Efficient Signaling Research Articles

Cell-to-Cell Adhesion and Neurogenesis in Human Cortical Development: A Study Comparing 2D Monolayers with 3D Organoid Cultures.

SummaryOrganoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process.

Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages.

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

Optimization of adiabatic pulses for pulsed arterial spin labeling at 7 tesla: Comparison with pseudo-continuous arterial spin labeling.

To optimize and evaluate adiabatic pulses for pulsed arterial spin labeling at ultrahigh field 7 tesla. Four common adiabatic inversion pulses, including hyperbolic secant, wideband uniform rate smooth truncation, frequency offset corrected inversion, and time-resampled frequency offset corrected inversion pulses, were optimized based on a custom-defined loss function that included labeling efficiency and inversion band uniformity. The optimized pulses were implemented in flow-sensitive alternating inversion recovery sequences and tested on phantom and 11 healthy volunteers with 2 constraints: 1) specific absorption rate normalized; and 2) equal peak RF amplitude, respectively. A pseudo-continuous arterial spin labeling sequence was implemented for comparison. Quantitative metrics such as perfusion and relative labeling efficiency versus residual tissue signal were calculated. Among the 4 pulses, the wideband uniform rate smooth truncation pulse yielded the lowest loss in simulation and achieved a good balance between labeling efficiency and residual tissue signal from both phantom and in vivo experiments. Wideband uniform rate smooth truncation-pulsed arterial spin labeling showed significantly higher relative labeling efficiency compared to the other sequences (P < .01), whereas the perfusion signal was increased by 40% when the highest amplitude was used. The 4 pulsed arterial spin labeling sequences yielded comparable perfusion signals compared to pseudo-continuous arterial spin labeling but with less than half the specific absorption rate. Optimized wideband uniform rate smooth truncation pulse with the highest amplitude allowed was recommended for 7 tesla pulsed arterial spin labeling.

Efficient IL-2R signaling differentially affects the stability, function, and composition of the regulatory T-cell pool

Signaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

An ultrasonic signal acquisition method of ferromagnetic materials based on similar matrix blind source separation

In order to overcome the problems of low signal-to-noise ratio, low efficiency and poor signal richness in ultrasonic signal acquisition of ferromagnetic materials, a new ultrasonic signal acquisit...

Hydrogenated Graphene Improves Neuronal Network Maturation and Excitatory Transmission.

Graphene is regarded as a viable bio-interface for neuroscience due to its biocompatibility and electrical conductivity, which would contribute to efficient neuronal network signaling. Here, monolayer graphene grown via chemical vapor deposition is treated with remote hydrogen plasma to demonstrate that hydrogenated graphene (HGr) fosters improved cell-to-cell communication with respect to pristine graphene in primary cortical neurons. When transferred to polyethylene terephthalate, HGr exhibits higher wettability than graphene (water contact angle of 83.7° vs 40.7°), while preserving electrical conductivity (≈3 kΩ □-1 ). A rich and mature network is observed to develop onto HGr. The intrinsic excitability and firing properties of neurons plated onto HGr appears unaltered, while the basic passive and active membrane properties are fully preserved. The formation of excitatory synaptic connections increases in HGr with respect to pristine graphene, leading to a doubled miniature excitatory postsynaptic current frequency. This study supports the use of hydrogenation for tailoring graphene into an improved neuronal interface, indicating that wettability, more than electrical conductivity, is the key parameter to be controlled. The use of HGr can bring about a deeper understanding of neuronal behavior on artificial bio-interfaces and provide new insight for graphene-based biomedical applications.

