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LYMPHOID NEOPLASIA
Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic
lymphocytic leukemia cells
Sergey Krysov,1 Samantha Dias,1 Alex Paterson,1 C. Ian Mockridge,2 Kathleen N. Potter,2 Kelly-Ann Smith,3
Margaret Ashton-Key,3 Freda K. Stevenson,2 and Graham Packham1
1Cancer Research UK Centre, Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton,
United Kingdom; 2Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals Trust, Southampton,
United Kingdom; and 3Department of Cellular Pathology, Southampton University Hospitals National Health Service Trust, Southampton General Hospital,
Southampton, United Kingdom
Although long considered as a disease of
failed apoptosis, it is now clear that
chronic lymphocytic leukemia (CLL) cells
undergo extensive cell division in vivo,
especially in progressive disease. Signaling via the B-cell receptor is thought to
activate proliferation and survival pathways in CLL cells and also has been
linked to poor outcome. Here, we have
analyzed the expression of the protooncoprotein MYC, an essential positive
regulator of the cell cycle, after stimula-
tion of surface IgM (sIgM). MYC expression was rapidly increased after sIgM
stimulation in a subset of CLL samples.
The ability of sIgM stimulation to increase
MYC expression was correlated with sIgMinduced intracellular calcium fluxes. MYC
induction was partially dependent on the
MEK/ERK signaling pathway, and MYC
and phosphorylated ERK1/2 were both
expressed within proliferation centers in
vivo. Although stimulation of sIgD also
resulted in ERK1/2 phosphorylation, re-
sponses were relatively short lived compared with sIgM and were associated
with significantly reduced MYC induction, suggesting that the kinetics of
ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of
MYC is likely to play an important role in
antigen-induced CLL cell proliferation.
(Blood. 2012;119(1):170-179)
Introduction
Chronic lymphocytic leukemia (CLL) is a relatively common
B-cell malignancy with a very variable clinical course;1,2 some
patients survive for many years, whereas others progress rapidly
despite aggressive therapy. Although considered for a long time as
a disease of failed apoptosis, it is now clear that increased cell
division plays a major role in accumulation of CLL cells. Metabolic
labeling experiments have demonstrated significant rates of cell
“birth” in vivo (up to ⬃ 1% of the malignant clone/day),3 and there
is also evidence for telomere erosion in CLL cells4-6 indicative of
extensive proliferation. Importantly, the extent of cell birth and
telomere erosion is associated with poor outcome or prognostic
markers, indicating that cell division is a determinant of disease
progression.
Cell division occurs predominantly within proliferation centers
(PCs) that are present within involved lymph nodes and to a lesser
extent in the bone marrow of CLL patients. PCs are thought to be
sites of antigen stimulation, implying a major role for ongoing
B-cell receptor (BCR) signaling in driving cell-cycle progression in
vivo, in the context of signals from soluble cytokines and
supporting cells with the PC microenvironment.7,8 Signaling responses after surface IgM (sIgM) stimulation are variable in CLL
samples, and retained signaling capacity is associated with markers
of poorer prognosis, including unmutated (U) immunoglobulin
heavy variable (IGHV) genes, ZAP-70, and CD38.9-14 For example,
in our study of intracellular Ca2⫹ responses, the majority of U-CLL
were responsive after sIgM stimulation, whereas in mutated
(M)–CLL responses were more variable, with ⬃ 40% retaining
signaling responses.9
The molecular mechanisms that drive cell division in CLL are
relatively poorly understood. Previous studies have shown that
CpG-containing oligodeoxynucleotides (CpG-ODNs; with or without IL-2) regulate components of the cell-cycle machinery in CLL
cells, including induction of cyclins A, D2, D3, and E and reduction
in p27kip1.15-18 sIgM stimulation also has been shown to result in
increased expression of cyclin D2 and cdk4 at the RNA and protein
levels in CLL cells.11,14 However, what links upstream signaling
pathways to these downstream effects on the cell-cycle machinery
is unclear.
One candidate mediator is the proto-oncoprotein MYC, a key
regulator of cell-cycle entry. MYC is a transcription factor that
is activated by mitogens and that regulates the expression of
proteins essential for cell-cycle progression and cell growth,
including ornithine decarboxylase 1 (ODC1), cyclin D2, and
cdk4.19-23 Recent immunoblotting and gene expression studies
have demonstrated that expression of MYC and its target genes
is increased in CLL lymph nodes compared with blood cells and
that increased basal expression of MYC in circulating CLL cells
is associated with progressive disease.24,25 MYC also was
identified as an anti-IgM–regulated gene in CLL cells as part of
a gene expression microarray study.26 However, the BCRdependent regulation of this critical cell-cycle protein in CLL
has not been studied in detail.
Submitted July 26, 2011; accepted November 2, 2011. Prepublished online as Blood
First Edition paper, November 15, 2011; DOI 10.1182/blood-2011-07-370403.
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 the article contains a data supplement.
