Association of Reactive Oxygen Species-Mediated Signal
Transduction with In Vitro Apoptosis Sensitivity in
Chronic Lymphocytic Leukemia B Cells
Adam L. Palazzo1., Erik Evensen1., Ying-Wen Huang1, Alessandra Cesano1, Garry P. Nolan2, Wendy J.
1Nodality Inc., South San Francisco, California, United States of America, 2Baxter Laboratory for Stem Cell Biology and Department of Microbiology and Immunology,
Stanford University, Palo Alto, California, United States of America
Background: Chronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable
response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in
signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify
proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately
linking these with clinical outcome.
Methodology/Principal Finding: SCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins
and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen
peroxide (H2O2), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL
samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR
network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B
cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic
proficiency was highly associated with the proficiency of CLL B cells to undergo H2O2-augmented signaling.
Conclusions/Significance: This linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis
increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients
for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also
the utility of this approach in relating mechanism to function of therapeutic agents.
Citation: Palazzo AL, Evensen E, Huang Y-W, Cesano A, Nolan GP, et al. (2011) Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro
Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells. PLoS ONE 6(10): e24592. doi:10.1371/journal.pone.0024592
Editor: Christopher Bunce, The University of Birmingham, United Kingdom
Received March 22, 2011; Accepted August 15, 2011; Published October 10, 2011
Copyright: ? 2011 Palazzo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by research funding from Nodality, Inc. to WJF. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: GPN is a consultant, Chairman of the SAB, and holder of
equity in Nodality, Inc. WJF, ALP, EE, Y-WH and AC are holders of equity in Nodality, Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on
sharing data and materials.
* E-mail: email@example.com
. These authors contributed equally to this work.
CLL is the most common adult leukemia in the Western world
and is characterized by aberrant accumulation of CD5+ B
lymphocytes in the peripheral blood, bone marrow and secondary
lymphoid organs. Clinical presentation, natural course of the
disease and response to treatment are all extremely variable, with
patient survival after diagnosis ranging from months to decades.
Although the biological mechanisms accounting for the unpre-
dictability of the disease are unknown, several biological indicators
including cytogenetics, presence or absence of somatic mutations
within the immunoglobulin heavy chain variable region (IgVH),
ZAP70 and CD38 expression have all been associated with
response to therapy and prognosis [1–3].
A greater understanding of CLL biology is needed to chart
disease progression as well as assist in selecting optimal therapeutic
strategies. Ideally, monitoring on an individual patient basis would
take into account differing inter-patient cell biology as well as shifts
in the intra-patient population biology of mutant cells within a
heterogeneous tumor cell population. SCNP studies in myeloid
leukemias and follicular lymphoma distinguished healthy from
diseased cells by their response to growth factors and cytokines [4–
10]. In these studies induced protein phosphorylation was shown
to be more informative than the frequently measured basal
phosphorylation state of a protein revealing signaling deregulation
consequent to the numerous cytogenetic, epigenetic and molecular
changes characteristic of transformed cells. Furthermore, SCNP
simultaneously measures multiple signaling proteins and assigns
their activation states to specific cell sub-sets within complex
primary cell populations .
Central to B cell development, and also believed to be
important in CLL progression, is the BCR signal complex
PLoS ONE | www.plosone.org1October 2011 | Volume 6 | Issue 10 | e24592
composed of membrane-bound immunoglobulin and the signal
transducing CD79a/CD79b heterodimer. In normal B cells,
antigen mediated BCR activation regulates cell survival, differ-
entiation, proliferation and migration [12,13]. Additional regu-
lation of BCR signaling involves phosphatase(s) whose activity is
regulated by NADPH-oxidase-generated reactive oxygen species
H2O2[14–16]. Furthermore, studies from the groups of Munroe
and Rajewsky have recognized that in conjunction with antigen-
driven responses, ligand-independent signaling (tonic signaling)
by both the pre-B cell receptor and BCR has an important role in
survival throughout B cell development [17–20]. Although the
molecular mechanisms governing tonic BCR signaling are not
well defined, recent studies suggest that tyrosine phosphatase
regulation by reactive oxygen species play a likely role
[14,16,17,21–23]. Furthermore, recent evidence has described
deregulated tonic BCR signaling in diffuse large B cell lymphoma
and CLL [24–28].
In CLL, associations have been observed between the clinical
course of the disease and functional alterations in the BCR and its
regulators, suggesting that both antigen-driven and tonic BCR
signaling play an important role in its pathogenesis. This is
corroborated by in vitro studies in which significant differences in
both ligand-mediated and ligand-independent BCR signaling were
found in primary CLL patient samples [25,29].
In a healthy physiological setting, apoptosis proceeds from
sensors that monitor cell stress and damage, to effectors that relay
the signals to activate programmed cell death pathways. Apoptosis
is also regulated by cell survival signals, and in B cells one such set
of signals emanates from the BCR via tonic signaling, as noted
above. The accumulation of malignant monoclonal B cells in CLL
has largely been attributed to defects in apoptosis cascades rather
than to aberrant proliferation . In some CLL patients
inactivation of cell death pathway proteins such as p53 (17p
deletion) is an example of how this mechanism can over-ride
benefit from a therapeutic agent . No relationship between
BCR signaling, cell survival and resistance of patient cells to
chemotherapy has yet been shown using existing analytical
methods. SCNP technology now provides an opportunity to re-
examine the global alterations in signaling that inevitably occur in
B cells in response to the genetic and molecular changes they have
Materials and Methods
All patients consented, in accordance with the Declaration of
Helsinki, for the collection and use of their samples for institutional
review board-approved research purposes.