Bilirubin oxidase labeling triggers an efficient signaling mechanism of oxygen reduction reaction for smart photocathodic immunoassay

Abstract Photocathodic immunoassays show brilliant prospect for practical detection in biological matrix owning to the inherent strength against signal interference from potential reductive molecules. However, their exploration with advanced and efficient signaling mechanism is still barren. We herein report an enhanced photocathodic immunoassay via utilizing bilirubin oxidase (BOD) labeling to trigger an illustrious signaling mechanism of oxygen reduction reaction (ORR). The target model of prostate-specific antigen (PSA, Ag) was employed to exemplify this platform. Typically, the fabricated NiO/CdSe/Au hybrid photocathode served as the substrate to anchor capture antibody (Ab1). In order to increase host loading, abundant BOD molecules were labeled on signal antibody (Ab2) with the assistance of carbon nanotube (CNT), forming the Ab2-BOD/CNT conjugate. When specific immunological recognition occurred, the BOD labeling could efficiently catalyze the reduction reaction of electron acceptor of dissolved oxygen, causing dramatically promoted cathodic photocurrent signal. By introducing this ingenious signaling mechanism triggered by BOD, enhanced photocathodic immunoassay with high sensitivity and accuracy in biological matrix was developed. This work pioneers an effective but general strategy for the design of advanced photocathodic bioassays for different targets of interest.

Electronic Bottleneck Suppression in Next‐Generation Networks with Integrated Photonic Digital‐to‐Analog Converters

Digital‐to‐analog converters (DAC) are indispensable functional units in signal processing instrumentation and wide‐band telecommunication links for both civil and military applications. As photonic systems are capable of high data throughput and low latency, an increasingly found system limitation stems from the required domain crossing such as digital to analog and electronic to optical. A photonic DAC implementation, in contrast, enables a seamless signal conversion with respect to both energy efficiency and short signal delay, often requiring bulky discrete optical components and electric–optic transformation, hence introducing inefficiencies. Herein, a novel coherent parallel photonic DAC concept along with a 4‐bit experimental prototype capable of performing this DAC without optic–electric–optic domain crossing is introduced. This new paradigm guarantees a linear intensity weighting among bits when operating at high sampling rates (50 GHz), featuring an exceptional sampling efficiency (&gt; 100 GS ) and small footprint (≈1 mm2) in an 8‐bit implementation. Importantly, this photonic DAC enables seamless interfaces of next‐generation data processing hardware with high relevance in data centers, task‐specific compute accelerators such as neuromorphic engines, and network edge processing applications.

Properties of primary cilia in melanin-concentrating hormone receptor 1-bearing hippocampal neurons in vivo and in vitro

The primary cilium is a solitary organelle that organizes a sensitive signaling hub in a highly ordered microenvironment. Cilia are plastic structures, changing their length in response to bioactive substances, and ciliary length may be regulated to ensure efficient signaling capacity. Mammalian brain neurons possess primary cilia that are enriched in a set of G protein-coupled receptors (GPCRs), including the feeding-related melanin-concentrating hormone (MCH) receptor 1 (MCHR1). We previously demonstrated a novel biological phenomenon, ciliary MCHR1-mediated cilia length shortening through Gi/o and Akt signaling, using a simple cell culture model of human retinal pigmented epithelial RPE1 cells exogenously expressing MCHR1. In the present study, we characterized the properties of endogenous MCHR1-expressing primary cilia in hippocampal neurons in rodents. Using cultured dissociated rat hippocampal neurons in vitro, we showed that MCH triggered cilia length reduction involved in MCHR1-Gi/o and -Akt signaling. In rat hippocampal slice cultures with preservation of the cytoarchitecture and cell populations, ciliary MCHR1 was abundantly located in the CA1 and CA3 regions, but not in the dentate gyrus. Notably, treatment of slice cultures with MCH induced Gi/o- and Akt-dependent cilia shortening in the CA1 region without influencing cilia length in the CA3 region. Regarding the in vivo mouse brain, we observed higher levels of ciliary MCHR1 in the CA1 and CA3 regions as well as in slice cultures. In the starved state mice, a marked increase in MCH mRNA expression was detected in the lateral hypothalamus. Furthermore, MCHR1-positive cilia length in the hippocampal CA1 region was significantly shortened in fasted mice compared with fed mice. The present findings focused on the hippocampus provide a potential approach to investigate how MCHR1-driven cilia shortening regulates neuronal activity and physiological function toward feeding and memory tasks.

