 induction in Ramos cells
Gene ID
Gene name
Fold change
Cell adhesion
IL32
Interleukin 32
CCL5
Chemokine (C-C motif) ligand 5
18.4
17.8
ICAM1
Intercellular adhesion molecule 1 (CD54)
13.5
FBLN5
Fibulin 5
6.3
ITGB2
Integrin, beta 2 (CD18)
5.7
ADAM8
ADAM metallopeptidase domain 8
5.2
ITGAM
Integrin, alpha M (CD11b)
3.4
FCGBP
Fc fragment of IgG binding protein
3.3
CD58
CD58 molecule
3.0
Apoptosis (proapoptotic)
FAS
Fas (TNF receptor superfamily, member 6)
BTG1
B-cell translocation gene 1
29.7
PLAGL1
Pleiomorphic adenoma gene-like 1
4.1
IFIH1
Interferon induced with helicase C domain 1
3.5
BID
BH3 interacting domain death agonist
2.3
RUNX3
Runt-related transcription factor 3
2.0
ID3
Inhibitor of DNA binding 3
0.5
4.5
Apoptosis (antiapoptotic)
BCL2A1
BCL2-related protein A1
84.2
SGK1
Serum/glucocorticoid regulated kinase 1
30.9
TNFRSF9
Tumor necrosis factor receptor superfamily, member 9
23.6
TNFAIP3
Tumor necrosis factor alpha-induced protein 3
17.1
CD40
CD40 molecule, TNF receptor superfamily member 5
6.7
IL1B
Interleukin 1 beta
6.3
NUP62
Nucleoporin 62 kDa
5.1
ATF5
Activating transcription factor 5
3.7
TRAF1
TNF receptor-associated factor 1
3.0
PIM1
Pim-1 oncogene
2.9
IL2RA
Interleukin 2 receptor alpha
2.7
BIR3
baculoviral IAP repeat-containing 3
2.6
IER3
Immediate early response 3
2.5
BCL2
B-cell CLL/lymphoma 2
2.5
BCL3
B-cell CLL/lymphoma 3
2.5
CFLAR
CASP8 and FADD-like apoptosis regulator
2.4
NFKB1
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)
2.4
NEK6
NIMA (never in mitosis gene a)–related kinase 6
2.3
BCL2L1
BCL2-like 1
2.1
RASSF4
Ras association (RalGDS/AF-6) domain family 4
2.9
PLEKHO1
Pleckstrin homology domain containing,
2.2
WEE1
WEE1 homolog (S. pombe)
2.0
MACF1
Microtubule-actin cross-linking factor 1
2.0
HGF
Hepatocyte growth factor (hepapoietin A; scatter factor)
0.3
Cell cycle (inhibiting)
7.8
Cell cycle (promoting)
STAG3
Stromal antigen 3
3.9
CYLD
Cylindromatosis (turban tumor syndrome)
2.6
TUBB2B
Tubulin, beta 2B
2.4
RNA from cells transfected with either pRTS-CA-IKK2 or pRTS-GFP and stimulated with doxycycline for 48 hours was analyzed by expression profiling. Genes were
identified as differentially expressed if the ratio between the 2 classes was at least 2-fold. Biologic significance was determined using Gene Ontology.20
We also observed a large number of differently expressed genes
related to cell survival and apoptosis. CA-IKK2 induced several
antiapoptotic genes such as members of the antiapoptotic BCL-2
family (BCL-2, BCL2L1 /BCL-X, BCL2A1), FLIP/CFLAR, and the
caspase inhibitor cIAP2/BIR3 (Table 1). However, we also detected
enhanced expression of several proapoptotic genes, with expression of Fas, a death receptor–inducing apoptosis via FADD and
caspase-8 activation, being strongly induced.
NF-␬B signaling sensitizes BL cells to Fas-induced apoptosis
Fas-receptor signaling plays a pivotal role for lymphocyte homeostasis, and Fas induction by CA-IKK2 could render BL cells
susceptible to apoptosis. We determined Fas expression on the cell
surface by flow cytometry. Upon induction of CA-IKK2 expression
by doxycycline, 70% to 80% of the cells became GFP positive.
This allowed us to compare levels of Fas surface expression in the
GFP⫺ and the GFP⫹ fractions. In control-transfected Ramos cells,
we found no differences in Fas expression between GFP⫺ and
GFP⫹ cells (Figure 6A). In contrast, cells transfected with the
pRTS-CA-IKK2 showed strong up-regulation in the majority of the
GFP⫹ cells (Figure 6A). Induction of Fas surface expression was
also seen in Namalwa cells, whereas there was no significant
influence of IKK2 expression in MedB1 and KM-H2 cells (supplemental Figure 11).