Cryopreserved, Ficoll-purified (Sigma Aldrich)  peripheral
blood mononuclear cells (PBMCs) from 23 CLL and 7 healthy
subjects were used in this study (Table 1). The majority of samples
were collected from patients previously treated. CLL diagnosis was
based on the Workshop on Chronic Lymphocytic Leukemia
Table 1. Clinical and molecular characteristics of patient samples.
Sample ID GenderAge at DX IgVH% ZAP 70FISHFirst Treatment (Rx) Type
CLL001M4493 0.4Not availableHDMP+Rituxan
CLL003F5392.60.6Not availableFR (2cycles) - good response
CLL004M5310092.511qdel(16%)HDMP Rituxan Frontline (3 cycles)
CLL005M6598.643.6tri12 (90%) Leukeran (4 mg/day)
CLL006F 6199.645.2 normal FISHF; then FR (4 cyc) - MRD on BM
CLL007M6699.379.1tri 12 (84%)Leukeran
CLL008M 7295.8313q del (73%)Leukeran
CLL009M81 95.29.813q del (100%)Chloroambucil
CLL010M 48 895Not available Fludarabine (3 cycles)
CLL011M 5793.3 1.1 tri 12(75%) ASCENTA-002
CLL012F 7296.8 1.2Not availableno Rx
CLL013M5999.662.8 tri 12(89%)Chlorambucil
CLL014M 54 90.50.5 tri 12 (karyotype)High dose chemoRx w/BM transplant
CLL015F 51 10091.9tri 12 (82%) ASCENTA-002
CLL016F53 930.1Normal FISHHDMP+R 04 Frontline
CLL017F3996.110.1 Normal Karyotype no Rx
CLL018M 55 100 91.1 13qdel(70%)HDMP+R 04 (3 cycles) Frontline
CLL019M 5010045.9Normal FISHRituximab (3 cycles) - 4 weeks
CLL020M4391.9 1.1 13 del (99%)Chlorambucil
CLL021M66 10065.3 13 del (99%) Solumedrol Rituxan
CLL022M 4810073.1 tri 12 (80%)no Rx
CLL023M 7294.7 1.3 13 del (17%)GMCSF and Rituxan
CLL024F49 10051.8 Not availableno Rx
IgVHmutational status and percent ZAP70-positive cells determined as previously described .
B Cell Signaling and In Vitro Apoptosis in CLL
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Samples were thawed at 37uC and suspended in RPMI 1640
1% FCS. Viability was measured for an aliquot immediately post
thaw with trypan blue. In this sample set viability was greater than
90% for all samples. Amine Aqua (Invitrogen) was used to
determine cell viability according to the manufacturer’s protocol.
Briefly, Amine Aqua was added to all samples before the 2 hour
rest and was present throughout the duration of the experimen-
tation. Cells were arrayed in duplicate in 96-deep-well plates at
6.06105cells or 8.06105cells per well for measurements of BCR
signaling and apoptosis respectively. More cells were used for
apoptosis assays to get a more reliable measurement over the
48 hours of exposure to F-ara-A. All measurements for signaling
and apoptosis were performed in duplicate for each sample and
assay performance characteristics noted (manuscript in prepara-
Ramos cell line control
The human Burkitt lymphoma Ramos cell line, a control for
BCR signaling, acquired from ATCC was cultured according to
the manufacturer’s protocol.
Treatment with extracellular modulators of BCR and
After a 2 hour rest at 37uC, each sample was treated in bulk for
10 minutes at 37uC with goat polyclonal (F(ab’)2human anti2m
(anti2m) or anti2c (Southern Biotech), final concentration 10 mg/
ml or with H2O2, final concentration 3.3 mM. For the
combination of anti2m and H2O2, anti2m was added first
followed by H2O2. Phorbol Myristate Acetate ((PMA) Sigma),
final concentration 400 nM, was used as a control to show that
cells were capable of signaling, in this case downstream of Protein
Kinase C and in a BCR-independent manner. For apoptosis
assays, samples were exposed either to 9-b-D-arabinosyl–2-
fluoroadenine (F-ara-A, Sigma-Aldrich), the free nucleoside of
fludarabine, final concentration 1mM, staurosporine (Sigma-
Aldrich), final concentration 5 mM for 48 and 6 hours respectively
or to vehicle (0.05% DMSO) for matching times at 37uC [34,35].
Cells were fixed with paraformaldehyde and permeabilized with
100% ice-cold methanol as previously described [5,16,36].