Rotated DFT‐s‐OFDM for transmitting PAPR‐minimised BPSK symbols

The third generation partnership project has recommended π/2-binary phase-shift keying (BPSK) in discrete-Fourier-transform-spread orthogonal frequency division multiplexing (DFT-s-OFDM) for low peak-to-average power ratio (PAPR) uplink transmission. Recently, the rotation angle of 7π/6 for rotated BPSK has been proposed by J. Kim et al. to achieve optimal PAPR performance. However, the authors discovered that this angle might negate the contribution to PAPR reduction in most number of allocated subcarriers owing to the discrete-Fourier-transform (DFT) boundary-matching problem. To solve this issue, they propose a novel scheme to fine-tune the rotation angle to approximately 7π/6 in a manner compatible with the long-term evolution/new radio specification, while ensuring that the data phase matches the periodicity. Simulations show that the proposed scheme can stably improve the PAPR by ∼0.2 dB compared to π/2-BPSK for any DFT spacing, thereby benefiting user equipment by providing better power efficiency and wider signal coverage without additional computational complexity.

Quality Signaling through Crowdfunding Pricing

Problem definition: This paper studies an entrepreneur’s pricing strategy in a reward-based crowdfund-ing campaign under asymmetric product quality information. We propose two signaling mechanisms and investigate the relative performance of these mechanisms under different market conditions. Academic/practical relevance: This problem is relevant to practice, as asymmetric quality information is a major concern in reward-based crowdfunding; high-quality entrepreneurs are looking for credible mechanisms to signal their quality to customers. Methodology: We develop a stylized game-theoretic signaling model with both funding and regular selling periods that captures asymmetric quality information between an entrepreneur and customers. Results: For a high-quality entrepreneur who lacks a strong fanbase, we propose a new theory on quality signaling. In many cases, a low funding price might be the only signaling tool needed (i.e., one-price signaling); a high-quality entrepreneur should offer a good deal to customers in the funding period to increase the chance of a successful campaign to reach the regular selling period. However, such an entrepreneur can increase his funding price, if he commits to his future price in the regular selling period (i.e., two-price signaling). We characterize financing target levels that allow entrepreneurs to signal quality through one- or two-price mechanisms. In particular, we show that two-price signaling is plausible for a broad range of financing target levels. When both one- and two-price signaling are plausible (i.e., when the financing target level is not large), the gap in potential high- and low-quality levels and the accuracy of the market signal on customers valuation of the product in the regular selling period determine the more efficient signaling mechanism. Managerial implications: Entrepreneurs should be mindful of pricing in both funding and regular selling periods, as it plays an essential role in signaling quality information. Our findings suggest practical quality signaling in crowdfunding. We demonstrate a trade-off between signaling costs and the value of learning of consumer preferences in reward-based crowdfunding. We demonstrate how price commitment significantly increases the possibility of quality signaling for high-quality entrepreneurs without serious funding price compromises, thus making crowdfunding platforms attractive to high-quality entrepreneurs.

NADPH oxidases and the evolution of plant salinity tolerance.

Soil salinization is a major threat to global food security and the biodiversity of natural ecosystems. To adapt to salt stress, plants rely on ROS-mediated signalling networks that operate upstream of a broad array of physiological and genetic processes. A key player in ROS signalling is NADPH oxidase, a plasma-membrane-bound enzyme encoded by RBOH genes. In this study, we have conducted a comprehensive bioinformatic analysis of over 50 halophytic and glycophytic species to link the difference in the kinetics of ROS signalling between contrasting species with the abundance and/or structure of NADPH oxidases. The RBOH proteins were predicted in all the tested plant lineages except some algae species from the Rhodophyta, Chlorophyta and Streptophyta. Within the glycophytic group, the number of RBOH copies correlated negatively with salinity stress tolerance, suggesting that a reduction in the number of RBOH isoforms may be potentially related to the evolution of plant salinity tolerance. While halophytes did not develop unique protein families during evolution, they evolved additional phosphorylation target sites at the N-termini of NADPH oxidases, potentially modulating enzyme activity and allowing more control over their function, resulting in more efficient ROS signalling and adaptation to saline conditions.

