Phospho-flow Cytometry Research Articles

191-P: Phospho-flow cytometry: A Novel Method to Measure Antibody Induced Activation in Endothelial Cells

191-P: Phospho-flow cytometry: A Novel Method to Measure Antibody Induced Activation in Endothelial Cells

Analysis of Signaling Events by Dynamic Phosphoflow Cytometry

Many proteins involved in cell signaling are phosphorylated. To determine the phosphorylation status of these signaling molecules at the single-cell level, we present a protocol for using state-specific antibodies to detect target phosphoproteins with fluorescence measurements by flow cytometry. To improve the signal intensity, a sandwich-labeling method for the analysis of signaling proteins is performed. By comparing the phosphorylation state of proteins in the presence and absence of sodium pervanadate, a nonspecific tyrosine phosphatase inhibitor, we determined the relative amount of tyrosine-phosphorylated protein in the samples, which reflects the activity of the signaling pathway. This dynamic approach, in combination with the signal amplification through a sandwich-labeling method, produces accurate and reproducible measurement of the activity of signaling pathways.

Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

Distinct Proteomic Signaling Networks Stratify Responses to Ara-C Based Induction Therapy in Patients with Acute Myeloid Leukemia (AML)

Background: AML represents a group of clonal hematopoietic stem cell disorders in which aberrantly regulated signaling pathways lead to oncogenic progression. In a prior study, a panel of 36 cytokine response nodes was measured in single cells by multi-parameter phosphoflow cytometry in newly diagnosed AML patient samples. Unique cancer network profiles were revealed that correlated with response to chemotherapy (Irish et al, Cell (2004) 118, p217).Objectives: Given the heterogeneity of AML, the current study was designed to expand the number of signaling nodes to 152 per patient sample. The signaling nodes were organized into 4 biological categories:Protein expression of receptors and drug transportersResponse to cytokines and growth factors,Phosphatase activity,Apoptotic signaling pathways.Methods: Multi-parameter flow cytometric analysis was performed on peripheral blasts taken at diagnosis from 33 AML patients who attained a complete response (CR, n=9) or no-remission (NR, n=24) to one cycle of standard 7 + 3 induction therapy (100–200mg/m2 cytarabine and 60mg/m2 daunorubicin).Results: The data show that expression of the receptors for c-Kit and FLT3L and the drug transporter ABCG2 were increased in patients who attained an NR versus CR. Readouts from the cytokine-Stat response panels and the growth factor-Map kinase and PI3-Kinase response panels (see Table 1) revealed increased signaling in blasts taken from NR patients versus blasts taken from patients who clinically responded to therapy. To determine the role of phosphatases, a physiologic phosphatase inhibitor, peroxide, (H2O2) revealed increased phosphatase activity in CRs versus NRs. In the absence of treatment with H2O2, CRs had lower levels of phosphorylated PLCƒ×2 and SLP-76 versus NRs, and attained higher levels of phosphorylated PLCƒ×2 and SLP-76 upon H2O2 treatment. Lastly, interrogation of the apoptotic machinery using agents such as staurosporine and etoposide showed that NR patient blasts failed to undergo cell death, as determined by cleaved PARP and cleaved Caspase 8. Of note, in NR patient blasts, these agents did promote an increase in phosphorylated Chk2 suggesting a communication breakdown between the DNA damage response pathway and the apoptotic machinery. In contrast, blasts from CR patients showed significant populations of cells with cleaved PARP and caspase 8 consistent with their clinical response outcomes.Conclusions: In this study, 152 signaling nodes per patient sample were measured by multi-parameter flow cytometry and revealed distinct signaling profiles that correlate with patient response to ara-C based induction therapy. Alterations were seen in expression for the c-Kit and Flt-3L receptors, the ABCG2 drug transporter, cytokine and growth factor pathway response, phosphatase activity and apoptotic response, all of which could stratify the NR from the CR patient subsets. Whether there is a combination of hierarchical nodes to best predict response to therapy is currently under investigation.