© 2012 by The American Society of Hematology
170
BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
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BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
Here, we have analyzed the regulation of MYC after stimulation
of sIgM in vitro. We demonstrate that sIgM stimulation results in
induction of MYC in some CLL samples. MYC induction was
partially dependent on the MEK1/2 3 ERK1/2 signaling pathway,
and MYC and phosphorylated ERK1/2 were both expressed within
PCs in vivo. Although stimulation of sIgD also resulted in ERK1/2
phosphorylation, responses were short lived compared with sIgM
and were associated with significantly reduced MYC expression,
suggesting that the kinetics of ERK1/2 activation is a critical
determinant of MYC induction. Our results suggest that ERK1/2dependent induction of MYC is likely to play an important role in
antigen-induced CLL proliferation.
Methods
Samples and reagents
This study was performed following ethical approval from the Southampton and South West Hampshire Research Ethics Committee. Informed
consent was provided in accordance with the Declaration of Helsinki.
Blood was obtained from 53 patients in total with typical CLL who attended
hematology outpatient clinics at the Leicester Royal Infirmary, Portsmouth
Hospital, Southampton General Hospital, the Royal Wolverhampton Hospitals NHS Trust, and the Royal Berkshire Hospital, Reading (all in the
United Kingdom). Clinical details for the patients studied are given in Table
1. IGHV gene mutation status and intracellular Ca2⫹ responses were
determined as described previously.9,27
PBMCs were isolated by Lymphoprep centrifugation (Axis-Shield
Diagnostics), washed and cryopreserved in RPMI-1640 (Invitrogen) supplemented with 10% (vol/vol) DMSO and 15% (vol/vol) FCS. CLL samples
were thawed in complete culture medium (RPMI-1640 supplemented with
10% [vol/vol] FCS, 2mM glutamine, and 1% [wt/vol] sodium pyruvate),
pelleted by centrifugation, and resuspended in complete medium. Cells
were allowed to recover by incubation for 1 hour at 37°C. Cell viability by
trypan blue exclusion was more than 90%. The proportion of contaminating
normal CD19⫹CD5⫺ B cells, as determined by flow cytometry, was less
than 1.0%. BCR signaling was evaluated after cells were treated with
20 ␮g/mL goat F(ab⬘)2 anti–human IgM or IgD (Southern Biotechnology)
at 37°C for various times.
Normal B cells were isolated from peripheral blood or buffy coats from
healthy donors using the B Cell Isolation Kit II (Miltenyi Biotec) according
to the manufacturer’s protocol. CD27⫺ cells were isolated by adding an
anti-CD27 antibody to the antibody cocktail provided with the kit.
MEK1/2 inhibitor U0126 was from Sigma-Aldrich.
Cell surface staining
Thawed lymphocytes were stained for 30 minutes at 4°C with anti-CD5
PerCP-Cy5.5 and anti-CD19 APC (both from BD Biosciences) and either
anti-CD38 PE (clone HB7; BD Biosciences), anti-IgM PE, or anti-IgD
FITC (both Dako UK). Data were acquired on an FACSCalibur flow
cytometer (BD Biosciences) with CellQuest Pro Version 3.3 software (BD
Biosciences). Mean fluorescence intensity and percentage of positive
staining within the CD5⫹CD19⫹ lymphocyte population were measured
relative to appropriate isotype controls. For CD38 analysis, samples with
greater than or equal to 30% expressing tumor cells were designated as
positive.28 Determination of ZAP-70 status was performed as described;
samples where greater than or equal to 20% of tumor cells expressed
ZAP-70 were designated ZAP-70 positive.29
Quantitative real-time PCR
CLL cells (1 ⫻ 107) in 1 mL of complete growth medium were treated with
20 ␮g/mL goat F(ab⬘)2 anti–human IgM or IgD or isotype control antibody
for 1 and 6 hours. Total RNA was isolated using the RNeasy Kit (QIAGEN)
according to the manufacturer’s instructions and converted to cDNA using
SIGM STIMULATION AND MYC EXPRESSION IN CLL
171
Table 1. Clinical details of samples
Sample
Stage*
IGHV mutation status†
ZAP-70, %
CD38, %
63
na
M
4
97
189
A
M
12
1
191
A
M
10
1
232
A
M
30
33
239
A
M
0
2
268
A
M
4
0
269
B
M
1
1
273
C
M
0
3
277
na
M
1
0
318
C
M
2
5
333
A
M
1
1
353
A
M
2
39
367
A
M
10
21
368
C
M
74
2
374
A
M
0
3
379
A
M
0
1
247
A
M
0
4
290
na
M
nd
5
296
na
M
nd
3
299
A
M
1
0
351
na
M
17
57
357
A
M
5
36
370
na
M
3
1
196
na
U
nd
38
199
C
U
32
22
221
A
U
16
51
231
B
U
11
82
233
A
U
48
34
256
B
U
5
15
284
B
U
48
12
285
A
U
35
100
288
A
U
61
71
291
na
U
88
18
298
C
U
26
92
304
B
U
24
74
305
C
U
65
9
306
A
U
23
49
328
A
U
16
23
343
A
U
16
54
346
B
U
51
89
361
A
U
5
98
376
A
U
9
63
383
A
U
17
24
385
A
U
11
36
238
A
U
82
6
293
A
U
29
16
352
B
U
4
3
358
na
U
1
7
359
na
U
1
10
409
B
U
77
56
410
A
U
8
56
421
B
U
94
42
422
B
U
62
48
na indicates not available; and nd, not defined.
*Binet stage at diagnosis.
