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Supplementary Figure 3 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Figure 5 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G1-S and G2-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis. [Cancer Res 2008;68(13):5132–42]

Supplementary Figure 2 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Figure Legends 1-6, Tables 1-4 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Figure 6 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Figure 4 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Figure 1 from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Supplementary Methods from Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

Theories of learning distinguish between elemental and configural stimulus processing depending on whether stimuli are processed independently or as whole configurations. Evidence for elemental processing comes from findings of summation in animals where a compound of two dissimilar stimuli is deemed to be more predictive than each stimulus alone, whereas configural processing is supported by experiments using similar stimuli in which summation is not found. However, in humans the summation effect is robust and impervious to similarity manipulations. In three experiments in human predictive learning, we show that summation can be obliterated when partially reinforced cues are added to the summands in training and tests. This lack of summation only holds when the partially reinforced cues are similar to the reinforced cues (experiment 1) and seems to depend on participants sampling only the most salient cue in each trial (experiments 2a and 2b) in a sequential visual search process. Instead of attributing our and others’ instances of lack of summation to the customary idea of configural processing, we offer a formal subsampling rule that might be applied to situations in which the stimuli are hard to parse from each other.

It has been suggested that there are two distinct and parallel mechanisms for controlling instrumental behavior in mammals: goal-directed actions and habits. To gain an understanding of how these two systems interact to control behavior, it is essential to characterize the mechanisms by which the balance between these systems is influenced by experience. Studies in rodents have shown that the amount of training governs the relative expression of these two systems: Behavior is goal-directed following moderate training, but the more extensively an instrumental action is trained, the more it becomes habitual. It is less clear whether humans exhibit similar training effects on the expression of goal-directed and habitual behavior, as human studies have reported contradictory findings. To tackle these contradictory findings, we formed a consortium, where four laboratories undertook a preregistered experimental induction of habits by manipulating the amount of training. There was no statistical evidence for a main effect of the amount of training on the formation and expression of habits. However, exploratory analyses suggest a moderating effect of the affective component of stress on the impact of training over habit expression. Participants who were lower in affective stress appeared to be initially goal-directed, but became habitual with increased training, whereas participants who were high in affective stress were already habitual even after moderate training, thereby manifesting insensitivity to overtraining effects. Our findings highlight the importance of the role of moderating variables such as individual differences in stress and anxiety when studying the experimental induction of habits in humans.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G1-S and G2-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis. [Cancer Res 2008;68(13):5132–42]

Autoimmune disease pathologies are multifactorial with complex interactive networks of cells and chemical messengers that initiate cascades of aberrant cellular activity. Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by systemic inflammation, destruction of the joints, and production of autoantibodies recognizing dozens of putative autoantigens. The presence of autoreactive T cells in individuals leads to pathological autoimmunity by activating additional cellular constituents to mediate inflammation and joint destruction. The etiology of RA is unknown, and knowledge is lacking of the molecular mechanisms underlying the production and subsequent regulation of autoreactive T cells and predicting patient responses to treatments. Biochemical investigations into mechanisms of the disease have relied on animal models that are helpful in dissecting elements of the disease but that are not necessarily reflective of human RA development. The study of multiple activated signaling pathways in complex populations of cells, such as peripheral blood, at the single-cell level has not previously been possible. This article describes how intracellular phosphoepitope staining methodology in conjunction with surface-cell immunophenotyping can be used to deconvolute cellular subsets and allow functional characterization of patient-derived material. Multiparameter flow cytometric analysis allows for small subpopulations-representing different cellular subsets and differentiation or activation states-to be discerned and simultaneously assessed for intracellular biochemical activities. This article also describes how single-cell signal network analysis can be used to stratify patients and may be useful for understanding mechanisms of disease progression, treatment resistance, and development of diagnostic indicators.

Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.

The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8–12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.

Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular oxidative stress, selectively induces apoptosis in HIV-1-infected CD4 + T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in vitro with HIV-1. This selective induction of apoptosis, which we detect by FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis marker expression, is abrogated by the glutathione precursor, N -acetyl- l -cysteine. Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected cells in the culture. These findings suggest that Gd-Tex may have therapeutic utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected cells in an infected host.