To analyze if Fas renders Ramos cells more sensitive to death
induction by Fas ligation, we treated the cells with agonistic
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100
pRTSGFP
80
60
60
50
40
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% of Max
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
KLAPPROTH et al
20
0
% of Max
100
pRTSCA-IKK2
80
60
CONTROL
APO-I
40
30
20
10
40
0
20
pRTS-GFP
0
pRTS-CA-IKK2
FAS Surface Expression
D
C
pRTS- GFP
CAIKK2
TD-IκBα
GFP
50
CAIKK2
Fas
Actin
dead cells (%)
empty
IEGZ empty
IEGZ-TD-IκBα
40
30
20
Figure 6. NF-␬B activation sensitizes Ramos cells to Fasmediated apoptosis. (A) Surface expression of Fas on Ramos
cells transfected with either pRTS-GFP (top panel) or pRTS-CAIKK2 (bottom panel). Cells were treated with doxycycline (0.5 ␮g/
mL) for 48 hours, incubated with APO-I antibodies and a corresponding secondary antibody conjugated to Alexa-647
fluorochrome. Stained cells were subjected to flow cytometry and
gates were set on GFP⫺ (gray line) and GFP⫹ (black line)
fractions. (B) Percentages of dead cells in Ramos transfected with
) or presence
pRTS-GFP or pRTS-CA-IKK2 in the absence (
) of agonistic anti-Fas antibody (APO-I). Cells were cultured
(
in the presence of doxycycline for 4 days to induce transgene
expression and as indicated treated with APO-I supernatant (5%
final concentration [f.c.]) for additional 48 hours. Viable and dead
cell numbers were determined by trypan blue exclusion. Error
bars represent SDs from triplicate experiments. (C) Fas mRNA
expression analyzed by reverse-transcription–PCR. Total RNA of
Ramos stably transduced with either IEGZ-empty or IEGZ-TDI␬B␣ and transfected with pRTS-GFP and pRTS-CA-IKK2, respectively, was isolated 48 hours after addition of doxycycline to the
culture medium. (D) Percentages of dead cells in cultures of
) or Ramos-IEGZ-TD-I␬B␣ (
) transRamos-IEGZ-empty (
fected with either pRTS-GFP or pRTS-CA-IKK2 after addition of
agonistic anti-Fas antibody (APO-I). Fas sensitivity of Ramos-CAIKK2 is abrogated by TD-I␬B␣ expression. Doxycycline (0.5 ␮g/
mL) was added to the culture medium for 4 days and APO-I
supernatant (5% f.c.) was added for additional 24 hours. Viable
and dead cell numbers were determined by trypan blue exclusion.
Error bars represent SDs from triplicate experiments.
10
0
pRTS-GFP
anti-Fas antibodies (APO-I). Whereas control cells were virtually
resistant to APO-I treatment, CA-IKK2–expressing cells showed
increased levels of dead cells (2- to 3-fold) in the absence of APO-I,
which was significantly enhanced by addition of APO-I (Figure
6B). Interestingly the constitutive increase in apoptosis of CAIKK2–expressing Ramos cells could not be prevented by addition
of neutralizing anti–human FasL mAbs (supplemental Figure 12),
suggesting a ligand-independent autoactivation of Fas receptors.
The strong induction of Fas mRNA expression by CA-IKK2 was
largely attenuated in TD-I␬B␣–expressing cells (Figure 6C). In
addition, APO-I–induced cell death in CA-IKK2–expressing cells
was completely blocked by TD-I␬B␣ (Figure 6D).
Discussion
MYC-induced apoptosis represents a major safeguard mechanism against unrestricted growth that has to be overcome in
tumorigenesis. A primary role for NF-␬B in malignant transformation is prevention of apoptosis caused by certain oncogenes.
NF-␬B–regulated genes in lymphocytes are associated with
cell-cycle progression, survival, and regulation of immune
responses.32 In many hematologic malignancies such as Hodgkin
lymphoma, mucosa-associated lymphatic tissue lymphoma, and
diffuse large B-cell lymphoma, NF-␬B is constitutively activated.33 NF-␬B itself is suggested to be an oncogenic factor in
lymphomagenesis or it might predispose malignant transformation by inhibiting apoptotic signals induced by other oncogenes.