A Novel CCR5-Specific Pharmacodynamic Assay in Whole Blood Using Phosphoflow Cytometry Highlights Different Ligand-Dependent Responses but Similar Properties of Antagonists in CD8+ and CD4+ T Lymphocytes

Chemokine CC motif receptor (CCR) 5 is a major drug target for both inflammation and virology indications. The primary function of CCR5 is to mediate the trafficking of CCR5-expressing lymphocytes to any of the CCR5 ligands, which are often increased during inflammatory responses. In addition, CCR5 is a coreceptor for HIV, mediating R5 tropic HIV infection of CCR5-expressing CD4 T cells. We report the use of a novel method to assay the pharmacodynamic (PD) properties of small-molecule and antibody inhibitors of CCR5 ligand-induced activation by measuring phosphorylation of serine residue 349 in the cytoplasmic tail of human CCR5 using phosphoflow cytometry in whole blood. This assay is highly specific and measures CCR5 phosphorylation in both CD8(+) and CD4(+) T cells and allows the calculation of inhibitor IC(50) values from both lymphocyte subsets in the presence of CCR5 antagonists. In addition, this assay is cross-reactive to nonhuman primates and allows PD analysis in whole blood from rhesus and cynomolgus macaque. Using this assay, we identified different ligand-dependent response properties between CD8(+) and CD4(+) T cells, although CCR5 antagonists behave with similar properties against both cell types. The use of this assay may be of particular benefit to monitor PD effects of CCR5 inhibitors during drug development, preclinical in vivo studies, and in patients currently being treated for HIV or CCR5-mediated inflammatory diseases with CCR5 inhibitors. Similar phosphoflow approaches to other GPCR targets on circulating lymphocytes may prove to be the most reliable PD assay for preclinical and potentially clinical development.

Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus

BackgroundOncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.Methods and FindingsIn vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.ConclusionsBy combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.

Dysregulated Signaling Networks in Chronic Myeloproliferative Disorders: Co-Operation of JAK2 and Src Family Kinases (SFK)

The JAK2-STAT5 pathway plays an key role in chronic MPD (cMPD) with the JAK2 V617F mutation. However, JAK2 also initiates several second messenger pathways, including the Ras-ERK and PI3K-AKT pathways. Since these essential second messenger pathways are also regulated by SFK activity, and crosstalk between JAK and SFK has been demonstrated, we investigated the potential SFK-dependent regulation of STAT5, Ras-ERK and PI3K-AKT in primary cells from patients with cMPD and myeloid cell lines with the JAK2 V617F mutation (HEL) or Epo-dependent (UT-7/Epo). In primary cells from JAK2 V617F patients, we show that SFK activity is required for optimal JAK2 induced phospho (p)-STAT5, nuclear targeting of p-STAT5, and STAT5-DNA binding. Both endogenous and Epo-induced colony formation in methylcellulose by peripheral blood cells derived from polycythemia vera (PV) patients was markedly suppressed with the SFK inihibitor dasatinib; endogenous colony formation was abrogated while both the number and size of Epo-induced colonies was markedly reduced (> 70% inhibition at 1 uM), suggesting that despite of the presence of activating JAK2 V617F in primary cells, SFK activity is required for optimal proliferation. Interestingly, a similar observation was also made in a PV patient lacking the JAK2 V617F mutation. When HEL or Epo-stimulated UT-7/Epo cells were treated with a JAK inhibitor (P6), there was significant inhibition of p-STAT5 induction and second messenger pathways, demonstrating the importance of this proximal kinase. Inhibition of JAK2 did not affect SFK activation however, suggesting that SFK may lie upstream of JAK2. Interestingly, when SFK activity was suppressed (PP2 or dasatinib), a marked inhibition of p-STAT5 (and STAT5 DNA-binding), p-AKT, p-ERK, p-90RSK, p-S6 ribosomal protein(rp), and global protein tyrosine phosphorylation was observed in cell lines and primary cells. These findings suggest that SFK activity is involved in the most proximal aspects of Epo signaling and facilitates JAK2 activation of key second messenger pathways essential for proliferation, differentiation, and survival. Since JAK2 and SFK coordinately regulate multiple second messenger pathways, we next examined the relative importance of each by specifically targeting either the Ras-ERK pathway (MEK inhibition) or the PI3K-AKT pathway (PI3K and mTOR inhibition) downstream from JAK2 and SFK in both primary cells and cell lines. Using intracellular phospho-flow cytometry and immunoblotting, we show that the SFK-dependent Ras-ERK-RSK axis can directly induce p-S6rp, a key component in translational control and cell cycle progression. To determine whether this finding can be reproduced in vivo, we examined p-S6rp expression in bone marrow biopsies from PV patients (n=16); a marked hyperphosphorylation S6rp was consistently seen in patients' megakaryocytes as compared to control specimens obtained from reactive processes, lymphoma staging, CMML, or even a case of hypoxia-induced polycythemia (all showing low/absent p-S6rp). Simultaneous staining for p-mTOR revealed that the level of p-mTOR was not increased. In sum, our findings suggest that SFK activation is necessary for optimal signal transduction along of the JAK2-STAT5 axis. In addition, the Ras-ERK-RSK pathway, requiring both JAK and SFK, may play a key role in S6rp hyperphosphorylation leading to increased cell cycling. Both findings point towards potential therapeutic targets, along with JAK2, for treating cMPD.

