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.
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.
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
SCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and
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.
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 unpredictability 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
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
Central to B cell development, and also believed to be important in CLL progression, is the BCR signal complex composed of membrane-bound immunoglobulin and the signal transducing CD79α/CD79β heterodimer. In normal B cells, antigen mediated BCR activation regulates cell survival, differentiation, proliferation and migration
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 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
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)
Sample ID | Gender | Age at DX | IgVH | % ZAP 70 | FISH | First Treatment (Rx) Type |
CLL001 |
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44 | 93 | 0.4 | Not available | HDMP+Rituxan |
CLL003 | F | 53 | 92.6 | 0.6 | Not available | FR (2cycles) - good response |
CLL004 |
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53 | 100 | 92.5 | 11qdel(16%) | HDMP Rituxan Frontline (3 cycles) |
CLL005 |
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65 | 98.6 | 43.6 | tri12 (90%) | Leukeran (4 mg/day) |
CLL006 | F | 61 | 99.6 | 45.2 | normal FISH | F; then FR (4 cyc) - MRD on BM |
CLL007 |
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66 | 99.3 | 79.1 | tri 12 (84%) | Leukeran |
CLL008 |
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72 | 95.8 | 3 | 13q del (73%) | Leukeran |
CLL009 |
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81 | 95.2 | 9.8 | 13q del (100%) | Chloroambucil |
CLL010 |
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48 | 89 | 5 | Not available | Fludarabine (3 cycles) |
CLL011 |
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57 | 93.3 | 1.1 | tri 12(75%) | ASCENTA-002 |
CLL012 | F | 72 | 96.8 | 1.2 | Not available | no Rx |
CLL013 |
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59 | 99.6 | 62.8 | tri 12(89%) | Chlorambucil |
CLL014 |
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54 | 90.5 | 0.5 | tri 12 (karyotype) | High dose chemoRx w/BM transplant |
CLL015 | F | 51 | 100 | 91.9 | tri 12 (82%) | ASCENTA-002 |
CLL016 | F | 53 | 93 | 0.1 | Normal FISH | HDMP+R 04 Frontline |
CLL017 | F | 39 | 96.1 | 10.1 | Normal Karyotype | no Rx |
CLL018 |
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55 | 100 | 91.1 | 13qdel(70%) | HDMP+R 04 (3 cycles) Frontline |
CLL019 |
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50 | 100 | 45.9 | Normal FISH | Rituximab (3 cycles) - 4 weeks |
CLL020 |
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43 | 91.9 | 1.1 | 13 del (99%) | Chlorambucil |
CLL021 |
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66 | 100 | 65.3 | 13 del (99%) | Solumedrol Rituxan |
CLL022 |
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48 | 100 | 73.1 | tri 12 (80%) | no Rx |
CLL023 |
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72 | 94.7 | 1.3 | 13 del (17%) | GMCSF and Rituxan |
CLL024 | F | 49 | 100 | 51.8 | Not available | no Rx |
IgVH mutational status and percent ZAP70-positive cells determined as previously described
Samples were thawed at 37°C 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 experimentation. Cells were arrayed in duplicate in 96-deep-well plates at 6.0×105 cells or 8.0×105 cells 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 preparation).
The human Burkitt lymphoma Ramos cell line, a control for BCR signaling, acquired from ATCC was cultured according to the manufacturer’s protocol.
After a 2 hour rest at 37°C, each sample was treated in bulk for 10 minutes at 37°C with goat polyclonal (F(ab’)2 human anti−μ (anti−μ) or anti−γ (Southern Biotech), final concentration 10 µg/ml or with H2O2, final concentration 3.3 mM. For the combination of anti−μ and H2O2, anti−μ was added first followed by H2O2
Cells were fixed with paraformaldehyde and permeabilized with 100% ice-cold methanol as previously described
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 (
Samples processed for cytometry
Cells were incubated with immunophenotypic cocktails (
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
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
The criteria used to assign apoptotic proficiency to a sample were a two-fold or greater increase in the number of CD3−−/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 CD3−/CD20+ cells at hours that were positive for c-PARP+and c-Caspase3+.
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 of 16 and 17 respectively among healthy B cells and 63 and 66 in CLL B cells (
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.
p-Lyn | p-Syk | p-PLCγ2 | p-BLNK | p-Erk | p-65/RelA | p-Akt | p-S6 | p-Stat 5 | ||
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Range | 66 | 62 | 57 | 50 | 94 | 68 | 63 | 1155 | 43 |
Mean | 52 | 79 | 63 | 35 | 153 | 56 | 105 | 579 | 51 | |
Stdev | 14 | 16 | 15 | 11 | 28 | 17 | 16 | 287 | 11 | |
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Range | 17 | 16 | 17 | 21 | 41 | 19 | 16 | 198 | 6 |
Mean | 66 | 55 | 51 | 53 | 157 | 57 | 94 | 377 | 35 | |
Stdev | 6 | 5 | 8 | 8 | 13 | 6 | 6 | 72 | 2 |
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 anti−μ 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
Consistent with previous reports, anti−µ-mediated BCR signaling was observed and further potentiated by H2O2 in B cells from healthy donors (
Analysis of the signaling responses showed that the CLL sample cohort was minimally responsive to anti–μ 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–μ-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-PLCγ2 population was 51%, 52% and 45% and 68% respectively, (
(A) CLL B cells were untreated or exposed to anti–µoranti–γalone, H2O2 alone 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 H2O2 treatment. The 2D plot has SHP-2 along the X-axis as the SHP-2 antibody was in the same antibody panel.