Flow cytometry determinations of BCR and apoptotic
Cells were incubated with panels of fluorochrome-conjugated
antibodies against a core set of B cell phenotypic markers combined
with antibodies recognizing intracellular signaling or apoptosis
molecules (Table S1). All antibody concentrations were optimized
to maximize signal to noise ratio and to minimize non-specific
binding. Eleven point titrations (two fold dilutions) were performed
with cryopreserved PBMCs from two healthy donors or with the
Ramos cell line. For surface antibodies, titrated in PBMCs, the
optimal concentration selected is one where the median fluores-
cence intensity (MFI) of the signal to noise ratio is maximal between
a cell immunophenotype that expresses an epitope versus one that
does not. For the antibodies against phospho-specific epitopes,
antibody titrations were performed to determine the concentration
that gave the maximal signal to noise ratio for fold increase of
stimulated over unstimulated signal (median fluorescence intensity
(MFI) of stimulated/ MFI unstimulated sample) in cell lines and
PBMCs (data not shown). Further determinations of phospho-
antibody specificity were determined by pre-blocking the antibody
withthe phospho-peptide epitope against which the antibodies were
generated (data not shown).
Samples processed for cytometry [16,36] were acquired on a
BD FACS Canto II flow cytometer equipped with a high
throughput sampler (HTS).
Expression of surface markers on unfixed cells
Cells were incubated with immunophenotypic cocktails (Table
S1) before FACS analysis.
Cells gated on light scatter characteristics were evaluated for
viability by exclusion of Amine Aqua. Live cells were gated as
CD3-/CD20+ to allow for direct comparison of B cells from CLL
and healthy samples. The gating scheme is shown in Figure S1.
Metrics included median fluorescence intensity (MFI), percentage
of positive cells, and mixture-model derived population content
and were extracted from CD3-/CD20+ cells. FCS files were
analyzed in FlowJo (Treestar, Ashland, OR) version 8.8.2. To
display and compare intensity values including negative numbers
and correct for large variance with some fluorophores we used the
inverse hyperbolic sine (arcsinh) with a cofactor instead of the
traditional log10scale as shown below.
The arcsinh transformation behaves linearly for small values
and log-like for larger values. This transformation for fluorescent
intensities was computed using the expression:
where x is the fluorescent intensity and a is a factor that
determines the linear region of the transform. The addition of
positive value even if x is negative.
The criteria used to assign apoptotic proficiency to a sample
were a two-fold or greater increase in the number of CD322/
CD20+ cells at 48 hours that were positive for cleaved PARP (c-
PARP+) and cleaved Caspase 3 (C-Caspase 3) after exposure to F-
ara-A compared to vehicle or to staurosporine compared to
vehicle. The criteria to assign apoptotic refractoriness to F-ara-A
were a less than two fold change in the number of of CD32/
CD20+ cells at hours that were positive for c-PARP+and c-
term guarantees that the log function will have a
Surface marker characterization and basal
phosphorylation states in healthy and CLL B cells
Comparison of MFI values of BCR signaling molecules in their
basal phosphorylation states showed greater variability in CLL
versus healthy B cells. MFI values for p-Akt and p-Lyn spanned a
range of16and17respectivelyamonghealthyB cellsand 63and 66
inCLL B cells(Figure 1 andTable 2).p-Erkandp-65/RelAshowed
no significant differences between healthy and CLL samples,
indicating that at their basal level the activation state of these
molecules did not reflect a CLL-dependent phenotype. Although
not statistically significant, some samples did have high basal levels
of p-65/RelA consistent with previous reports documenting a wide
degree of variability for the phosphorylation status of this
transcription factor  . Similarly, expression (determined
by MFI) of B cell lineage markers (CD5, CD19 and CD20) and
tyrosine phosphatases (CD45, SHP-1 and SHP-2) also showed
greater variability in CLL B cells (Table S2). The expected 2:1
B Cell Signaling and In Vitro Apoptosis in CLL
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kappa/lambda ratio was evident in healthy B cells and contrasted
with the exclusive expression of either kappa or lambda chain in
CLL B cells (data not shown). In 4 samples no light chain, IgM or
IgG were detected. Although technical limitations cannot be
excluded, the presence of CLL B cell markers such as CD5,
CD19 and CD20 suggest these cells to be a clonal B cell population
witha CLLphenotype.Expected hallmarks ofCLL wereseen inthe
low expression of IgM and CD79bin individual patient samples
(Table S2). For CD38 expression most samples were negative but in
seven samples, there was a bimodal profile for CD38 expression
(data not shown and . No significant separation of CLL samples
into distinct subgroups could be made based on expression levels of
the measured surface markers or the tyrosine phosphatases.
Modulated signaling responses distinguish subgroups of
CLL patient samples
To test whether differences in CLL physiology could be
discerned based on intracellular signaling responses, cells were
treated with extracellular modulators. The modulators chosen
were anti2m to cross link and activate the BCR and H2O2,a mild
oxidant produced naturally by healthy B cells to control the
strength of antigen receptor signaling by reversible inhibition of
tyrosine phosphatases [5,16]  The 10-minute time point for
modulator treatment was chosen based on kinetic analyses (data
not shown) and produced robust, but not necessarily maximal
phosphorylation, of all the BCR pathway signaling molecules
under study. The H2O2concentration chosen was one in which
minimal effects were seen on intracellular signaling molecules in
healthy B cells (Figure S2) and was consistent with H2O2
concentrations used in other studies [5,7,16,40].
