Synthetic cytokine circuits that drive T cells into immune-excluded tumors.

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

De novo design of tyrosine and serine kinase-driven protein switches.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

Multi-kinase control of environmental stress responsive transcription.

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.

Model-guided optogenetic study of PKA signaling in budding yeast.

In eukaryotes, protein kinase A (PKA) is a master regulator of cell proliferation and survival. The activity of PKA is subject to elaborate control and exhibits complex time dynamics. To probe the quantitative attributes of PKA dynamics in the yeast Saccharomyces cerevisiae, we developed an optogenetic strategy that uses a photoactivatable adenylate cyclase to achieve real-time regulation of cAMP and the PKA pathway. We capitalize on the precise and rapid control afforded by this optogenetic tool, together with quantitative computational modeling, to study the properties of feedback in the PKA signaling network and dissect the nonintuitive dynamic effects that ensue from perturbing its components. Our analyses reveal that negative feedback channeled through the Ras1/2 GTPase is delayed, pinpointing its time scale and its contribution to the dynamic features of the cAMP/PKA signaling network.

Delayed Ras/PKA signaling augments the unfolded protein response.

During environmental, developmental, or genetic stress, the cell's folding capacity can become overwhelmed, and misfolded proteins can accumulate in all cell compartments. Eukaryotes evolved the unfolded protein response (UPR) to counteract proteotoxic stress in the endoplasmic reticulum (ER). Although the UPR is vital to restoring homeostasis to protein folding in the ER, it has become evident that the response to ER stress is not limited to the UPR. Here, we used engineered orthogonal UPR induction, deep mRNA sequencing, and dynamic flow cytometry to dissect the cell's response to ER stress comprehensively. We show that budding yeast augments the UPR with time-delayed Ras/PKA signaling. This second wave of transcriptional dynamics is independent of the UPR and is necessary for fitness in the presence of ER stress, partially due to a reduction in general protein synthesis. This Ras/PKA-mediated effect functionally mimics other mechanisms, such as translational control by PKR-like ER kinase (PERK) and regulated inositol-requiring enzyme 1 (IRE1)-dependent mRNA decay (RIDD), which reduce the load of proteins entering the ER in response to ER stress in metazoan cells.

Multicellular architecture of malignant breast epithelia influences mechanics.

Cell-matrix and cell-cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the "perceived stiffness" of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing.

Conformational biosensors reveal GPCR signalling from endosomes.

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the ß2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

Stochastic modeling of cellular networks.

Noise and stochasticity are fundamental to biology because they derive from the nature of biochemical reactions. Thermal motions of molecules translate into randomness in the sequence and timing of reactions, which leads to cell-cell variability ("noise") in mRNA and protein levels even in clonal populations of genetically identical cells. This is a quantitative phenotype that has important functional repercussions, including persistence in bacterial subpopulations challenged with antibiotics, and variability in the response of cancer cells to drugs. In this chapter, we present the modeling of such stochastic cellular behaviors using the formalism of jump Markov processes, whose probability distributions evolve according to the chemical master equation (CME). We also discuss the techniques used to solve the CME. These include kinetic Monte Carlo simulations techniques such as the stochastic simulation algorithm (SSA) and method closure techniques such as the linear noise approximation (LNA).

Cellular noise regulons underlie fluctuations in Saccharomyces cerevisiae.

Stochasticity is a hallmark of cellular processes, and different classes of genes show large differences in their cell-to-cell variability (noise). To decipher the sources and consequences of this noise, we systematically measured pairwise correlations between large numbers of genes, including those with high variability. We find that there is substantial pathway variability shared across similarly regulated genes. This induces quantitative correlations in the expression of functionally related genes such as those involved in the Msn2/4 stress response pathway, amino-acid biosynthesis, and mitochondrial maintenance. Bioinformatic analyses and genetic perturbations suggest that fluctuations in PKA and Tor signaling contribute to pathway-specific variability. Our results argue that a limited number of well-delineated "noise regulons" operate across a yeast cell and that such coordinated fluctuations enable a stochastic but coherent induction of functionally related genes. Finally, we show that pathway noise is a quantitative tool for exploring pathway features and regulatory relationships in un-stimulated systems.

Tuning the activation threshold of a kinase network by nested feedback loops.

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness. GSK-3beta engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.