Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Gß Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration.

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gß and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gß. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gß regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gß and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.

Interaction of motility, directional sensing, and polarity modules recreates the behaviors of chemotaxing cells.

Chemotaxis involves the coordinated action of separable but interrelated processes: motility, gradient sensing, and polarization. We have hypothesized that these are mediated by separate modules that account for these processes individually and that, when combined, recreate most of the behaviors of chemotactic cells. Here, we describe a mathematical model where the modules are implemented in terms of reaction-diffusion equations. Migration and the accompanying changes in cellular morphology are demonstrated in simulations using a mechanical model of the cell cortex implemented in the level set framework. The central module is an excitable network that accounts for random migration. The response to combinations of uniform stimuli and gradients is mediated by a local excitation, global inhibition module that biases the direction in which excitability is directed. A polarization module linked to the excitable network through the cytoskeleton allows unstimulated cells to move persistently and, for cells in gradients, to gradually acquire distinct sensitivity between front and back. Finally, by varying the strengths of various feedback loops in the model we obtain cellular behaviors that mirror those of genetically altered cell lines.

Self-organizing glycolytic waves fuel cell migration and cancer progression.

Glycolysis has traditionally been thought to take place in the cytosol but we observed the enrichment of glycolytic enzymes in propagating waves of the cell cortex in human epithelial cells. These waves reflect excitable Ras/PI3K signal transduction and F-actin/actomyosin networks that drive cellular protrusions, suggesting that localized glycolysis at the cortex provides ATP for cell morphological events such as migration, phagocytosis, and cytokinesis. Perturbations that altered cortical waves caused corresponding changes in enzyme localization and ATP production whereas synthetic recruitment of glycolytic enzymes to the cell cortex enhanced cell spreading and motility. Interestingly, the cortical waves and ATP levels were positively correlated with the metastatic potential of cancer cells. The coordinated signal transduction, cytoskeletal, and glycolytic waves in cancer cells may explain their increased motility and their greater reliance on glycolysis, often referred to as the Warburg effect.

Reconstructing Signaling Networks Using Biosensor Barcoding.

Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.

Salmonella immunotherapy engineered with highly efficient tumor antigen coating establishes antigen-specific CD8+ T cell immunity and increases in antitumor efficacy with type I interferon combination therapy.

Bacteria-based cancer therapy employs various strategies to combat tumors, one of which is delivering tumor-associated antigen (TAA) to generate specific immunity. Here, we utilized a poly-arginine extended HPV E7 antigen (9RE7) for attachment on Salmonella SL7207 outer membrane to synthesize the bacterial vaccine Salmonella-9RE7 (Sal-9RE7), which yielded a significant improvement in the amount of antigen presentation compared to the previous lysine-extended antigen coating strategy. In TC-1 tumor mouse models, Sal-9RE7 monotherapy decreased tumor growth by inducing E7 antigen-specific immunity. In addition, pairing Sal-9RE7 with adjuvant Albumin-IFNß (Alb-IFNß), a protein cytokine fusion, the combination significantly increased the antitumor efficacy and enhanced immunogenicity in the tumor microenvironment (TME). Our study made a significant contribution to personalized bacterial immunotherapy via TAA delivery and demonstrated the advantage of combination therapy.

Cells navigate with a local-excitation, global-inhibition-biased excitable network.