Consistent with previous reports, anti2m-mediated BCR
signaling was observed and further potentiated by H2O2in B
cells from healthy donors (Figure S2) .
Analysis of the signaling responses showed that the CLL sample
cohort was minimally responsive to anti–m treatment but could be
broadly segregated into two patient groups based on their
responsiveness to H2O2. In Group l a significant subpopulation of
cells was responsive to H2O2 such that there was an anti–m-
independent increase in phosphorylation of signaling molecules
downstream of the BCR (the mean percentage of a cell subset with
activated p-Lyn, p-Syk, p-BLNK or p-PLCc2 population was 51%,
52% and 45% and 68% respectively, (Table 3 and Figure 2(A)).
Signaling was coordinated in that all these components of the
proximal B cell receptor network were augmented in concert. In all
Figure 1. Basal phosphorylation levels of signaling molecules downstream of the BCR. Box and whisker plots comparing magnitude and
range of basal signaling between CLL (blue) and healthy donors (green). Notch and red horizontal line indicates median signaling for parameter, box
drawn from lower to upper quartiles of data, and whiskers extend to 1.5 times the interquartile range. p-values from Student’s t-test comparing
Arcsinh transformed MFI values from CLL and healthy B cells.
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but three cases, the addition of anti–m did not mediate a further
increase in measured signaling responses, consistent with the notion
that aberrant phosphatase activity might be regulating signaling
downstream of the BCR in CLL.
In Group ll a greatly reduced subpopulation of cells had a
signaling response after exposure to H2O2compared to Group l
samples. For example, the mean percentage of cells in a
subpopulation with activated p-Lyn, p-Syk, p-BLNK or p-PLCc2
was 14%, 17%, 13% and 33% respectively (Table 3 and
Interestingly, the H2O2-mediated p-Akt response was similar
between the two groups (a mean cell subpopulation of 58% for
Group l and 52% for Group ll, Table 3), suggesting that an
alternative phosphatase such as the H2O2–sensitive PTEN  is
not differentially regulated in CLL and healthy B cells. The mean
number of cells with activation of Erk in Group ll was less than in
Group I (44% and 71% respectively). In healthy B cells, all
signaling molecules except Akt were minimally responsive to H2O2
treatment alone (Table 3). Given that H2O2is a known inhibitor
of phosphatase activity, and that phosphatase activation is a
physiological regulator of proximal BCR signaling activities [5,14–
17,21–23], these data suggest that deregulation of phosphatase
activity could explain some of the differences observed between
CLL and healthy B cell signaling responses.
Unexpectedly, in 9/23 CLL samples an increase in p-Stat5 was
observed in response to H2O2within a subset of cells in individual
samples inGroup 1 (Table3,Figure 2(C)(lefthand panels)and Figure
S2(A)). In 11/23 CLL samples a minimal number of cells exhibited a
H2O2-mediated increase in phosphorylated Stat5 and this was also
true forhealthyB cells(Table3,Figure 2(C)(right hand panels,Figure
S3(B) and (C)). This observation suggests either that there is a
significant re-wiring event downstream of ligand-independent BCR
signaling or that an alternative pathway is activated in response to
H2O2, and either could be connected to Stat5 activity.
Interestingly, in many patient samples at least two prominent
CLL cell populations with unique and definable signaling
responses were observed. For example, a sample in which a
dominant cell subset demonstrated augmented signaling in
response to H2O2, other subsets could be identified with marginal
responses (Figure 2(A and C)). No such distinctions were observed
using basal phosphorylation states, underscoring that activation of
BCR signaling molecules highlights the differences in pathway
biology between and within samples.
Lack of responsiveness of the Lyn/Syk/BLNK/PLCc2
signaling proteins to H2O2treatment associated with lack
of apoptotic response to F-ara-A in CLL B cells
There has long been a presumed link between ligand-dependent
and independent BCR signaling with B cell survival [17–20]. If
such links are critical, then it might be further postulated that in
CLL and other B cell malignancies, associations may exist between
signaling potential downstream of the BCR and apoptotic
competence. To test this, apoptotic responses of CLL samples
and healthy donors were enumerated by SCNP after in vitro
exposure to F-ara-A for 48 hours. Representative CLL samples
responsive or refractory (Criteria described in Materials and
Methods) to in vitro F-ara-A exposure show simultaneous
measurement of cleaved caspase 3 and cleaved PARP in each
cell (Figure 3 and Figure S4(B) (C)). Measurements of loss of
mitochondrial cytochrome C in the same cells are consistent with
these apoptotic responses (data not shown).
Within apoptotically responsive samples there were at least two
cell subpopulations, with a second cell subset that was refractory
to in vitro F-ara-A exposure (Figure 3 (Left hand panels). This is
reminiscent of the signaling data described above in which cell
subsets with heterogeneous signal transduction responses were
seen within the same sample (Figure 2(A)). At 48 hours 5
leukemic samples (in the absence of F-ara-A) had high
background values for cleaved PARP and cytochrome C and
were therefore excluded from the analysis (Figure S3). DNA
damage was assessed using an antibody against the phospho-
threonine 68 epitope on Chk2, the ATM phosphorylation site
. Although differences in p-Chk2 levels were seen in cell
subsets within F-ara-A responsive and refractory samples, these
differences were not statistically significant (data not shown).