†M indicates mutated; and U, unmutated.
oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase
(Promega). PCR reactions were performed using a 7500 Real-Time PCR
System and TaqMan Universal PCR Master Mix (Applied Biosystems) and
the following TaqMan probes: Human B2M (␤2-microglobulin) Endogenous Control (4333766T), ODC1 (Hs00159739_m1), CCND2
(Hs00277041_m1), CDK4 (Hs00262861_m1), and MYC (Hs00153408_m1;
all Applied Biosystems). Relative RNA quantities were calculated with the
equation RQ ⫽ 2⫺(⌬⌬CT) using B2M expression as an internal control.
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172
KRYSOV et al
Immunoblot analysis
Cells were lysed on ice for 30 minutes using lysis buffer (1% [vol/vol]
Nonidet P-40, 10mM Tris-HCl, pH 8.0, 150mM NaCl, and 0.02% [wt/vol]
sodium azide). Samples were centrifuged, and the protein content of the
supernatant was measured using the BCA Protein Assay Kit (Pierce
Chemical). Immunoblotting was performed using 30 ␮g and 50 ␮g of
protein lysate for analysis of ERK1/2 and MYC/MAX, respectively. The
following antibodies were used: anti–T202/Y204-phosphorylated (p)ERK1/2,
anti-ERK1/2, anti-p38 MAPK, anti-MAX (all from Cell Signaling Technology) and anti-MYC (clone 9E10; Cancer Research UK, Research Monoclonal Antibody Service). Secondary HRP–conjugated antibodies were from
GE Healthcare. Images were collected using a Fluor-S MultiImager
(Bio-Rad Laboratories) and quantified using Fluor-S software Quantity One
Version 4.6.3 (Bio-Rad Laboratories Inc). All values were normalized to the
relevant loading control, and relative fold-change was calculated with the
isotype control antibody–treated cells taken as a 100% of expression.
PhosFlow analysis
The kinetics of ERK1/2 phosphorylation was determined using flow
cytometry.30 PBMCs were thawed, washed, and resuspended in complete
culture medium at 1 ⫻ 107 cells/mL. Aliquots (100 ␮L) were treated with
F(ab⬘)2 anti–human IgM or IgD for various times at 37°C, fixed with BD
Cytofix (BD Biosciences) according to the manufacturer’s instructions, and
stored at ⫺80°C. Immediately before staining, cells were pelleted and
resuspended in 90% (vol/vol) methanol and incubated on ice for 30 minutes. After washing twice with FACS buffer (BD Biosciences), the cells
were labeled with 5 ␮L anti–pERK1/2 (pT202/pY204) AlexaFluor 488 (BD
Biosciences) for 30 minutes at room temperature. For analysis of normal
CD20⫹CD27⫺ B cells, cells were additionally labeled with 5 ␮L of CD20
PerCP-Cy5.5 and 5 ␮L of CD27 PE (both from BD Biosciences), and
AlexaFluor 488 fluorescence within the CD20⫹CD27⫺ lymphocyte population was measured. Data were acquired on a FACScalibur flow cytometer
and analyzed with CellQuest Pro Version 3.3 software.
Analysis of CLL cell-cycle entry
CLL cell-cycle entry was stimulated by treating cells for 3 or 48 hours with
CpG-containing ODNs (7 ␮g/mL, ODN-2006; Invitrogen).15 S-phase entry
was quantified using bromodeoxyuridine (BrdU) staining and flow cytometry (FITC BrdU Flow Kit; BD Biosciences, PharMingen). Samples were
gated to exclude dead cells, and the proportion of BrdU⫹ cells in S-phase
was determined as a proportion of all viable cells.
Immunohistochemistry
Immunohistochemical analysis was performed using formalin-fixed and
paraffin-embedded lymph node tissue sections obtained from 8 cases of
CLL/small lymphocytic lymphoma (SLL). Immunostaining was performed
using the Bond autostainer and reagents (Leica Microsystems) and an
MYC-specific monoclonal antibody (N262; Santa Cruz Biotechnology) at a
dilution of 1:50 using the Bond ER1 protocol for 20 minutes or a T202/Y204
pERK1/2-specific antibody (Cell Signaling Technology) at a dilution of
1:100 using the Bond ER1 protocol for 30 minutes. A Burkitt lymphoma
and colon cancer biopsy samples were used as a positive control for MYC
and pERK1/2 staining, respectively.
Statistics
Statistical analyses were performed using Prism Version 4.03 software
(GraphPad). All tests were 2-tailed with 95% confidence interval values.
Results
Induction of MYC protein after stimulation of sIgM in CLL cells
We performed immunoblotting to determine whether sIgM stimulation increased MYC protein expression in CLL cells (Figure 1A).
We first analyzed induced MYC expression in 18 samples, all of
which were considered as anti-IgM–responsive based on the ability
BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
of anti-IgM to promote intracellular Ca2⫹ mobilization.9 This
cohort comprised 8 U-CLL samples, as well as 10 M-CLL samples
representative of the subset of M-CLL that retain sIgM intracellular
Ca2⫹ responses.9 We selected a greater than 20% increase in MYC
expression as an arbitrary cut-off to separate positive and negative
responses, based on consideration of the ability of immunoblotting
to reproducibly detect increases. Using this cut-off, MYC expression was increased in 16/18 (89%) of these samples at 3 hours after
stimulation with anti-IgM relative to control cells. On average,
MYC expression was increased by 2.0-fold at this time (range, 1.0to 4-fold) and was statistically significantly higher in anti-IgM
stimulated compared with control cells (P ⫽ .0001; Figure 1B).