Pharmacologic Inhibition of Src Family Kinases Differentially Affects T Cell Cytokine Production and Proliferation: Implications for Novel Immunomodulatory Therapies.

It is well-recognized that the T cell receptor (TCR) signaling pathway, catalyzed by the Src family kinase (SFK) Lck, is the essential first step in T cell immune responses. The long-held goal of specifically targeting SFK activity for immunomodulatory therapy is becoming more of a reality as SFK inhibitors are entering into clinical use. In particular, dasatinib is an oral small molecule inhibitor of Abl and SFK, including Lck. Given the central importance of Lck in transmitting signals from the TCR signaling complex, and the potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent could provide a novel route of immunomodulation via targeted inhibition of antigen-induced signaling, particularly in combination with currently available immunomodulatory agents. Using intracellular phospho-flow cytometry and western blotting, we show that low nanomolar concentrations of dasatinib potently inhibit TCR induced global protein tyrosine phosphoryation, including the key signaling elements CD3, ZAP-70, LAT, and PLC. In addition, the second messenger pathway Ras/Raf/MEK/ERK is potently blocked, whereas the PI3K-AKT pathway is minimally affected. Furthermore, induction of many pro-inflammatory cytokines, including TNF-α, IFN-γ, IL-2, GM-CSF, IL-17, IP-10, RANTES, and IL-6 (measured by cytokine antibody array) was significantly inhibited when the TCR was triggered in the presence of dasatinib. T cell proliferation, assessed by flow cytometric CFSE dilution analysis, was inhibited in a dose-dependent manner with dasatinib, showing complete inhibtion at 10 nM. Cell cycle analysis based on DNA content by PI staining revealed that dasatinib-treated T cells failed to enter the cell cycle. Considering that the PI3K-AKT pathway was only minimally affected by dasatinib, we examined the ability to achieve enhanced inhibition by using sub-optimal doses of dasatinib and rapamycin that individually produce mild-moderate inhibition of proliferation (20% and 40% inhibition, respectively). When dasatinib and rapamycin are combined at these lower doses, there was a consistently enhanced inhibitory effect on T cell proliferation (80–95% inhibition). Concurrent CD28 stimulation failed to overcome the inhibitory effects of SFK inhibition when the inhibitor was present throughout the stimulation. Interestingly, when SFK inhibition was delayed for at least 24 hours after the initiation of T cell activation with concurrent CD28 stimulation, we observed a divergent effect with regard to cytokine production and proliferation. Pro-inflammatory cytokine production was significantly decreased even if SFK activity was inhibited 48 hours after stimulating T cells, suggesting that ongoing TCR-dependent SFK activity is essential for cytokine production. However, late inhibition of SFK activity with dasatinib increased proliferation, with greater numbers of T cells achieving more rounds of cell division. Thus, while necessary for cytokine production, once the cells have entered the cell cycle, SFK activity regulates negative feedback in CD28-mediated proliferation. In conclusion, targeted inhibition of SFK activity is a promising approach for novel immunomodulatory therapy, particularly in combination with other signaling pathway inhibitors, but timing of SFK inhibition in relation to T cell activation could have an important impact on the immunomodulatory effect.