Internal quantum efficiency and time signals from intensity-modulated photocurrent spectra of perovskite solar cells

Intensity Modulated Photocurrent Spectroscopy (IMPS) is a small-perturbation optoelectronic technique that measures the quantum efficiency of a photoelectrochemical device as a function of optical excitation frequency. Metal Halide Perovskites (MHPs) are mixed electronic–ionic semiconductors with an extraordinary complex optoelectronic behavior and a record efficiency surpassing 25%. In this paper, we propose a simplified procedure to analyze IMPS data in MHPs based on the analysis of the internal quantum efficiency and the time signals featuring in the frequency spectra. In this procedure, we look at the change of each signal when optical excitation wavelength, photon flux, and temperature are varied for an archetypical methyl ammonium lead iodide solar cell. We use drift-diffusion modeling and comparison with relatively simpler dye-sensitized solar cells (DSC) with viscous and non-viscous electrolytes to help us to understand the origin of the three signals appearing in MHP cells and the measurement of the internal quantum efficiency.

Investor behavior under the Covid-19 pandemic: the case of Indonesia

This study begins with the assumption that the existence of abnormal circumstances will force investors to take measures to protect their investments in the capital market. Recently, the stock index in the Indonesian market has been declining and continued to fall until the end of April 2020 due to the impact of the Covid-19 pandemic. In terms of efficient market theory, prospect theory and signaling theory, this study aims to analyze the relationship between risk and return in the Indonesian capital market during the Covid-19 pandemic as a manifestation of investor behavior. To test hypotheses, the correlation test, the independent sample t-test and the Cohen test for 629 public firms with 52,836 observable data are used. The findings show that for financial sectors and non-financial sectors, the fourth period differs from previous periods when the relationship between systematic risk and stock returns is positive, although only non-financial sectors have a significant effect. The results show that efficient market theory, prospect theory and signaling theory are consistent with the phenomena around the Covid-19 pandemic in Indonesia. In addition, Cohen’s test results suggest that government policies in the face of the pandemic are successful in stimulating the market.

Dual Epitope Targeting and Enhanced Hexamerization by DR5 Antibodies as a Novel Approach to Induce Potent Antitumor Activity Through DR5 Agonism.

Higher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an equimolar mixture of two DR5-specific IgG1 antibodies with an Fc-domain mutation that augments antibody hexamerization after cell surface target binding. The two antibodies do not compete for binding to DR5 as demonstrated using binding competition studies, and binding to distinct epitopes in the DR5 extracellular domain was confirmed by crystallography. The unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent DR5 agonist activity by inducing efficient DR5 outside-in signaling and caspase-mediated cell death. Preclinical studies in vitro and in vivo demonstrated that maximal DR5 agonist activity could be achieved independent of Fc gamma receptor-mediated antibody crosslinking. Most optimal agonism was observed in the presence of complement complex C1, although without inducing complement-dependent cytotoxicity. It is hypothesized that C1 may stabilize IgG hexamers that are formed after binding of HexaBody-DR5/DR5 to DR5 on the plasma membrane, thereby strengthening DR5 clustering and subsequent outside-in signaling. We observed potent antitumor activity in vitro and in vivo in large panels of patient-derived xenograft models representing various solid cancers. The results of our preclinical studies provided the basis for an ongoing clinical trial exploring the activity of HexaBody-DR5/DR5 (GEN1029) in patients with malignant solid tumors.

Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources.