There was no significant difference in the fold increase in MYC
expression between U-CLL and M-CLL samples (P ⬎ .05; data not
shown). MYC protein levels were either maintained or decreased at
6 hours after stimulation, although in the 2 cases with the highest
levels of MYC induction, MYC expression continued to increase.
Both the MYC1 and MYC2 isoforms, generated by alternate
translation initiation, were detected and all CLL samples expressed
MAX (MYC associated factor X), the obligate dimerization partner
for MYC-dependent transcriptional regulation (Figure 1A).
We next studied MYC regulation in 9 samples (3 U-CLL and
6 M-CLL) all of which were considered to be sIgM intracellular
Ca2⫹ nonresponders (Figure 1C). There was no increase in MYC
expression in any of the samples at 3 hours, and MYC expression
was increased by greater than 20% in only 1/9 (11%) samples at
6 hours after stimulation of sIgM (Figure 1D). On average, MYC
was not differentially expressed after sIgM stimulation in these
samples (P ⬎ .05). Overall, there was a very strong association
between the ability of sIgM stimulation to increase MYC expression and induce intracellular Ca2⫹ mobilization (P ⫽ .0001, Fisher
exact test; Figure 1E).
Analysis of MYC target gene expression
To determine whether the transcriptional activity of MYC also was
increased after sIgM stimulation, we analyzed the expression of
ODC1, CDK4, and CCND2 RNAs, well-characterized MYC target
genes associated with proliferation.19-23 ODC1 and CCND2 RNAs
were induced within 1 hour (mean induction, 2.04- and 2.54-fold,
respectively), whereas CDK4 RNA was more strongly induced
(mean induction, 2.3-fold) at 6 hours after stimulation with antiIgM (Figure 2). Thus, MYC is active in sIgM-stimulated CLL cells
because its induction is associated with increased MYC target gene
expression.
Activation of MEK1/2 in anti-IgM–stimulated CLL cells
The MEK1/2 3 ERK1/2 signaling pathway is activated after sIgM
stimulation in normal B cells and in signaling responsive CLL
cells,31,32 and is known to play a major role in controlling MYC
expression via both transcriptional and posttranscriptional pathways.33,34 We therefore investigated the activation of ERK1/2 in
CLL cells and its role in induction of MYC expression. Phosphorylation of ERK1/2 at T202/Y204 was quantified using single-cell flow
cytometry in 37 CLL samples. This cohort comprised 16 M-CLL
and 21 U-CLL, of which 31 samples were considered as sIgM
responsive based on intracellular Ca2⫹ responsiveness. The kinetics of ERK1/2 activation are important in determining downstream
responses;35 therefore, we investigated ERK1/2 phosphorylation
for up to 45 or 60 minutes after sIgM stimulation.
ERK1/2 phosphorylation increased after sIgM stimulation in
29/31 (94%) of the intracellular Ca2⫹ responsive samples
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BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
SIGM STIMULATION AND MYC EXPRESSION IN CLL
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Figure 1. Induction of MYC protein after sIgM stimulation in CLL samples. (A,C) CLL samples were incubated
with anti-IgM for 3 or 6 hours or for 6 hours with the
isotype control antibody (IC). Expression of MYC and
MAX protein was analyzed by immunoblotting. Results
shown are representative of those obtained from analysis
of 18 intracellular Ca2⫹-responsive (A) and 9 intracellular
Ca2⫹ nonresponsive (C) CLL samples. MAX expression
demonstrates equal loading of protein samples.
(B,D) Quantitation of the fold increase in MYC protein
expression (relative to isotype-control treated cells;
C) measured by densitometry analysis of immunoblots at
3 or 6 hours after stimulation with anti-IgM for intracellular
Ca2⫹-responsive (B) and -nonresponsive (D) samples.
Graphs show data for individual samples, and any statistically significant differences (Student matched paired
t test) between control and anti-IgM–stimulated cells (NS
indicates not significant, P ⬎ .05). (E) Comparison between MYC induction (⬎ 20% increase compared with
control cells) and positive (䡺) and negative (f) intracellular Ca2⫹ responses (Fisher exact test) in anti-IgM–treated
CLL samples (n ⫽ 27).
(Figure 3A shows representative responsive examples). ERK1/2
phosphorylation was rapidly induced reaching a peak at ⬃ 5 to
15 minutes after stimulation, and there was a positive correla-
Figure 2. Effect of anti-IgM on expression of MYC target genes. CLL samples
(n ⫽ 8) were stimulated with anti-IgM for 1 or 6 hours or with isotype control antibody.
Expression of ODC1, CDK4, and CCND2 RNA were analyzed by quantitative
real-time PCR. Expression values for the isotype control antibody (C) were set to
1.0 for each time point. Graphs show the fold induction for each sample and
statistically significant differences are indicated (Student matched paired t test; NS
indicates not significant, P ⬎ .05).
tion between sIgM-induced signaling responses measured by
increased ERK1/2 phosphorylation and intracellular Ca2⫹ mobilization within this cohort (Figure 3B; P ⫽ .0127; R2 ⫽ 0.16).