Indeed, the tumor-promoting role of NF-␬B signaling is widely
acknowledged. It therefore came as a surprise when 2 recent
studies using gene expression profiling demonstrated that very
low NF-␬B signatures are a hallmark of BL.13,14 Our finding that
MYC-transformed mouse and human lymphomas are insensitive
pRTS-CA-IKK2
to NF-␬B inhibition extends these observations and proves that
canonical NF-␬B activation is dispensable for proliferation or
cell survival. Apparently, in MYC-driven lymphomas NF-␬B is
not involved in tumor promotion. This absence of NF-␬B
signaling has been described for GC centroblasts, the normal
counterparts of malignant BL. Therefore, the low NF-␬B profile
in MYC-driven lymphomas could reflect the normal differentiation program of GC-derived B cells. However, we now present
evidence that NF-␬B inhibition provides a selective advantage
to MYC-transformed lymphomas and its activation actually
interferes with both cell survival and proliferation. These
observations might have implications for the development of
therapies against MYC-positive tumors.
Components of the NF-␬B signaling pathway are present in
these cells, as NF-␬B activity can be restored by turning off
MYC expression. Importantly, most lymphoma cell lines were
resistant to NF-␬B induction by conventional extracellular
stimuli. We did, however, find a few lines showing some
inducible NF-␬B activation upon either PMA/ionomycin or LPS
treatment (data not shown). Furthermore, we found NF-␬B
activation after anti-CD40 stimulation in Ramos and 1 murine
MYC-driven lymphoma line, whereas Namalwa and other tested
murine lines showed no NF-␬B activation. This suggests that
although decreased inducibility of NF-␬B is a common feature
in these lymphoma cells, the actual pathways leading to the
block in NF-␬B activation in individual tumors vary. MYCmediated NF-␬B suppression has been described in several
different cell systems, but it was typically associated with
increased sensitivity to apoptosis induction. In mouse primary
fibroblasts, inducible MYC expression impairs TNF-induced
activation of NF-␬B, thereby rendering them sensitive to
TNF-induced cell death. Apoptosis could be inhibited by overexpression of p65/RelA.34 In TRAIL-resistant human colon
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BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
cancer cell lines, expression of MYC sensitized cells to
TRAIL-induced apoptosis through inactivation of NF-␬B.35
Expression of CA-IKK2 in murine lymphoma cells as well as
in human BL led to enhanced cell adhesion, reduced proliferation, and apoptosis. We can exclude toxic effects of CA-IKK2
overexpression, because tested Hodgkin and primary mediastinal B-cell lymphoma lines were not sensitive to CA-IKK2
expression and blocking of NF-␬B in CA-IKK2–expressing
Ramos prevented cell death. The physiologic activation of
NF-␬B in a murine MYC-driven lymphoma line or human BL
Ramos by CD40 stimulation resulted in comparable effects of
cell clumping and cell death.
NF-␬B stimulates the expression of several antiapoptotic
proteins. Among them are CFLAR, inhibitors of apoptosis
(IAPs), and members of the antiapoptotic BCL-2 family.36-38
Our gene expression profiling suggests that NF-␬B indeed
regulates both proapoptotic and antiapoptotic genes in BL. The
net consequence is, however, induction of apoptosis. Our results
therefore indicate a tumor-suppressive role for NF-␬B activity
in MYC-induced lymphomas. Apoptosis as a safeguard mechanism against tumors operates at 2 levels. (1) It operates as an
immune surveillance mechanism by instructing cytotoxic cells
of the immune system to eliminate transformed cells. For this
level of protection, cell-cell contact of the malignant cell with
cytotoxic T cells and often presentation of tumor antigens are
necessary. (2) It operates on the level of cell intrinsic pathways
because deregulation of oncogenic proteins simultaneously
induces apoptosis. Tumor cells escape cytotoxic T cell–mediated
elimination by down-regulating accessory molecules or by blocking intracellular processing of tumor-specific antigens. BL cells
show low expression of LFA-1 (CD11/CD18) and LFA-3 (CD58)39
as well as ICAM-1.40 Antigen presentation is also affected due to
down-regulation of MHC molecules as well as transporters associated with antigen presentation (TAP1/TAP2).41 This nonimmunogenic phenotype is imposed by MYC expression, as it was shown
in a conditional model that MYC inactivation results in upregulation of accessory molecules and antigen presentation.42
However, the mechanism of this MYC-induced repression of
immunogenicity was not clear. Our results demonstrate that
IKK2/NF-␬B is controlling expression of several of these such
as LFA-1, LFA-3, ICAM-1, TAP1, HLA-I, and HLA-2 (supplemental Table 1). The nonimmunogenic phenotype in BL cells is
largely the result of low NF-␬B.