Furthermore, staurosporine a global kinase inhibitor, and
mechanistically distinct from F-ara-A, mediated apoptosis in all
except 3 samples (Figure S5(A) and S5(B)).
Given the pro-survival role BCR signaling molecules play in
healthy and tumorigenic B cell biology [5,12,16,43], the data
were analyzed for any associations between H2O2-modulated
signaling and apoptotic response to in vitro F-ara-A exposure.
To evaluate the CLL cohort for trends, all cell events from
gated B cells of all CLL samples and, separately, all healthy
samples were combined into respective ‘virtual’ samples that
represented a composite of the fluorescence intensities for each
modulated signaling molecule. The histograms show a greater
spread in the fluorescence intensities in CLL versus healthy B
cells treated with H2O2 for each intracellular signaling
molecule (Figure 4(A) CLL B cells (cyan) versus healthy B cells
(pink)). Combining H2O2 with anti2m did not produce
additional substantial changes compared to H2O2 alone in
the B cell population distribution of CLL B cells, suggesting
that H2O2was defining the signaling potential of these CLL B
cell populations. This contrasted with healthy B cells in which
the combination of H2O2and anti2m, but not H2O2alone
resulted in an enhanced population distribution based on
signaling (Figure 4(A), fourth column ).
Table 2. Range of basal phosphorylation levels (MFI) of BCR signaling molecules in CLL and healthy B cells.
p-Lynp-Syk p-PLCc2 p-BLNK p-Erkp-65/RelAp-Aktp-S6p-Stat 5
CLLRange 66 6257 5094 68 631155 43
Mean 5279 633515356105 579 51
Stdev1416 151128 1716 28711
HealthyRange 17 16 1721 41 1916 1986
Mean 66 5551 5315757 9437735
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Defining cell populations by mixture models in CLL B
Ontheassumptionthat atleasttwosubpopulationsofcellscould be
driving the distribution of expression in the combined samples, the
underlying "subpopulations" were decomposed via mixture modeling
for the CLL samples to represent the underlying probability
distributions (Figure 4(B)). The trends in the mixture models
emphasize the patterns (as expected) of the individual patient samples:
the presence of an H2O2de-repressed cell subpopulation and a cell
subset non-responsive to H2O2. The mixture model has the benefit of
showing,atleastforthiscohortof patients,the averaged boundariesof
where such subpopulations of cells lay on the histograms. The relative
numbers of cells in each population defined by these curves were used
as metrics that may be linked to the presence or absence of these
observed cell subsets to apoptosis response.
Receiver operating characteristic (ROC) curves (Figure 5(A))
were generated to demonstrate whether presence of either or both
of the populations defined by the mixture models (Figure 4(B)) was
associated with apoptotic response to in vitro F-ara-A exposure. No
such associations were observed for healthy B cells, as expected,
since the H2O2concentration was selected to give no response in
healthy B cells as previously reported .
Table 3. BCR and apoptosis responses in CLL and healthy B cells.
Sample GroupCLL Sample p-Lynp-Syk p-BLNK p-Stat 5p-PLC–c
Group I CLL02480 657347 77 4688
Group 1CLL003 73 7464 60 81 7486
Group 1CLL008 57 57 4736776784
Group 1CLL01056543930 858090
Group 1CLL009 476354 2979 5669
Group 1CLL01443 525236 675957
Group 1CLL0014041 2034514352
Group 1 CLL002393936 21564070
Group 1 CLL016 27 26 1723 39 5641
Group 1Mean5152453568 5871
Group 2CLL01924 29 19 124066 53
Group 2CLL0042023 1913453176
Group 2CLL018 20 211815 3345 61
Group 2 CLL01319 25 19 1337 26 27
Group 2 CLL0121732237 4861 34
Group 2 CLL0051518109 357441
Group 2 CLL02010557 33 40 44
Group 2CLL023 10 1614 10 4387 83
Group 2 CLL0078704 19 6640
Group 2 CLL02168967810
Group 2Mean1417 139 3352 44
Group 2 SD69741123 22
Healthy CON19546 10 3.4 185121
+ + (42)
HealthyCON1963560.16 12 48 15
Healthy CON21913.5 30.6254426
HealthyCON240113 0.64.538 10
HealthyCON202 0.635 0.65 45 17
HealthyCON193 0.51.22 0.34 336
Healthy SD1.3 1.7 2.5 1.0 7.7 7.1 7.7
Percentage of B cells from CLL and healthy samples that undergo H2O2-mediated phosphorylation of intracellular signaling molecules. Values are taken from the 2D
cytometry plots (Figures S3(A) and (B), S4(B) and (C) and data not shown). Mean and standard deviations are shown for each CLL and healthy sample. CLL samples were
segregated into Group 1 and Group 2 based their H2O2-mediated p-Stat5 response which was statistically significant (p-value 1.6610–4) between the two groups. There
was statistical significance for all other signaling molecules between each group (p-values within the range of 3.8610–5–9610–3). The exception was H2O2-mediated p-
Akt signaling for which the p-value was 0.5. Apoptosis responses to in vitro F-Ara-A exposure are shown as+for proficient and – for refractory according to the criteria
described in the Data Analysis section on page 11. The percentage of cells that are positive for cleaved caspase 3 and cleaved PARP at 48 hours after background
subtraction are in parentheses.