Sample Group | CLL Sample | p-Lyn | p-Syk | p-BLNK | p-Stat 5 | p-PLC–γ | p-Akt | p-Erk | Apoptosis |
Group I | CLL024 | 80 | 65 | 73 | 47 | 77 | 46 | 88 |
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Group 1 | CLL003 | 73 | 74 | 64 | 60 | 81 | 74 | 86 |
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Group 1 | CLL008 | 57 | 57 | 47 | 36 | 77 | 67 | 84 |
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Group 1 | CLL010 | 56 | 54 | 39 | 30 | 85 | 80 | 90 |
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Group 1 | CLL009 | 47 | 63 | 54 | 29 | 79 | 56 | 69 |
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Group 1 | CLL014 | 43 | 52 | 52 | 36 | 67 | 59 | 57 |
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Group 1 | CLL001 | 40 | 41 | 20 | 34 | 51 | 43 | 52 |
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Group 1 | CLL002 | 39 | 39 | 36 | 21 | 56 | 40 | 70 |
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Group 1 | CLL016 | 27 | 26 | 17 | 23 | 39 | 56 | 41 |
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Group 1 |
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Group 1 |
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Group 2 | CLL019 | 24 | 29 | 19 | 12 | 40 | 66 | 53 |
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Group 2 | CLL004 | 20 | 23 | 19 | 13 | 45 | 31 | 76 |
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Group 2 | CLL018 | 20 | 21 | 18 | 15 | 33 | 45 | 61 |
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Group 2 | CLL013 | 19 | 25 | 19 | 13 | 37 | 26 | 27 |
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Group 2 | CLL012 | 17 | 32 | 23 | 7 | 48 | 61 | 34 |
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Group 2 | CLL005 | 15 | 18 | 10 | 9 | 35 | 74 | 41 |
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Group 2 | CLL020 | 10 | 5 | 5 | 7 | 33 | 40 | 44 |
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Group 2 | CLL023 | 10 | 16 | 14 | 10 | 43 | 87 | 83 |
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Group 2 | CLL007 | 8 | 7 | 0 | 4 | 19 | 66 | 40 |
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Group 2 | CLL021 | 6 | 8 | 9 | 6 | 7 | 8 | 10 |
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Group 2 | CLL011 | 5 | 7 | 5 | 3 | 28 | 68 | 19 |
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Group 2 |
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Group 2 |
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Healthy | CON195 | 4 | 6 | 10 | 3.4 | 18 | 51 | 21 |
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Healthy | CON196 | 3 | 5 | 6 | 0.16 | 12 | 48 | 15 |
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Healthy | CON219 | 1 | 3.5 | 3 | 0.6 | 25 | 44 | 26 |
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Healthy | CON240 | 1 | 1 | 3 | 0.6 | 4.5 | 38 | 10 |
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Healthy | CON228 | 1 | 4 | 5 | 1 | 4 | 30.5 | 29 |
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Healthy | CON202 | 0.6 | 3 | 5 | 0.6 | 5 | 45 | 17 |
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Healthy | CON193 | 0.5 | 1.2 | 2 | 0.3 | 4 | 33 | 6 |
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Healthy |
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Healthy |
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Ramos | 50 | 53 | 88 | 38 | 77 | 85 | 64 |
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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 (
In Group ll a greatly reduced subpopulation of cells had a signaling response after exposure to H2O2 compared to Group l samples. For example, the mean percentage of cells in a subpopulation with activated p-Lyn, p-Syk, p-BLNK or p-PLCγ2 was 14%, 17%, 13% and 33% respectively (
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,
Unexpectedly, in 9/23 CLL samples an increase in p-Stat5 was observed in response to H2O2 within a subset of cells in individual samples in Group 1 (
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 (
There has long been a presumed link between ligand-dependent and independent BCR signaling with B cell survival
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).
Within apoptotically responsive samples there were at least two cell subpopulations, with a second cell subset that was refractory to
Given the pro-survival role BCR signaling molecules play in healthy and tumorigenic B cell biology
(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 H2O2 treatment. By contrast healthy B cells (pink) demonstrate a single cell subset with minimal activation of signaling after H2O2 treatment. (B) Mixture models comprised of two normal distributions
On the assumption that at least two subpopulations of cells could 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 (
Receiver operating characteristic (ROC) curves (
(A) AUROC curves were expressed with 95% confidence limits in order to evaluate how statistically significant H2O2–induced signaling is in predicting an
Area under the ROC curves (AUC of ROC curve)
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 (
The areas under the ROC curves demonstrated significant associations between H2O2-mediated signaling and apoptotic proficiency for the entire CLL sample cohort (
Although, several molecular and cytogenetic lesions have emerged as potential prognostic indicators for CLL many disparities and confounding issues limit their clinical utility
The data in this study have highlighted a link between H2O2-induced changes in phosphorylation of signaling proteins downstream of the BCR and
Although the CLL sample cohort was obtained from CLL patients receiving different treatments, it was striking that their H2O2 response was able to segregate the samples into two groups. The concentration of H2O2 used 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
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
Although not definitively proven, the data in this study potentially support a mechanism whereby H2O2 through 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, H2O2 was 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 (
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 H2O2 generation with consequent increases in p-Stat5 in several hematopoietic growth factor cascades in cell lines
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 (
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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.