Cells have an internal compass that enables them to move along shallow chemical gradients. As amoeboid cells migrate, signaling events such as Ras and PI3K activation occur spontaneously on pseudopodia. Uniform stimuli trigger a symmetric response, whereupon cells stop and round up; then localized patches of activity appear as cells spread. Finally cells adapt and resume random migration. In contrast, chemotactic gradients continuously direct signaling events to the front of the cell. Local-excitation, global-inhibition (LEGI) and reaction-diffusion models have captured some of these features of chemotaxing cells, but no system has explained the complex response kinetics, sensitivity to shallow gradients, or the role of recently observed propagating waves within the actin cytoskeleton. We report here that Ras and PI3K activation move in phase with the cytoskeleton events and, drawing on all of these observations, propose the LEGI-biased excitable network hypothesis. We formulate a model that simulates most of the behaviors of chemotactic cells: In the absence of stimulation, there are spontaneous spots of activity. Stimulus increments trigger an initial burst of patches followed by localized secondary events. After a few minutes, the system adapts, again displaying random activity. In gradients, the activity patches are directed continuously and selectively toward the chemoattractant, providing an extraordinary degree of amplification. Importantly, by perturbing model parameters, we generate distinct behaviors consistent with known classes of mutants. Our study brings together heretofore diverse observations on spontaneous cytoskeletal activity, signaling responses to temporal stimuli, and spatial gradient sensing into a unified scheme.

Targeting Ras signaling excitability in cancer cells through combined inhibition of FAK and PI3K.

The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.

A frequent kinase domain mutation that changes the interaction between PI3Kalpha and the membrane.

Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110alpha, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kalpha), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110alpha in complex with two interacting domains of its regulatory partner (p85alpha), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85alpha is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110alpha. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110alpha His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

Imaging and analysis for simultaneous tracking of fluorescent biosensors in barcoded cells.

We recently developed a biosensor barcoding approach for highly multiplexed tracking of molecular activities in live cells. In this protocol, we detail the labeling of cells expressing different genetically encoded fluorescent biosensors with a pair of barcoding proteins and parallel imaging. Signals from cells with the same barcodes are then pooled together to obtain the dynamics of the corresponding biosensor activity. We describe the steps involved in cell barcoding, image acquisition, and analysis by deep learning models. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Structural effects of oncogenic PI3Kα mutations.

Physiological activation of PI3Kα is brought about by the release of the inhibition by p85 when the nSH2 binds the phosphorylated tyrosine of activated receptors or their substrates. Oncogenic mutations of PI3Kα result in a constitutively activated enzyme that triggers downstream pathways that increase tumor aggressiveness and survival. Structural information suggests that some mutations also activate the enzyme by releasing p85 inhibition. Other mutations work by different mechanisms. For example, the most common mutation, His1047Arg, causes a conformational change that increases membrane association resulting in greater accessibility to the substrate, an integral membrane component. These effects are examples of the subtle structural changes that result in increased activity. The structures of these and other mutants are providing the basis for the design of isozyme-specific, mutation-specific inhibitors for individualized cancer therapies.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody.

Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.

Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer.

Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells.

The Ras/PI3K/extracellular signal-regulated kinases (ERK) signaling network plays fundamental roles in cell growth, survival, and migration and is frequently activated in cancer. Here, we show that the activities of the signaling network propagate as coordinated waves, biased by growth factor, which drive actin-based protrusions in human epithelial cells. The network exhibits hallmarks of biochemical excitability: the annihilation of oppositely directed waves, all-or-none responsiveness, and refractoriness. Abrupt perturbations to Ras, PI(4,5)P2, PI(3,4)P2, ERK, and TORC2 alter the threshold, observations that define positive and negative feedback loops within the network. Oncogenic transformation dramatically increases the wave activity, the frequency of ERK pulses, and the sensitivity to EGF stimuli. Wave activity was progressively enhanced across a series of increasingly metastatic breast cancer cell lines. The view that oncogenic transformation is a shift to a lower threshold of excitable Ras/PI3K/ERK network, caused by various combinations of genetic insults, can facilitate the assessment of cancer severity and effectiveness of interventions.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The original version of this Article contained an error in the spelling of the author Jr-Ming Yang, which was incorrectly given as J.-Ming Yang. This has now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

In the original version of this Article, the label "RTK" in Figure 6a was inadvertently changed to "RTE". This has now been corrected in the PDF and HTML versions of the Article.

Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.