Efficacy of TG101348, a Selective JAK2 Inhibitor, in Treatment of a Murine Model of JAK2V617F-Induced Polycythemia Vera.

The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Targeting syk kinase for the treatment of B-cell lymphoma

3600 Background: B-Cells require constitutive signaling from the B-Cell Receptor (BCR). Elimination of the BCR induces cell death. Since the BCR lacks enzymatic activity and relies upon the non-receptor tyrosine kinase Syk to initiate a signal transduction cascade, we hypothesized that R406; a small molecule Syk kinase inhibitor might eliminate the constitutive BCR signal and therefore have selective cytotoxicity for B-cells. Methods: Signal transduction changes were evaluated in cell lines and primary normal B-Cells via western blotting and phosphoflow cytometry. Effects on B-Cell, T-Cell, and Myeloid lineage cell lines were evaluated by 3H-thymidine proliferation assays, BRDU cell cycle analysis, and Annexin-V staining. Primary tumor samples were evaluated for B-Cell selective toxicity by evaluation of active caspase activity. Results: In cell culture R406 significantly alters the basal phosphorylation status of multiple proteins. Proliferation of B-Cell lines is reduced with an IC50 range of 625nM to 2.5uM without effect on T-Cell or Myeloid lineage cell lines. Cell cycle analysis reveals a reduction in B-Cell S-Phase with increase in G1/G0. Apoptosis is also increased in the B-cell lines without significant increase in the non-B cell lines. Primary normal B-Cells show reduced phosphorylation of BTK, Erk, and p38 in response to BCR cross linking. Primary tumor samples reveal activity against B-Cell neoplasms. In samples containing both malignant B-Cells and normal T-Cells, the induction of active caspase-3 is restricted to the malignant B-Cell population. Conclusions: 1) R406 alters signal transduction in B-Cells. 2) R406 demonstrates selective anti-B-Cell activity among cell lines. 3) Primary tumor samples reveal cytotoxic activity against malignant B-Cells without toxicity to T-Cells within the same specimen. 4) This compound has been safely tested in humans for non-malignant diseases with an appropriate pharmacokinetic profile; therefore these results support the initiation of an upcoming multicenter phase I/II clinical trial in patients with lymphoma. No significant financial relationships to disclose.

Deeper Insights into Hematological Oncology Disorders via Single-Cell Phospho-Signaling Analysis

An understanding of hematological cancer cell signaling processes poses one of the most complex and intractable problems in modern biomedical inquiry. While we understand some of the fundamental players that contribute to oncogenic processes, significant effort is focused upon determining how these individual players relay information to each other to create the composite functions of a cancer cell. Efforts designed to understand these processes at the single cell level will undoubtedly allow for understanding of the heterogeneity of hematological tumors as well as, simultaneously, the function of the 'responding' immune system. I will relate some of the insights our laboratory has developed over the last several years applying single-cell phospho-flow cytometry to the study of signaling in primary patient material and murine models. While it is clear that this analysis now allows us to accomplish phospho-signaling biochemistry at the single cell level with primary cell material, we are only beginning to develop some of the bioinformatics tools to appropriately display the vast amount of information collected by such approaches. These approaches, however, have already allowed us to develop approaches that prognosticate patient outcomes based on signaling status, prior to any treatment, as well as subgroup patient subtypes according to signaling states. The modest efforts to date presage a time where it should be possible to provide far more tailored therapies specific to the varied diseases represented by the hematological malignancies.