Matthew R. Clutter

Coordinate Analysis of Murine Immune Cell Surface Markers and Intracellular Phosphoproteins by Flow Cytometry

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, ∼80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.

Single Cell Mass Cytometry Adapted to Measurements of the Cell Cycle

Mass cytometry is a recently introduced technology that utilizes transition element isotope‐tagged antibodies for protein detection on a single‐cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5‐iodo‐2‐deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.

Fluorescent Cell Barcoding for Multiplex Flow Cytometry

Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10‐ to 100‐fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed. Curr. Protoc. Cytom. 55:6.31.1‐6.31.15.

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry

Intracellular flow cytometry permits quantitation of diverse molecular targets at the single‐cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10‐fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high‐throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal‐to‐noise ratios.

Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide–Gated Channels

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat–containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel’s function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression.

Phospho-specific flow cytometry in drug discovery.

Here we present phospho-specific flow cytometry as a new tool for drug discovery with applications throughout the drug development pipeline, from target identification to library screening, disease model assessment and clinical screening and diagnostics. The single cell, multiparameter nature of flow cytometry generates high-content datasets, and current improvements in the technology are rapidly increasing its high-throughput capacity, making it a valuable platform in modern drug discovery.:

Novel Hematopoietic Progenitor Populations Revealed by Direct Assessment of GATA1 Protein Expression and cMPL Signaling Events

Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self‐renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte‐lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone‐marrow cKit(+) Lineage (−) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein‐level expression of GATA1, a critical megakaryocyte/erythrocyte‐promoting transcription factor. GATA1‐high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed “FSE cells.” GATA1‐low progenitors were identified as KLS Flk2(+), CD150(−), and cMPL(−), and were termed “Flk(+) CD150(−) cells.” FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony‐forming unit–spleen activity compared with Flk(+) CD150(−) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal‐transduction cascades was observed by intracellular flow cytometry in long‐term HSCs and FSE cells, but not in Flk(+) CD150(−) cells in response to stimulation with thrombopoietin, an important megakaryocyte‐promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis. STEM CELLS 2011;29:1774–1782

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.

Abstract 5394: A high-throughput screening platform for the identification of small molecule inhibitors of MAP2K4

Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed two in vitro MAP2K4 kinase assays to evaluate kinase inhibitory function, one a western-blot based assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, and the other an Alphascreen based activity assay. In this manner, we validated the performance of our initial FTS screen. Finally, by coupling our structure-activity relationship data to MAP2K49s crystal structure, we constructed a model for inhibitor binding. It predicted binding of our identified inhibitors to MAP2K49s ATP pocket. We next applied this approach in a high-throughput fashion to over 2200 chemically diverse compounds and identified new inhibitors of MAP2K4. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. Citation Format: Sankar Narayan Krishna, Chi-Hao Luan, Matthew R. Clutter, Rama K. Mishra, Karl A. Scheidt, Wayne F. Anderson, Raymond C. Bergan. A high-throughput screening platform for the identification of small molecule inhibitors of MAP2K4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2014-5394