We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, and FKHR. ICAM-2 clustering protected primary human CD19+ cells from TNFα- and Fas-mediated apoptosis as determined by single-cell analysis. ICAM-2 engagement by CD19+ cells of its natural receptor, LFA-1, on CD4+ naive cells specifically induced AKT activity in the absence of an MHC-peptide interaction. These results attribute a novel signaling function to ICAM-2 that might suggest mechanisms by which ICAM-2 signals intracellular communication at various immunological synapses.

Myoseverin, a trisubstituted purine, inhibits microtubule assembly in vitro, interferes with normal mitotic spindle assembly, and arrests the cell cycle in mitosis in U937 cells. We synthesized a variety of myoseverin derivatives and screened them for inhibition of spindle assembly in Xenopus egg extracts and for microtubule disassembly in vitro. Selected compounds were tested against 60 cancer cell lines at the National Cancer Institute as possible anticancer drug candidates.

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Many disease processes are characterized by cellular processes that have gone awry. Identification of these specific cellular processes within the heterogeneous cellular systems can provide insights into determining resistant cell populations that arise to a therapeutic agent. Phosphoflow has emerged as a powerful tool to measure and monitor intracellular networks within the native context of the cell. When applied to clinical material, it is possible to discern the subtle phospho-protein differences existent in the subcellular compartments. In this chapter, it is discussed how phosphoflow can be used to characterize the signaling differences as a function of both disease and therapeutic treatment and be used for identification of resistant subpopulations.

Objectives. RA is a common, relapsing autoimmune disease primarily affecting the joints. Fibroblast-like synovial (FLS) cells are thought to be responsible for pannus formation and secretion of factors that recruit leucocytes to affected joints, thereby promoting bone and cartilage destruction. Fibrocytes are multipotent circulating stem cells that may have a role in RA pathogenesis, perhaps as the precursors of the FLS cells, or by regulating FLS cell function. Methods. We utilized multidimensional phospho-specific flow cytometry to characterize the activation status of peripheral blood (PB) fibrocytes derived from human RA patients at different stages of disease and from mice with CIA. Results. Human PB fibrocytes from RA patients exhibited phosporylation activation of the p44/42 and p38 MAP kinases (MAPKs), and STAT3 (signal transducer and activator of transcription) and STAT-5 early in disease, within the first year of diagnosis. Similarly, in murine CIA, an increase in the total number of PB phosphoSTAT5-positive fibrocytes was observed at early time points in disease. Notably, in the affected paws of mice with CIA, we identified an increased number of fibrocytes, in contrast to the paws of control mice. Conclusions. These data suggest that activated fibrocytes may influence the disease process in RA and may serve as surrogate markers for disease in the PB of affected patients.

Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.

Background Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules. Results Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFβ1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNγ producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3α. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology. Conclusion Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications.

Understanding a molecular basis for cellular function is a common goal of biomedicine. The complex and dynamic cellular processes underlying physiological processes become subtly or grossly perturbed in human disease. A primary objective is to demystify this complexity by creating and establishing relevant model systems to study important aspects of human disease. Although significant technological advancements over the last decade in both genomic and proteomic arenas have enabled progress, accessing the complexity of cellular interactions that occur in vivo has been a difficult arena in which to make progress. Moreover, there are extensive challenges in translating research tools to clinical applications. Flow cytometry, over the course of the last 40 years, has revolutionized the field of immunology, in both the basic science and clinical settings, as well as having been instrumental to new and exciting areas of discovery such as stem cell biology. Multiparameter machinery and systems exist now to access the heterogeneity of cellular subsets and enable phenotypic characterization and functional assays to be performed on material from both animal models and humans. This review focuses primarily on the development and application of using activation-state readouts of intracellular activity for phospho-epitopes. We present recent work on how a flow cytometric platform is used to obtain mechanistic insight into cellular processes as well as highlight the clinical applications that our laboratory has explored. Furthermore, this review discusses the challenges faced with processing high-content multidimensional and multivariate data sets. Flow cytometry, as a platform that is well situated in both the research and clinical settings, can contribute to drug discovery as well as having utility for both biomarker and patient-stratification.