However, there were several individual samples where intracellular Ca2⫹ and ERK1/2 phosphorylation responses did not seem
to be closely correlated and ERK1/2 phosphorylation increased
after sIgM stimulation in 5/6 (83%) of the intracellular Ca2⫹
nonresponsive samples.
After the initial peak activation of ERK1/2 phosphorylation,
2 patterns were observed in responsive CLL samples. In most
samples (24/31, 77%), anti-IgM stimulation resulted in a protracted
activation of ERK1/2 phosphorylation, defined here as being
maintained at greater than or equal to 1.2-fold over background at
30 minutes after stimulation. The protracted activation of ERK1/2
phosphorylation was confirmed by immunoblotting in a subset of
samples (supplemental Figure 1A, available on the Blood Web site;
see the Supplemental Materials link at the top of the online article).
A further 5 samples showed a transient induction of ERK1/2
phosphorylation that rapidly returned to baseline (see sample 63 in
Figure 3A).
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KRYSOV et al
BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
Figure 3. Activation of ERK1/2 phosphorylation in
sIgM-stimulated CLL B cells. (A) CLL samples were
stimulated with anti-IgM for up to 60 minutes and ERK1/2
phosphorylation analyzed by flow cytometry. Graphs
show the fold increase in ERK1/2 phosphorylation after
stimulation with anti-IgM relative to untreated cells for
5 representative samples. (B) Correlation between the
maximal percentage of cells showing increased intracellular Ca2⫹ and maximal fold induction of ERK1/2 phosphorylation after sIgM stimulation. Results of linear regression
are shown (n ⫽ 37).
Overall, these data demonstrate that there is a strong
tendency for coactivation of ERK1/2 and intracellular Ca2⫹
responses in CLL samples after sIgM stimulation, and sIgM
stimulation generally results in protracted activation of ERK1/2
phosphorylation.
Induction of MYC protein is partially dependent on activation of
the ERK1/2 pathway
The MEK1/2 kinase mediates ERK1/2 phosphorylation and
activation in response to BCR stimulation.36,37 We used the
MEK1/2 inhibitor U0126 to determine whether activation of
ERK1/2 was directly involved in regulation of MYC expression.
Analysis of ERK1/2 phosphorylation confirmed that MEK1/2
was effectively inhibited in U0126-treated cells. The induction
of MYC protein expression by anti-IgM was significantly
reduced (by ⬃ 50%) in cells pretreated with U0126 (Figure
4A-B). Therefore, MEK1/2 is required for optimal MYC
expression in CLL cells after activation of sIgM, although other
pathways seem to contribute.
MEK1/2 activity is required for optimal MYC expression and
cell-cycle entry in CLL cells treated with CpG-ODN
We examined the effects of U0126 on MYC expression and cellcycle entry in cells treated with CpG-ODN, a well-studied model
for cell-cycle entry in CLL cells.15,16 Our analysis focused on
U-CLL samples, because previous studies have demonstrated that
CpG-ODN predominantly induce a proliferative response in these
cells, whereas CpG-ODN generally promote apoptosis in MCLL.15,38,39 Consistent with this, we found that CpG-ODN stimulation of U-CLL samples for 48 hours resulted in a greater than or
equal to 2-fold increase in the proportion of BrdU-positive cells in
all 6 samples analyzed, although the proportion of cells entering
cell cycle varied considerably between samples (Figure 5A-B).
Treatment with CpG-ODN also slightly reduced levels of spontaneous cell death; the average proportion of dead cells in these
6 samples was 21% in untreated and 15% in CpG-ODN stimulated
cases (data not shown).
Stimulation with CpG-ODN increased ERK1/2 phosphorylation and MYC expression in all samples tested (Figure 5C).
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BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
SIGM STIMULATION AND MYC EXPRESSION IN CLL
175
also was possible to detect an inhibitory effect of U0126 on
S-phase entry (Figure 5B-C).
Effect of sIgD stimulation on ERK1/2 phosphorylation and MYC
expression in CLL cells
Figure 4. Effect of U0126 on MYC induction. Cells were stimulated with anti-IgM for
3 hours in the presence of U0126 (10␮M) or DMSO as a control. (A) Expression of
MYC and phosphorylated and total ERK1/2 was analyzed by immunoblotting. Two
representative samples are shown of a total of 5 samples analyzed. Note that
intervening lanes were removed for clarity, and the complete blot is shown in
supplemental Figure 1D. (B) Quantitation of MYC expression. Graphs show mean
(⫾ SEM) MYC expression relative to untreated cells (set to 1.0), derived from
5 independent experiments. The reduction in MYC expression in cells treated with
anti-IgM and U0126 was statistically significant compared with cells treated with
anti-IgM alone (Student matched pairs t test).