The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen.IMPORTANCEAspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

Mouse models for V103I and I251L gain of function variants of the human MC4R display decreased adiposity but are not protected against a hypercaloric diet

ObjectiveThe melanocortin 4 receptor (MC4R) is a G protein-coupled receptor that plays major roles in the central control of energy balance. Loss-of-function mutations of MC4R constitute the most common monogenic cause of early-onset extreme obesity in humans, whereas gain-of-function mutations appear to be protective. In particular, two relatively frequent alleles carrying the non-synonymous coding mutations V103I or I251L are associated with lower risks of obesity and type-2 diabetes. Although V103I and I251L MC4Rs showed more efficient signalling in transfected cells, their specific effects in live animals remain unexplored. Here, we investigated whether the introduction of V103I and I251L mutations into the mouse MC4R leads to a lean phenotype and provides protection against an obesogenic diet. MethodsUsing CRISPR/Cas9, we generated two novel strains of mice carrying single-nucleotide mutations into the mouse Mc4r which are identical to those present in V103I and I251L MCR4 human alleles, and studied their phenotypic outcomes in mice fed with normal chow or a high-fat diet. In particular, we measured body weight progression, food intake and adiposity. In addition, we analysed glucose homeostasis through glucose and insulin tolerance tests. ResultsWe found that homozygous V103I females displayed shorter longitudinal length and decreased abdominal white fat, whereas homozygous I251L females were also shorter and leaner due to decreased weight in all white fat pads examined. Homozygous Mc4rV103I/V103I and Mc4rI251L/I251L mice of both sexes showed improved glucose homeostasis when challenged in a glucose tolerance test, whereas Mc4rI251L/I251L females showed improved responses to insulin. Despite being leaner and metabolically more efficient, V103I and I251L mutants fed with a hypercaloric diet increased their fasting glucose levels and adiposity similar to their wild-type littermates. ConclusionsOur results demonstrate that mice carrying V103I and I251L MC4R mutations displayed gain-of-function phenotypes that were more evident in females. However, hypermorphic MC4R mutants were as susceptible as their control littermates to the obesogenic and diabetogenic effects elicited by a long-term hypercaloric diet, highlighting the importance of healthy feeding habits even under favourable genetic conditions.

Market Implications of Short-Term Reserve Deliverability Enhancement

Midcontinent Independent System Operator (MISO) plans to introduce 30-minute short-term reserve (STR) to address 30 minutes system flexibility needs. Transmission bottlenecks in the power system may inhibit the deliverability of STR. Insufficient deliverable reserve may require additional operator manual adjustments, which may be uneconomic and distort the market price signal. To improve reserve deliverability, event-based zonal STR clearing model and nodal STR clearing model are proposed to distribute reserve across the grid. This paper compares reserve deliverability and market clearing prices between the zonal STR model and the nodal STR model. A new design on penalty function is proposed in this paper to reflect the true value of security constraints for reserve deployment with the consideration of multiple events. Analysis of MISO test cases shows that the zonal approach is imprecise and may not guarantee the actual post-event flows within the physical limits. Nodal STR model can improve the reserve deliverability, market efficiency, and price signal.

Abstract 1426: A PI3K/AKT scaffolding protein, IQ motif-containing GTPase associating protein 1 (IQGAP1), promotes head and neck carcinogenesis

Abstract Purpose: Head and neck cancer (HNC) is the sixth most common cancer worldwide with a 5-year survival rate of less than 50%. The PI3K/AKT/mTOR signaling pathway is frequently implicated in HNC. Recently, IQ motif-containing GTPase activating protein 1 (IQGAP1) was discovered to scaffold the PI3K/AKT signaling pathway. IQGAP1 gene expression is increased in HNC, raising the hypothesis that IQGAP1 contributes to HNC. Experimental Design: We performed a combination of in vitro studies using human cancer cell lines treated with a cell permeable peptide that interferes with IQGAP1's ability to bind to PI3K, and in vivo studies utilizing mice genetically knocked out for the Iqgap1 (Iqgap1-/-). In vivo EGF-stimulation assays were used to evaluate PI3K signaling. To study the role of IQGAP1 in HNC, we used a well-validated mouse model that drives HNC via a synthetic oral carcinogen, 4-nitroquinoline 1-oxide (4NQO). Results: IQGAP1 is necessary for efficient PI3K signaling in vitro and in vivo. Disruption of IQGAP1-scaffolded PI3K/AKT signaling reduced HNC cell survival. Iqgap1-/- mice had significantly lower cancer incidences, lesser disease severity and fewer cancer foci. IQGAP1 protein levels were increased in HNC arising in Iqgap1+/+ mice. The level of PI3K signaling in 4NQO-induced HNC arising in Iqgap1-/- mice was significantly reduced, consistent with the hypothesis that IQGAP1 contributes to HNC at least partly through PI3K signaling. High IQGAP1 expression correlated with reduced survival, and high pS6 levels correlated with high IQGAP1 levels in HNC patients. Conclusions: These data demonstrate that IQGAP1 contributes to head and neck carcinogenesis. Citation Format: Tao Wei, Suyong Choi, Darya Buehler, Richard A. Anderson, Paul F. Lambert. A PI3K/AKT scaffolding protein, IQ motif-containing GTPase associating protein 1 (IQGAP1), promotes head and neck carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1426.