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Area under the ROC curves (AUC of ROC curve)  for
signaling induced by H2O2treatment showed that p-Lyn (AUC
0.84), p-Syk (AUC 0.75), p-BLNK (AUC 0.79), p-PLCc2 (AUC
0.81), p-Erk (AUC 0.77) and p-Stat5 (AUC 0.84) signaling
stratified patient samples according to their apoptotic pathway
response (Figure 5(A)). Using the metrics derived from the mixture
models, the ROC curves showed that samples in which H2O2
exposure revealed signaling were more likely to undergo F-ara-A
mediated apoptosis (Figure 4(B), 5(A)). By contrast, samples in
which H2O2failed to induce signaling were largely non-responsive
to F-ara-A (Figures 2(A–C), Figure 3 and Table 3).
Of note, although the range of expression observed for SHP-1,
SHP-2 and CD45 tyrosine phosphatases was greater in CLL
compared to healthy B cells (Table S2) no association could be
seen between their expression with induced signaling or apoptosis.
Thus levels of these phosphatases alone were not surrogates for
these pathway functions. No associations could be made between
the IgVHmutational status or ZAP70 expression status and in vitro
response to F-ara-A (Fisher’s exact test for association between
IgVHstatus and apoptosis F-ara-A responder/F-ara-A refractory,
p value=1, odds ratio=0.84).
The areas under the ROC curves demonstrated significant
associations between H2O2-mediated signaling and apoptotic
proficiency for the entire CLL sample cohort (Figure 5(A)).
However, in order to predict response to in vitro F-ara-A treatment
for an individual sample, an un-scaled mixture model of for
example, H2O2-induced phosphorylation of Stat5 was established
for all the CLL samples (Figure 5(B), top panels). Samples CLL007
and CLL021 have one population distribution of cells and are
refractory to F-ara-A exposure. Samples CLL014 and CLL024
show population distributions of cells that span both subpopula-
tions and CLL B cells and these samples are responsive to F-ara-A
exposure. CLL009 has a signaling profile predictive of apoptotic
sensitivity but was refractory to in vitro F-ara-A. This latter sample
does not fit the model presumably due to alternative pathways that
confer refractoriness to apoptosis (Figure 5(B)). By contrast
CLL013 had a reduced H2O2-mediated signaling response but
yet had a strong apoptotic response (Table 3, Figure S3 and data
not shown) suggesting that in this sample a different biology may
be driving CLL.
Although, several molecular and cytogenetic lesions have
emerged as potential prognostic indicators for CLL many
disparities and confounding issues limit their clinical utility
[33,45,46]. For example, although primary resistance to fludara-
bine has been shown to occur in patients harboring p53 deletions,
a recent study reported that treatment-naı ¨ve patients with p53
deletions exhibit clinical heterogeneity with some patients
experiencing an indolent course [47,48]. These published clinical
studies suggest that there are underlying differences in CLL
biology, which if understood, could provide more reliable
prognostic information for individual patients.
The data in this study have highlighted a link between H2O2-
induced changes in phosphorylation of signaling proteins down-
stream of the BCR and in vitro F-ara-A-mediated apoptosis in CLL
B cells. Specifically, the data showed: (1) The sample cohort could
Figure 2. H2O2treatment segregates CLL samples into two sub-groups based on magnitude of their signaling responses. (A) CLL B
cells were untreated or exposed to anti–moranti–calone, H2O2alone or the combination for 10 minutes. Representative 2D flow plots show CLL B cell
subsets which exhibit (A) robust H2O2–mediated signaling (B) a reduced H2O2–mediated signaling response for proximal BCR signaling molecules.(C)
phosphorylation of Stat5 demonstrates either an increased (left-hand columns) or a marginal response (right-hand columns) to H2O2treatment. The
2D plot has SHP-2 along the X-axis as the SHP-2 antibody was in the same antibody panel.
Figure 3. Effect on apoptosis of In vitro exposure of CLL B cells to F-ara-A. Representative 2D flow plots (cleaved caspase 3 (X-axis) and
cleaved PARP (Y-axis)) show that samples CLL014 and CLL024 undergo F-ara-A-mediated apoptosis (left-hand panels, (red arrows). By contrast
samples CLL021 and CLL007 were relatively refractory to F-ara-A treatment (right-hand panels).
B Cell Signaling and In Vitro Apoptosis in CLL
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B Cell Signaling and In Vitro Apoptosis in CLL
PLoS ONE | www.plosone.org9 October 2011 | Volume 6 | Issue 10 | e24592
be divided into two groups based on the size of a cell subset within
each sample that was responsive to the reactive oxygen species
H2O2based on increased phosphorylation of p-Lyn, p-Syk, p-
BLNK, p-PLCc2 and p-Stat5. (2) In most cases in which the H2O2
responsive subpopulation was greater than 30%, a cell subset
proficient for F-ara-A mediated apoptosis was seen in the same
sample. (3) A mixture modeling metric was derived that was linked
to the presence or absence of these observed cell subsets to
apoptosis response. (4) AUC values for this model were above 0.75
for p-Lyn, p-Syk, p-BLNK, p-PLCc2 and p-Stat5 and stratified
patient samples according to their apoptotic pathway response.