Side-by-side comparison of MYC expression in 4 samples
demonstrated that, on average, the levels of MYC induced by
CpG-ODN were slightly higher than those induced by anti-IgM
(2.1 ⫾ 0.3-fold and 1.6 ⫾ 0.1-fold, respectively [mean ⫾ SD],
Student t test, P ⫽ .027). Pretreatment with U0126 partially
suppressed the induced MYC expression paralleling the effects
of sIgM stimulation (Figure 4B). For CpG-ODN stimulation, it
CLL cells generally express both sIgM and sIgD. In contrast to
sIgM, sIgD is not down-modulated by antigen engagement in vivo,
and the majority of CLL samples retain signaling responses in vitro
to sIgD stimulation.9 We therefore compared the effects of sIgM
and sIgD in individual samples of CLL. We analyzed the effects of
sIgD stimulation on ERK1/2 phosphorylation using the same
31 sIgM-responsive samples, all of which were considered sIgDresponsive as assessed by intracellular Ca2⫹ mobilization. Rapid
increases in ERK1/2 phosphorylation were detected in 29/31 (93%)
of these samples (Figure 6A). However, in contrast to sIgM, sIgD
responses were almost always transient and rapidly returned to
background. Using the cut-off of greater than or equal to 1.2-fold
increase at 30 minutes, only 3/31 (10%) samples showed protracted
ERK1/2 responses (see supplemental Figure 1B for a direct
comparison of sIgM and sIgD responses). The transient responses
to sIgD stimulation were confirmed by immunoblotting in a subset
of samples (supplemental Figure 1A). The difference in the
proportion of transient or protracted responses after stimulation of
sIgM or sIgD was highly significant (Fisher exact test, P ⫽ .0001;
Figure 6B). We also investigated ERK1/2 phosphorylation in
6 samples that were considered sIgM nonresponsive but that
retained responsiveness to anti-IgD. Similar to the retained intracellular Ca2⫹ responsiveness, 5/6 (83%) of these samples also
demonstrated a greater than 1.2-fold increase in ERK1/2 phosphorylation after sIgD stimulation (data not shown). The response was
transient in 4/5 responding samples.
MYC induction was analyzed by immunoblotting in the
18 samples shown in Figure 1B, all of which were considered as
sIgD responsive in terms of intracellular Ca2⫹ responses. In
contrast to sIgM responses, some of these samples did not show
any evidence of increased MYC protein expression after stimulation of sIgD at any time point, and in those cases that did, the
increase was clearly lower than after sIgM stimulation (Figure 6C
and supplemental Figure 1C). Overall, the induction of MYC
protein at both 3 and 6 hours was significantly lower following
stimulation of sIgD compared with sIgM (Student t test,
Figure 5. Effect of U0126 on CpG-ODN–treated CLL cells.
CLL cells (n ⫽ 6) were pretreated with 10␮M U0126 or DMSO
for 15 minutes, before stimulation with CpG-ODN (7␮g/mL) for
3 or 48 hours. (A) Representative analysis of BrdU and 7AAD
staining in untreated (top) and CpG-ODN (bottom)–treated CLL
cells (48 hours). S-phase and dead cells are gated in the bottom
panel. (B) Quantitation of S-phase (48 hours, in the presence or
absence of CpG-ODN ⫾ U0126. (C) Immunoblot analysis of
MYC, phosphorylated ERK1/2, and ␤-actin expression at
3 hours.
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KRYSOV et al
BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
Figure 6. Activation of ERK1/2 phosphorylation and
MYC expression in sIgD-stimulated CLL and normal B
cells. (A) CLL samples were stimulated with anti-IgD for up
to 60 minutes and analyzed for ERK1/2 phosphorylation by
flow cytometry. Graphs show the fold increase in ERK1/2
phosphorylation after stimulation with anti-IgD relative to
untreated cells for 4 representative samples. Note that the
same samples are shown in Figure 3A (after sIgM stimulation), and direct comparison of sIgM and sIgD responses is
shown in supplemental Figure 1B. (B) Comparison between
protracted/transient ERK1/2 responses after stimulation of
sIgM (䡺) or sIgD (f; Fisher exact test, P ⫽ .0001; n ⫽ 37).
(C) Quantitation of MYC protein induction after stimulation
of sIgM or sIgD, relative to isotype antibody-treated controls
(n ⫽ 18). Note values for anti-IgM–treated cells are the
same as those shown in Figure 1B and are shown again
here to allow direct comparison with anti-IgD–treated cells.
See supplemental Figure 1C for side-by-side immunoblot
analysis of MYC expression in sIgM- or sIgD-stimulated
CLL cells. (D) Normal B cells were stimulated with anti-IgM
(䡺) or anti-IgD (f) for up to 60 minutes and ERK1/2
phosphorylation analyzed by flow cytometry. Graph shows
mean fold increase in ERK1/2 phosphorylation for
CD20⫹CD27⫺ cells. Data are mean values ⫾ SEM (n ⫽ 8).
(E) CD19⫹CD27⫺ B cells isolated from healthy donors were
stimulated with anti-IgM or anti-IgD for 3 or 6 hours or for
6 hours with the isotype control. MYC and MAX expression
was analyzed by immunoblotting. Results are shown for
cells isolated from 2 separate donors.
P ⫽ .002 and P ⫽ .003, respectively; Figure 6C). Thus, in contrast
to sIgM, sIgD engagement generally triggers a transient activation
of ERK1/2 that is associated with a relatively weak induction of
MYC protein expression.