Abstract 1076: Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies

Abstract TNF Receptor Superfamily members (TNF-R-SF), including CD40, are key regulators of the immune system and have been immunotherapeutic targets for over 20 years. CD40 signaling serves as an important co-stimulatory signal for antigen-presenting cells (APC). For the induction of a CD40 driven anti-tumor immune response multiple approaches, - most of them antibody based - are currently under investigation. However, the structural necessity of trimerization within the TNF-R-SF, defines bivalent antibodies generally as unfeasible inducers of signaling events within this protein family. To overcome the known inadequacies of antibodies, we developed HERA-CD40L, composed of two trivalent CD40L-receptor-binding domains, fused to a silenced human IgG1 Fc-domain. This hexavalent CD40 agonist mimics the natural ligand and enables efficient receptor clustering and superior signaling. HERA-CD40L treatment increased the pro-inflammatory state of all CD40-expressing cells examined. It promoted the licensing of dendritic cells (DC), macrophages, B cells and other APC. Comparison to benchmark antibodies revealed that HERA-CD40L elicited a stronger and more rapid activation of NFκB signaling in primary B cells. HERA-CD40L treatment, but not clinical benchmark antibodies triggered immediate NFκB, MAPK, PI3K and STAT-1 signaling in primary monocyte-derived immature DC. As a result, HERA-CD40L induced upregulation of activation markers and co-stimulatory molecules in B cells and DC. Using SEC fractionation followed by a CD40 reporter assay, we could furthermore demonstrate that the activity of a clinical benchmark antibody derived exclusively from antibody aggregates. In vitro HERA-CD40L treatment converted immature phagocytic macrophages into mature/professional APC and induced repolarization of M2- to M1-like macrophages. These findings were confirmed in vivo using a mouse surrogate (mmHERA-CD40L). Upon treatment of MC38-CEA and CT26wt syngeneic mouse models, we observed single agent anti-tumor efficacy. In the CT26wt model, mmHERA-CD40L treatment converted cold into hot tumors by increasing T cell infiltration. Furthermore, mmHERA-CD40L induced a strong dose dependent decrease of tumor associated pro-tumorigenic M2-macrophages indicating a profound reorganization of the tumor microenvironment. Both in vitro (human) and in vivo (mouse), HERA-CD40L increased antigen-specific immune system activation without affecting the non-specific immune cells. These data, together with pilot PD/safety results, demonstrate that the activity of HERA-CD40L is both potent and safe. In conclusion, HERA-CD40L is a potent agonist able to show single agent anti-tumor activity. The biological activity is distinct from and superior to clinical benchmark “agonistic” antibodies. HERA-CD40L has a well-defined mechanism of action, does not depend on Fc gamma receptor-mediated crosslinking and hence functions as a true agonist. Citation Format: Christian Gieffers, David M. Richards, Jaromir Sykora, Christian Merz, Julian P. Sefrin, Katharina Billian-Frey, Karl Heinonen, Mauricio Redondo Müller, Matthias Schröder, Meinolf Thiemann, Oliver Hill. Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1076.