Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcεRI Aggregation and Structural Changes.

Immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptors are critical components of the innate and adaptive immune systems. FcεRI mediates the allergic response via crosslinking of IgE-bound receptors by multivalent antigens. Yet, the underlying molecular mechanisms that govern the response of FcεRI to specific antigens remain poorly understood. We compared responses induced by two antigens with distinct geometries, high valency DNP-BSA and trivalent DF3, and found unique secretion and receptor phosphorylation profiles that are due to differential recruitment of Lyn and SHIP1. To understand how these two antigens can cause such markedly different outcomes, we used direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging combined with Bayesian Grouping of Localizations (BaGoL) analysis to compare the nanoscale characteristics of FcεRI aggregates. DF3 aggregates were found to be smaller and more densely packed than DNP-BSA aggregates. Using lifetime-based Förster resonance energy transfer (FRET) measurements, we discovered that FcεRI subunits undergo structural rearrangements upon crosslinking with either antigen, and in response to interaction with monovalent antigen presented on a supported lipid bilayer. The extent of conformational change is positively correlated with signaling efficiency. Finally, we provide evidence for forces in optimizing FcεRI signaling, such that immobilizing DF3 on a rigid surface promoted degranulation while increasing DNP-BSA flexibility lowered degranulation. These results provide a link between the physical attributes of allergens, including size, shape, valency, and flexibility, and FcεRI signaling strength. Thus, the antigen modulates mast cell outcomes by creating unique aggregate geometries that tune FcεRI conformation, phosphorylation and signaling partner recruitment.

Spatial Stochastic Model of the Pre-B Cell Receptor.

Survival and proliferation of immature B lymphocytes requires expression and tonic signaling of the pre-B cell receptor (pre-BCR). This low level, ligand-independent signaling is likely achieved through frequent, but short-lived, homo interactions. Tonic signaling is also central in the pathology of precursor B acute lymphoblastic leukemia (B-ALL). In order to understand how repeated, transient events can lead to sustained signaling and to assess the impact of receptor accumulation induced by the membrane landscape, we developed a spatial stochastic model of receptor aggregation and downstream signaling events. Our rule- and agent-based model builds on previous mature BCR signaling models and incorporates novel parameters derived from single particle tracking of pre-BCR on surfaces of two different B-ALL cell lines, 697 and Nalm6. Live cell tracking of receptors on the two cell lines revealed characteristic differences in their dimer dissociation rates and diffusion coefficients. We report here that these differences affect pre-BCR aggregation and consequent signal initiation events. Receptors on Nalm6 cells, which have a lower off-rate and lower diffusion coefficient, more frequently form higher order oligomers than pre-BCR on 697 cells, resulting in higher levels of downstream phosphorylation in the Nalm6 cell line.

Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1.

The high-affinity immunoglobulin E (IgE) receptor, FcεRI, is the primary immune receptor found on mast cells and basophils. Signal initiation is classically attributed to phosphorylation of FcεRI ß- and γ-subunits by the Src family kinase (SFK) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. Here, we investigate the mechanistic contributions of Lyn, Syk, and SHIP1 to the formation of the FcεRI signalosome. Using Lyn-deficient RBL-2H3 mast cells, we found that another SFK can weakly monophosphorylate the γ-subunit, yet Syk still binds the incompletely phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). Once recruited, Syk further enhances γ-phosphorylation to propagate signaling. In contrast, the loss of SHIP1 recruitment indicates that Lyn is required for phosphorylation of the ß-subunit. We demonstrate two noncanonical Syk binding modes, trans γ-bridging and direct ß-binding, that can support signaling when SHIP1 is absent. Using single particle tracking, we reveal a novel role of SHIP1 in regulating Syk activity, where the presence of SHIP1 in the signaling complex acts to increase the Syk:receptor off-rate. These data suggest that the composition and dynamics of the signalosome modulate immunoreceptor signaling activities.

VpreB surrogate light chain expression in B-lineage ALL: a report from the Children's Oncology Group.