ERK1/2 phosphorylation and MYC expression in normal B cells
Because responses to sIgM and sIgD stimulation differed in CLL,
we also analyzed responses in normal B cells to determine whether
these differences are CLL specific. We focused on naive (CD27⫺)
B cells as a comparator because these cells, unlike CD27⫹ cells,
coexpress sIgM and sIgD at relatively high homogeneous levels
and are thought to represent the normal counterpart of U-CLL.40 In
contrast to CLL cells, stimulation of either sIgM or sIgD on normal
CD27⫺ B cells induced protracted ERK1/2 phosphorylation responses and equivalent induction of MYC protein (Figure 6D-E).
Thus, reduced induction of MYC in sIgD stimulation CLL cells
seems to be a specific feature of these cells.
Expression of MYC and phosphorylated ERK1/2 in vivo
To confirm the relevance of our findings, we analyzed MYC
expression in vivo by immunohistochemistry in lymph node
biopsies from 8 patients with CLL/SLL. MYC expression was
detected within the malignant cells in 7 (88%) of these samples
(Figure 7 illustrates 2 representative samples). MYC-positive
cells were mostly confined to PCs that were visible as areas
comprising larger, CD5⫹, CD23⫹ CLL cells (Figure 7A,C).
Typically 20% to 50% of cells in the PCs expressed MYC.
Expression patterns were similar to that of the proliferation
marker Ki67 (data not shown). Phosphorylated ERK1/2 also was
detected in CLL cells in PCs (Figure 7). Because PCs are
considered the likely site of antigen engagement, these data are
consistent with the idea that induction of MYC downstream of sIgM and ERK1/2 is a key proliferation-promoting
pathway in CLL.
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BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
SIGM STIMULATION AND MYC EXPRESSION IN CLL
177
Figure 7. Expression of phosphorylated ERK1/2 and
MYC in vivo. Immunohistochemical analysis of MYC
(A,C) and phospho-ERK1/2 (B,D) in 2 CLL/SLL lymph
node biopsy samples. The images show expression
within representative PCs comprising larger, less densely
stained cells. (E-F) MYC expression in Burkitt lymphoma
and phospho-ERK1/2 expression in colon cancer, respectively. Original magnification ⫻600.
Discussion
Recent in vivo labeling studies and analysis of telomeres have
challenged the long-held view that CLL is predominantly a disease
of failed apoptosis and demonstrated a key role for cell proliferation in driving disease progression.3-6 It is critical therefore to
identify the pathways that control cell-cycle entry in CLL because
this will provide novel opportunities for therapeutic targeting. The
key observation in this work is that activation of sIgM signaling
pathways leads to induction of the MYC proto-oncoprotein, an
essential positive regulator of cell-cycle entry.
Several recent studies have investigated MYC expression in
CLL. MYC was identified as a sIgM-regulated gene, as part of a
global gene expression array analysis reported by Gribben and
colleagues.26 We have confirmed this and have now shown that
MYC also is induced at the protein level. Similar to intracellular
Ca2⫹, it is likely that MYC induction in vitro may occur only in a
subset of the malignant clone. However, the absence of highquality antibodies has prevented us from investigating this directly.
We also have shown that MYC is transcriptionally active because
its induction is associated with increased expression of downstream
target genes ODC1, CDK4, and CCDN2, required for cell-cycle
entry. Consistent with our findings, a previous immunoblotting
study revealed increased expression of MYC in lymph nodes
tissues, compared with circulating CLL cells.25 Importantly, we
studied MYC expression in situ and demonstrated activation of
MYC (as well as ERK1/2 phosphorylation) specifically within the
PCs of CLL/SLL lymph nodes, sites of malignant cell proliferation
and probably antigen engagement and sIgM stimulation.
Overall, the ability of sIgM stimulation to increase MYC
expression was associated with intracellular Ca2⫹ mobilization,
another readout of signaling capacity that is linked to poor
prognostic markers.9 Thus, MYC is one part of a program of
downstream events linked to sIgM stimulation and one that is likely
to play a critical role in cell-cycle entry. However, there was
variation between intracellular Ca2⫹ responses and ERK1/2 phosphorylation responses in some individual samples. Heterogeneity
within sIgM signaling responses has been described previously,31
and it will be important to further investigate the functional and
clinical significance of these variable signaling responses. Because
MYC plays a direct role in proliferation, its induction may provide
a useful marker of functionally relevant responses to antigen
engagement, and hence of clinical behavior.
Our results demonstrate that MEK1/2 3 ERK1/2 signaling
plays an important role in MYC induction, in both anti-IgM and
CpG-ODN–treated cells. This is consistent with the finding that the
MYC regulating transcription factor Elk1 is phosphorylated and
activated downstream of ERK1/2 after pre-B cell receptor activation, where transcriptional induction of MYC downstream of
ERK1/2 has been shown to be critical for the expansion of early
B cells.41 However, effects of U0126 were partial, consistent with
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178
BLOOD, 5 JANUARY 2012 䡠 VOLUME 119, NUMBER 1
KRYSOV et al
the idea that additional pathways are involved in MYC induction, acting
either in parallel or in series. MYC induction after sIgM stimulation of
normal mature mouse B cells in the presence of lipopolysaccharide is
partly dependent on NFKB1 and REL,42 and treatment of CLL cells with
BAFF also has been shown to increase MYC expression via canonical
NF-␬B signaling.24 Because sIgM stimulation induces NF-␬B activation, NF-␬B also may contribute to optimal MYC expression.43,44
Recent studies also have demonstrated that inhibition of PI3K␦ by
CAL-101 interferes with sIgM-induced ERK1/2 activation,45 suggesting that proliferation promoting effects of PI3K activation15 may be at
least partly mediated via ERK1/2 3 MYC signaling. Future studies will
focus on uncovering the molecular circuitry linking these signaling molecules, their downstream pathways and MYC expression
in CLL cells.