However, immune surveillance cannot account for the cell
death we observed in vitro. CA-IKK2 induced several proapoptotic
genes in Ramos cells. BTG1 (B-cell translocation gene 1), a
Bcl-2–regulated mediator of apoptosis in breast cancer cells,43
PLAGL1, IFIH1, and RUNX3 are all considered tumor suppressors
because of their apoptosis-inducing capacities.44-46 BID, a BH3only protein promoting cytochrome c release, had been connected
to MYC-induced cell-death priming.47 Most significantly, we
detected strong up-regulation of Fas, a death receptor previously
linked to MYC-induced apoptosis. MYC has been shown to
sensitize cells to Fas-induced death.48 Insensitivity to Fas-mediated
apoptosis is a common attribute of BL cells and correlates with low
Fas surface expression.49,50 Ramos and Namalwa cells are completely resistant to agonistic Fas-antibody and blocking canonical
NF-␬B activation did not enhance sensitivity. However, NF-␬B
activation itself renders the cells highly susceptible to Fasmediated apoptosis.
The high expression of Fas after NF-␬B activation can contribute to apoptosis of the cells in vitro via 2 ways. (1) Apoptosis can
IKK2 SUPPRESSES MYC-INDUCED LYMPHOMAGENESIS
2457
be the result of Fas-ligand (FasL) expression by the Ramos cells
that would result in Fas receptor ligation in an autocrine manner.
(2) Apoptosis could be induced by a ligand-independent selfassembly of the receptors, which has been described.51 MYCdependent FasL expression has been reported at least for some
cell types.52 However, using the described method of metaanalysis of multiple cancer studies within the Oncomine database,53,54 we found no consistent pattern of FasL up-regulation
in BL compared with other lymphomas, and with real-time PCR
we found FasL mRNA to be below detection levels in Ramos
and Namalwa cells. We observed constitutive apoptosis in response
to IKK2 expression in Ramos, which was reduced by CFLAR
overexpression or caspase-8 knockdown, pointing toward death
receptor–mediated cell killing. CFLAR has been reported to be
down-regulated by MYC, rendering cells sensitive to extrinsic
death-inducing signals via caspase-8 processing.28 Indeed, we
found CFLAR levels in Ramos cells to be very low and only
slightly up-regulated after IKK2/NF-␬B activation. Low CFLAR
expression due to MYC repression could explain the toxicity of
CA-IKK2, because NF-␬B–mediated death receptor induction
cannot be balanced by sufficient expression of the antiapoptotic
adaptor molecule. NF-␬B–induced apoptosis could be further
enhanced by APO-I stimulation, indicating high sensitivity of the
cells toward Fas signaling. Activation of self-assembling Fas
receptors would explain the death-inducing activity in vitro even
in the absence of enhanced FasL expression. In vivo, FasL is
expressed by several cell types, especially T cells and natural
killer cells. The Fas-FasL apoptosis pathway is critical for
activation-induced cell death, limiting proliferation of lymphocytes in the periphery. Furthermore, Fas-mediated apoptosis is
involved in negative selection of B cells in germinal centers. Fas
is a known tumor suppressor also for BL. Induction of Fas
expression and restoration of Fas sensitivity upon NF-␬B
activation impede immune escape in vivo and contribute to
tumor suppression. The fact that mouse and human MYCtransformed lymphoma cells show no evidence of NF-␬B
activation argues for a strong selective pressure against NF-␬B
in lymphomagenesis.
Acknowledgments
We thank K. Holzmann and the Microarray Facilities of the
University of Ulm. We thank D. Eick and G. W. Bornkamm for the
inducible gene expression vector system, S. Fulda and G. Strauss
for providing caspase-8 shRNA and the APO-I antibody, and
P. Krammer for the CFLAR expression vector.
Authorship
Contribution: K.K. designed the research, performed experiments,
analyzed results, made the figures, and wrote the paper; S.S., D.M.,
and B.B. performed experiments; and T.W. designed the research
and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Thomas Wirth, Institute of Physiological
Chemistry, University of Ulm, Albert-Einstein-Allee 11, 89081
Ulm, Germany; e-mail: thomas.wirth@uni-ulm.de.
From www.bloodjournal.org by guest on September 29, 2016. For personal use only.
2458
KLAPPROTH et al
BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
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From www.bloodjournal.org by guest on September 29, 2016. For personal use only.
2009 114: 2448-2458
doi:10.1182/blood-2008-09-181008 originally published
online July 23, 2009
The IKK2/NF-κB pathway suppresses MYC-induced lymphomagenesis
Kay Klapproth, Sandrine Sander, Dragan Marinkovic, Bernd Baumann and Thomas Wirth
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