Immunotherapies directed against B-cell surface markers have been a common developmental strategy to treat B-cell malignancies. The immunoglobulin heavy chain surrogate light chain (SLC), comprising the VpreB1 (CD179a) and Lamda5 (CD179b) subunits, is expressed on pro- and pre-B cells, where it governs pre-B-cell receptor (BCR)-mediated autonomous survival signaling. We hypothesized that the pre-BCR might merit the development of targeted immunotherapies to decouple "autonomous" signaling in B-lineage acute lymphoblastic leukemia (B-ALL). We used the Children's Oncology Group (COG) minimal residual disease (MRD) flow panel to assess pre-BCR expression in 36 primary patient samples accrued to COG standard- and high-risk B-ALL studies through AALL03B1. We also assessed CD179a expression in 16 cases with day 29 end-induction samples, preselected to have ≥1% MRD. All analyses were performed on a 6-color Becton-Dickinson flow cytometer in a Clinical Laboratory Improvement Amendment/College of American Pathologist-certified laboratory. Among 36 cases tested, 32 cases were at the pre-B and 4 cases were at the pro-B stages of developmental arrest. One or both monoclonal antibodies (mAbs) showed that CD179a was present in ≥20% of the B-lymphoblast population. All cases expressed CD179a in the end-induction B-lymphoblast population. The CD179a component of the SLC is commonly expressed in B-ALL, regardless of genotype, stage of developmental arrest, or National Cancer Institute risk status.

EGFR transactivates RON to drive oncogenic crosstalk.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation - arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Differential epidermal growth factor receptor (EGFR) phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single-molecule pull-down technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predominantly phosphorylated at multiple sites, yet phosphorylation at specific tyrosines is variable and only a subset of receptors share phosphorylation at the same site, even with saturating ligand concentrations. We found distinct populations of receptors as soon as 1 min after ligand stimulation, indicating early diversification of function. To understand this heterogeneity, we developed a mathematical model. The model predicted that variations in phosphorylation are dependent on the abundances of signaling partners, while phosphorylation levels are dependent on dimer lifetimes. The predictions were confirmed in studies of cell lines with different expression levels of signaling partners, and in experiments comparing low- and high-affinity ligands and oncogenic EGFR mutants. These results reveal how ligand-regulated receptor dimerization dynamics and adaptor protein concentrations play critical roles in EGFR signaling.

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ.

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αßγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk-FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk's bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.

Leukemia-derived exosomes and cytokines pave the way for entry into the brain.

Infiltration of acute lymphoblastic leukemia (ALL) blasts into the CNS remains as a major clinical problem, with high risk for chemotherapy-resistant relapse and treatment-related morbidity. Despite the common inclusion of CNS prophylaxis treatments in therapy regimens, there are significant gaps in understanding the mechanisms that mediate leukemia cell entry into the CNS as well as roles for resident cells in the brain. In this study, we employ a xenograft model of human B cell precursor (BCP)-ALL in immunocompromised mice. This model system recapitulates key pathological characteristics of leptomeningeal involvement seen in patients and provides insights into rare cases that involve parenchymal invasion. We examine the infiltration of engrafted leukemia blasts into brains of recipient mice and provide evidence that the interaction between blasts and brain resident cells causes aberrant activation of host cells in the brain microenvironment. BCP-ALL blasts also release multiple cytokines and exosomes containing IL-15 that bind and are internalized by astrocytes and brain vessel endothelial cells. Leukemic invasion is linked to production of VEGF-AA by astrocytes and disruption of the blood-brain-barrier (BBB) integrity. Knockdown of either IL-15 or IL-15Rα in the NALM6 cell line decreases CNS infiltration in engrafted mice. These results provide important insights into the multiple mechanisms by which lymphoblasts modulate the brain microenvironment to breach the BBB for metastatic invasion.

Timescale Separation of Positive and Negative Signaling Creates History-Dependent Responses to IgE Receptor Stimulation.

The high-affinity receptor for IgE expressed on the surface of mast cells and basophils interacts with antigens, via bound IgE antibody, and triggers secretion of inflammatory mediators that contribute to allergic reactions. To understand how past inputs (memory) influence future inflammatory responses in mast cells, a microfluidic device was used to precisely control exposure of cells to alternating stimulatory and non-stimulatory inputs. We determined that the response to subsequent stimulation depends on the interval of signaling quiescence. For shorter intervals of signaling quiescence, the second response is blunted relative to the first response, whereas longer intervals of quiescence induce an enhanced second response. Through an iterative process of computational modeling and experimental tests, we found that these memory-like phenomena arise from a confluence of rapid, short-lived positive signals driven by the protein tyrosine kinase Syk; slow, long-lived negative signals driven by the lipid phosphatase Ship1; and slower degradation of Ship1 co-factors. This work advances our understanding of mast cell signaling and represents a generalizable approach for investigating the dynamics of signaling systems.

Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens.

Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher ß2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.

Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics.

Cross-linking of immunoglobulin E-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.

Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia.

Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ∼2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcεRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Science Signaling Podcast for 29 November 2016: Pre-B cell receptor signaling in leukemia.

This Podcast features an interview with Bridget Wilson, author of a Research Article that appears in the 29 November 2016 issue of Science Signaling, about pre-B cell receptor (pre-BCR) signaling in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Signaling through the pre-BCR, an immature form of the BCR, promotes the survival of B cell progenitors and has been implicated in the pathology of BCP-ALL. Erasmus et al found that pre-BCRs formed transient homomeric complexes that correlated with pro-survival signaling. Preventing homotypic interactions between pre-BCRs sensitized B cells to chemotherapeutic agents, suggesting that interfering with such interactions may improve the efficacy of existing chemotherapies for BCP-ALL.Listen to Podcast.

Dynamic pre-BCR homodimers fine-tune autonomous survival signals in B cell precursor acute lymphoblastic leukemia.

The pre-B cell receptor (pre-BCR) is an immature form of the BCR critical for early B lymphocyte development. It is composed of the membrane-bound immunoglobulin (Ig) heavy chain, surrogate light chain components, and the signaling subunits Igα and Igß. We developed monovalent quantum dot (QD)-labeled probes specific for Igß to study the behavior of pre-BCRs engaged in autonomous, ligand-independent signaling in live B cells. Single-particle tracking revealed that QD-labeled pre-BCRs engaged in transient, but frequent, homotypic interactions. Receptor motion was correlated at short separation distances, consistent with the formation of dimers and higher-order oligomers. Repeated encounters between diffusing pre-BCRs appeared to reflect transient co-confinement in plasma membrane domains. In human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, we showed that frequent, short-lived, homotypic pre-BCR interactions stimulated survival signals, including expression of BCL6, which encodes a transcriptional repressor. These survival signals were blocked by inhibitory monovalent antigen-binding antibody fragments (Fabs) specific for the surrogate light chain components of the pre-BCR or by inhibitors of the tyrosine kinases Lyn and Syk. For comparison, we evaluated pre-BCR aggregation mediated by dimeric galectin-1, which has binding sites for carbohydrate and for the surrogate light chain λ5 component. Galectin-1 binding resulted in the formation of large, highly immobile pre-BCR aggregates, which was partially relieved by the addition of lactose to prevent the cross-linking of galectin-BCR complexes to other glycosylated membrane components. Analysis of the pre-BCR and its signaling partners suggested that they could be potential targets for combination therapy in BCP-ALL.

Effect of Spatial Inhomogeneities on the Membrane Surface on Receptor Dimerization and Signal Initiation.

Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as "clusters" by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones ("domains") for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.

Spatial Modeling of Drug Delivery Routes for Treatment of Disseminated Ovarian Cancer.

In ovarian cancer, metastasis is typically confined to the peritoneum. Surgical removal of the primary tumor and macroscopic secondary tumors is a common practice, but more effective strategies are needed to target microscopic spheroids persisting in the peritoneal fluid after debulking surgery. To treat this residual disease, therapeutic agents can be administered by either intravenous or intraperitoneal infusion. Here, we describe the use of a cellular Potts model to compare tumor penetration of two classes of drugs (cisplatin and pertuzumab) when delivered by these two alternative routes. The model considers the primary route when the drug is administered either intravenously or intraperitoneally, as well as the subsequent exchange into the other delivery volume as a secondary route. By accounting for these dynamics, the model revealed that intraperitoneal infusion is the markedly superior route for delivery of both small-molecule and antibody therapies into microscopic, avascular tumors typical of patients with ascites. Small tumors attached to peritoneal organs, with vascularity ranging from 2% to 10%, also show enhanced drug delivery via the intraperitoneal route, even though tumor vessels can act as sinks during the dissemination of small molecules. Furthermore, we assessed the ability of the antibody to enter the tumor by in silico and in vivo methods and suggest that optimization of antibody delivery is an important criterion underlying the efficacy of these and other biologics. The use of both delivery routes may provide the best total coverage of tumors, depending on their size and vascularity.