Although anti-IgM stimulation enhances MYC expression, this
does not seem to be sufficient to promote efficient CLL cell S-phase
entry or division. For this reason, we focused on a cell-cycle model
whereby CLL cells are treated with CpG-containing ODN to
activate TLR9. This model has been used previously to investigate
pathways of cell-cycle control and responses are linked to important clinical parameters, including time to treatment and overall
survival.16,17,38 Using this approach, we confirmed that MEK1/2
activity was required for optimal MYC induction and S-phase
entry. The modestly higher levels of MYC induced by CpG-ODN
compared with anti-IgM may contribute to effective cell-cycle
entry in CpG-ODN– but not anti-IgM–stimulated cells. However, it
is also likely that other pathways contribute to determine proliferative responses. In CLL cells, anti-IgM has been shown to increase
expression of cyclin D2 and cdk4 but not to substantially decrease
the negative cell-cycle regulator p27kip1.14 Additional signals from
supporting immunocytes or stromal cells are presumably required
to down-modulate p27kip1 and other negative regulators, supporting
efficient sIgM-induced cell-cycle entry in vivo.14
Interestingly, consequences of stimulation of sIgD and sIgM
were distinct in CLL cells. Whereas sIgM stimulation generally led
to protracted ERK1/2 phosphorylation and MYC induction in
responsive samples, sIgD stimulation generally triggered transient
ERK1/2 phosphorylation with relatively modest effects on MYC.
These observations suggest it is the kinetics of ERK1/2 activation
that is critical for regulation of MYC and are consistent with
findings from other systems where protracted ERK1/2 activation is
required for effective MYC induction and cell-cycle entry. Transient ERK1/2 activation leads to transcriptional activation of the
MYC gene but does not effectively increase MYC protein expression because it fails to prevent MYC proteolysis,33,34 whereas
protracted ERK1/2 activation lead to increased MYC gene transcription and stabilization of MYC via phosphorylation.35,46 Interestingly, similar differences in the kinetics of signaling responses have
been observed for intracellular Ca2⫹ fluxes, at least in CD38
positive CLL cells, where sIgM and sIgD stimulation induced
protracted and transient intracellular Ca2⫹ fluxes, respectively.13
These observations also may explain why retained sIgM responsiveness but not sIgD correlates with prognostic markers and outcome
in CLL; although signal competent, sIgD stimulation does not
effectively engage downstream pathways driving cell-cycle progression. Although sIgM responses were similar between responsive
CLL samples and normal CD27⫺ B cells, sIgD responses were
clearly much weaker in CLL cells compared with normal CD27⫺
B cells, indicating that this is a specific feature of CLL cells.
In summary, our results demonstrate that sIgM activation leads
to ERK1/2-dependent induction of MYC expression in CLL cells.
MYC induction seems to be dependent on protracted ERK1/2
activation because MYC was not induced in cells treated with
anti-IgD, which induced transient ERK1/2 phosphorylation responses. Pharmacologic inhibition of signaling pathways activated
by sIgM and leading to induction of MYC, including MEK1/2, may
be an attractive therapeutic strategy, especially in progressive
disease.
Acknowledgments
The authors are very grateful to Drs Andrew Duncombe, Vlad
Malykh, Helen McCarthy, Abe Jacob, Ben Kennedy, and Henri
Grech and to Richard Palmer and colleagues for providing CLL
samples and associated data and to the patients who donated
clinical samples. They are also very grateful for the help of Isla
Henderson for characterization of CLL samples, the support of
Professor Christian Ottensmeier, and the helpful comments of Dr
Andrew Steele.
This work was supported by the Kay Kendall Leukaemia Fund,
Cancer Research UK, the Southampton Experimental Cancer
Medicine Center, and Tenovus Solentside.
Authorship
Contribution: S.K., S.D., A.P., C.I.M., K.N.P., and K.-A.S. performed the research and analyzed data; S.K., K.N.P., M.A.-K.,
F.K.S., and G.P. designed the research and analyzed data; S.K. and
G.P. wrote the initial draft of the manuscript, and all authors
contributed to the modification of the draft and approved the final
submission.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Sergey Krysov, Cancer Research UK
Centre, Somers Cancer Research Building (MP824), Cancer Sciences Unit, University of Southampton School of Medicine,
Southampton General Hospital, Tremona Road, Southampton
SO16 6YD, United Kingdom; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2012 119: 170-179
doi:10.1182/blood-2011-07-370403 originally published
online November 15, 2011
Surface IgM stimulation induces MEK1/2-dependent MYC expression in
chronic lymphocytic leukemia cells
Sergey Krysov, Samantha Dias, Alex Paterson, C. Ian Mockridge, Kathleen N. Potter, Kelly-Ann
Smith, Margaret Ashton-Key, Freda K. Stevenson and Graham Packham
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