Although the CLL sample cohort was obtained from CLL
patients receiving different treatments, it was striking that their
H2O2response was able to segregate the samples into two groups.
The concentration of H2O2used in study was in the millimolar
range and was chosen based on its ability to mediate a response in
leukemic B cells but not in healthy B cells as previously reported
. It is difficult to ascertain exact intracellular concentrations of
H2O2as its production tends to be localized either in the plasma
membrane or in endosomes and inactivating antioxidant enzymes
prevent indiscriminate oxidation of intracellular molecules by
H2O2 [14,49]. H2O2 is produced by NADPH Oxidase (Nox)
enzymes that are activated by cell surface receptors including BCR
[14,15,50]. To function as an intracellular signaling molecule,
H2O2must be imported into the cytosol and reported intracellular
concentrations range from micromolar to millimolar levels
[49,51]. As an intracellular second messenger, H2O2,results in
amplification of receptor tyrosine kinase signaling by transiently
and reversibly inactivating tyrosine phosphatases (PTPS) through
reversible oxidation of the catalytic cysteine to sulfenic acid
[14,49,52]. A likely role for PTPs in both ligand-dependent and
independent BCR signaling was revealed in several studies. In
healthy B cells and follicular lymphoma H2O2participates in anti-
m mediated signaling [5,7,15,16]. Other reports showed that Syk
was activated by pervanadate/H2O2 in the absence of BCR
That activated BCR signaling molecules, in the absence of
ligand, play an important survival role in CLL and other B cell
malignancies is substantiated by recent studies. One study showed
that in CLL B cells where Lyn protein is over-expressed, its
inhibition by small molecule inhibitors in vitro in the absence of a
BCR ligand, induced apoptosis . Corroborating these findings,
in vitro treatment of DLBCL and CLL cells with R406 a small
molecule inhibitor of Syk (a substrate of Lyn) also induced
apoptosis [25,26,54]. More recently, a phase I/II clinical trial of
fostamatinib disodium, an oral Syk inhibitor showed clinical
activity in CLL and non-Hodgkins lymphoma . Another study
showed a negative correlation between the expression of ZAP70
with the phosphorylation state of Syk and a positive correlation
between p-Syk with p21cip, a cell cycle inhibitor . Further
insights into the relationship of phosphatase activity with BCR
signaling molecules and apoptosis could be determined by
experiments including specific tyrosine phosphatase inhibitors
specifically targeting SHP-1 and/or CD45. A priori, such
inhibitors would be predicted to promote CLL blast cell survival.
Consistent with this hypothesis, ectopic expression of protein
tyrosine phosphatase, PTPRO, (silenced in CLL by DNA
methylation) increased growth inhibition in response to F-ara-A
. In DLBCL, PTPROt was identified as a tumor suppressor
with a role in tonic BCR signaling . Furthermore, additional
studies will be required to determine whether lymphoid tyrosine
phosphatase (Lyp) also known as PTPN22, whose expression was
reported to be increased in CLL B cells, plays a role in the
response of CLL B cells to therapeutic agents (Negro et al., Blood
(ASH Annual Meeting Abstracts) 2009 114: Abstract 800).
Although not definitively proven, the data in this study
potentially support a mechanism whereby H2O2
inhibition of tyrosine phosphatases relieves a negative feedback
loop that results in activation of signaling proteins within the BCR
network. Regardless of its exact mechanism of action, H2O2was
able to reveal differential signaling within CLL samples and these
signaling differences appear to be associated with a signaling
posture that either drives, or is driven by the ability of these cells to
undergo apoptotic induction by, in this case F-ara-A.
In this study, SCNP analysis, combined with mixture modeling
identified at least two phenotypes of CLL B cells based on their
H2O2– mediated response of signaling molecules (Figure 2(A),
(B), (C), Table 3). Notably, some samples demonstrated
simultaneous presence of both cell subsets, suggesting co-
evolution of signaling phenotypes, a common precursor of these
cell subsets, ora lineage relationship between
subpopulations of cells (Figure 2(A), (C)). Interestingly, and in
contrast to studies where the presence of ZAP70 and unmutated
IgVH correlated with greater anti2m-mediated-BCR signaling
[25,29], the signaling responses described here were unrelated to
the IgVHmutational status or to ZAP70 expression and spanned
a range of cytogenetic abnormalities. Although further studies are
warranted to investigate this issue, it is important to note that the
above studies [25,29] were accomplished via indirect assay of
total phospho-tyrosine on signaling proteins. In our study, we
undertook direct assay of phosphorylation sites using antibodies
directed against known, functional, epitopes on a per cell basis.
Additionally, no associations were observed between SHP-1,
SHP-2, CD22 or CD45 expression levels with H2O2-mediated
signaling (data not shown).