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent-based permeabilizations, staining protocol for adherent cells, methodology for whole-blood staining, and multiparameter staining procedures for surface and intracellular antigens.

Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

Intracellular assays of signaling systems have been limited by an inability to correlate functional subsets of cells in complex populations on the basis of active kinase states. Such correlations could be important in distinguishing changes in signaling status that arise in rare cell subsets during functional activation or in disease manifestation. Here we demonstrate the ability to simultaneously detect activated kinase members of the mitogen-activated protein kinases family (p38 MAPK, p44/42 MAPK, JNK/SAPK), members of cell survival pathways (AKT/PKB), and members of T-cell activation pathways (TYK2), among others, in subpopulations of complex cell populations by multiparameter flow-cytometric analysis. We demonstrate the utility of these probes in identifying distinct signaling cascades for (1) both artificial and physiological stimulatory conditions of peripheral blood mononuclear cells (PBMCs), (2) cytokine stimulation in human memory and naive lymphocyte subsets as identified by five differentiation markers, and (3) ordering of kinase activation in potential signaling hierarchies. Polychromatic flow-cytometric active kinase measurements demonstrate that multidimensional analysis of signaling pathways can provide functional signaling pathway assessment on a single-cell level and allow for potential correlation with biological and clinical parameters.

2101 Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have a poor prognosis and current treatments are only palliative. Gene therapy approaches using replication-deficient viral vectors have been investigated previously for GBM with limited efficacy, likely due to poor gene delivery throughout the tumor. Methods: Toca 511 is a novel retroviral replicating vector (RRV) that is engineered to deliver a modified cytosine deaminase prodrug activator gene (CD) selectively to cancer cells. In those cancer cells expressing the CD gene, the antifungal prodrug 5-FC is converted to 5-FU. We are currently evaluating the safety and tolerability of a single dose of Toca 511 administered by direct intratumoral injection to subjects with recurrent high grade glioma (rHGG). After a period of time during which the vector is allowed to spread through the tumor, the patient begins oral 5-FC which is repeated cyclically. Results: Three subjects have been enrolled in each of the first two dose groups. No dose limiting toxicities (DLTs) have been identified in these groups and the treatment was well tolerated. There have been no grade 3 or 4 AEs considered related to either Toca 511 or 5-FC. A further 9 subjects were then enrolled at the 3rd dose level and are being followed. A second study has been initiated in which Toca 511 is injected into the walls of the resection cavity at the time of surgery for rHGG and followed by cyclic treatment with oral 5-FC. Conclusions: These clinical studies are the first to investigate a RRV to deliver genes to human brain tumors and further dose escalation is anticipated.

Unique among replicating viruses being developed as oncolytic agents, replicating murine leukemia virus (RMLV) retroviruses propagate without immediate lysis of host cells and maintain viral persistence through stable integration. Due to its intrinsic inability to infect quiescent post-mitotic cells and susceptibility to innate antiviral defenses that exist in normal cells but are inactivated in many cancers, RMLV vector replication has been shown to be highly tumor-selective. We have previously demonstrated that RMLV vectors are capable of highly efficient replication in tumor cells, and we have found significantly enhanced survival benefit in a variety of cancer models in vivo when RMLV vectors are employed for delivery of prodrug activator genes such as yeast cytosine deaminase (yCD), which mediates the conversion of inactive prodrug 5-fluorocytosine (5FC) to the anti-cancer drug, 5-fluorouracil (5FU). In the present study, we tested a newly developed RMLV vector with modified virus backbone sequences and a codon-optimized yCD gene (Toca511). First, we examined in vitro cytotoxicity using human (U87) and murine (TU-2449) glioma cell lines by MTS assay. The cell viability of Toca511-transduced cells was reduced by >80% after 5 or 8 days of exposure to the prodrug, respectively. Second, we examined the therapeutic efficiency of this newly developed vector in subcutaneous U87 human glioblastoma models and TU2449 syngeneic mouse models. Toca511-mediated prodrug activator therapy strongly inhibited tumor growth in both subcutaneous models. Third, therapeutic efficacy was evaluated, and the minimum effective doses of 5FC prodrug and Toca511 vector determined in an intracranial U87 xenograft model. Systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with Toca511 but not in control mice, associated with significant survival benefit even after injecting only 1×1e3 total vector units (p=0.0015). These data suggest that we have been successful in developing an improved retrovirus vector that shows strong therapeutic efficacy when used for prodrug activator gene therapy in both subcutaneous and intracranial xenograft models of human and/or murine glioma. This vector has now entered Phase I/II clinical trials for glioblastoma. Now, with the use of RMLV vectors such as Toca511, the original promise of retrovirus-mediated gene therapy strategies for cancer may finally be fulfilled. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2011-5386

Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have poor prognosis and current treatments remain only palliative. Previous gene therapy approaches using replication-deficient viral vectors have demonstrated limited efficacy likely due to poor gene delivery to the tumor. Methods: We used a novel replication-competent retroviral vector (Toca 511) designed and engineered to efficiently deliver a modified cytosine deaminase (CD) prodrug activating gene to glioma cells. Previous studies showed that such a replicating vector can preferentially infect intracranial human xenografts in nude mice (Tai et al. Mol Ther. 2005;12:842) and lead to increased survival in treatment groups compared to controls. In preparation for clinical trials we have improved the CD gene activity and further stabilized the vector structure to create Toca 511. We investigated the functionality of Toca 511 using stereotactic, intratumoral delivery in a syngeneic, orthotopic glioma model in immune-competent B6C3F1 mice. Freshly excised tumors were analyzed by quantitative PCR, Western, and HPLC. Results: Brain tumors isolated from mice treated with Toca 511 and fluorocytosine (5FC) had observable expression of CD protein and vector copy numbers per genome that demonstrate efficient tumor infection. Extensive tumor specific viral spread with almost no spread of vector from the site of injection was observed in the first 24 days. Highly efficient intratumoral conversion of the 5FC prodrug into the anticancer drug 5-fluorouracil (5FU) was demonstrated after intraperitoneal or oral dosing with 5FC. Studies with Toca 511 at high and mid dose levels in combination with 5FC resulted in prolonged survival compared to controls (> 5 months, p Conclusions: Toca 511 efficiently delivers a functional CD gene for expression in glioma cells in a syngeneic, orthotopic model in immune-competent mice. Toca 511 increases survival through the delivery of CD gene and subsequent efficient conversion of 5FC into 5FU within the brain tumor with no safety related findings. A phase 1 ascending dose trial investigating the safety and tolerability of Toca 511 in combination with 5FC in patients with recurrent GBM is about to initiate. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B33.

e13541 Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM) have poor prognosis and few palliative options. Gene therapy approaches using rep...

Background The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality. Methods and Findings PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed. Conclusions Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules. Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFβ1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNγ producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3α. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology. Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications.

Using a muscle cell differentiation screen, we have identified myoseverin from a 2,6,9-trisubsituted purine library as a purine-based microtubule binding molecule [1]. Structure-activity relation studies of myoseverin identify positions N2 and N6 to be critical for inhibiting muscle differentiation. Inhibition of microtubule polymerization induced the reversion of terminally differentiated myotubes to mononucleated cells that were responsive to both growth and differentiation conditions, without any observable cytotoxicity. Comparison of myoseverin derivatives to taxol, vinblastine, nocodazole, and colchicine identify myoseverin's effect as being selectively reversible in addition to lacking the cytotoxic effects of these non-purine-based microtubule-disrupting molecules. Myoseverin, as a purine-based microtubule inhibitor, reverted terminal muscle-differentiated cells to a state that was responsive to environmental cues. These results suggest that myoseverin may have applications in muscle regeneration and stem cell differentiation.