Although not a member of the canonical BCR signaling
network, the increase seen in H2O2–mediated p-Stat5 could be
due to a bystander effect resulting from phosphatase inhibition
with consequent increases in kinase activities for which Stat5 is a
substrate. Interestingly, Sattler et al, showed the importance of
H2O2generation with consequent increases in p-Stat5 in several
hematopoietic growth factor cascades in cell lines . A pivotal
role was also demonstrated for activated Stat5 in hematopoietic
stem cell self- renewal and expansion of multi-potential progen-
itors in myeloid disease . In addition, highly distinctive
cytokine responses in Stat5 phosphorylation were reported in both
normal and leukemic stem/progenitor cells . Furthermore, in
a recent study phospholipase C-b3 was shown to be a tumor
suppressor by acting as a scaffold for simultaneous interaction with
p-Stat5 and SHP-1 and by doing so promoted the dephosphor-
ylation of p-Stat5 . Whether these mechanisms regulate p-
Stat5 in CLL awaits further study.
Figure 4. Population distributions of all CLL and all healthy B cells based on their fluorescence intensities. (A) Arcsinh transformed
fluorescence intensities either from all gated CLL and healthy B cells in all samples were used to derive the histograms. CLL samples (cyan)
demonstrate multiple examples of bimodal activation (arrows), revealed after H2O2treatment. By contrast healthy B cells (pink) demonstrate a single
cell subset with minimal activation of signaling after H2O2treatment. (B) Mixture models comprised of two normal distributions  were generated
from the histograms of CLL B cells in (A) . These metrics were termed ‘MixMod1’ and ‘MixMod2’ representing the areas under the curve for the
distributions with lower (red) and higher (blue) fluorescence intensities, respectively.
B Cell Signaling and In Vitro Apoptosis in CLL
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B Cell Signaling and In Vitro Apoptosis in CLL
PLoS ONE | www.plosone.org11October 2011 | Volume 6 | Issue 10 | e24592
The clinical complexity (and unpredictability) of CLL as well as
the many components governing cell proliferation and survival
mechanisms, suggest a diversity of mechanisms that give rise to
CLL. Nonetheless, the current studies, although mechanistically
incomplete demonstrate a convergence of signaling patterns in
CLL that lead to a remarkably limited set of phenotypic cell
signaling outcomes. This suggests that despite the underlying
molecular and clinical heterogeneity that maintains cellular
homeostasis in CLL, only a limited number of signaling pathway
variations exist and these may be exploited for therapeutic benefit.
Although the sample set was limited, the encouraging AUC values
(Figure 5(A)) endorse follow-up studies with expanded sample
cohorts, both cryopreserved and fresh, to determine whether
SCNP of individual samples can predict treatment outcome and
stratify patients who might gain the most benefit from fludarabine-
based treatment regimens.
and healthy donors.
Gating scheme applied to B cells from CLL
healthy B cells. PBMCs from healthy donors were either
untreated or stimulated for 10 minutes with anti-malone, H2O2
alone or the combination. 2D flow plots of gated B cells show
exemplary samples in which H2O2potentiates anti-m mediated
signaling of proximal BCR effectors as previously reported .
H2O2 amplifies BCR-mediated signaling in
two groups based on p-Stat 5 signaling in cell subsets.
Changes in Stat 5 phosphorylation are shown in 2D flow plots and
panels (A) and (B) show samples organized by their apoptotic
response to in vitro F-ara-A exposure. (A) Stat 5 is phosphorylated
in response to H2O2alone in a CLL B cell subset within this CLL
sample sub-group. All samples with these Stat 5 responsive cells
undergo F-ara-A-induced apoptosis. (B) Minimal Stat 5 phos-
H2O2treatment segregates CLL samples into
phorylation is seen in response to H2O2alone within this CLL
sample sub-group. All samples except for CLL009 fail to undergo
H2O2–mediated Stat 5 phosphorylation. (C) Stat 5 is not
phosphorylated in healthy B cells in response to H2O2.
exposure of all samples from CLL and healthy donors to
F-ara-A. (A) 2D flow plots show that healthy B cells undergo
apoptosis in response to F-ara-A exposure. (B) 2D flow plots in
which CLL B cells subsets undergo apoptosis after exposure to F-
ara-A. (C) 2D flow plots in which CLL B cells subsets are
refractory to F-ara-A exposure.
Measurements of apoptosis after In vitro
exposure of CLL samples to staurosporine (5mM) for
6 hours. (A) 2D flow plots showing response of samples that were
recorded as F-ara-A responders (Table 3 and Figure S4 (A). (B) 2D
flow plots showing response of samples that were recorded as F-
ara-A non-responders (Table 3 and Figure S4 (B)).
Measurements of apoptosis after in vitro
The authors thank J. M. Irish for intellectual input, T.J. Kipps and L.
Rassenti for providing clinical samples and R. Lin, D. Soper, and D.R.
Parkinson for critical reading of the manuscript.
Conceived and designed the experiments: ALP EE Y-WH AC GPN WJF.
Performed the experiments: ALP EE Y-WH. Analyzed the data: ALP EE
Y-WH GPN WJF. Contributed reagents/materials/analysis tools: ALP EE
Y-WH. Wrote the paper: EE GPN WJF.
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