Viral Kinetics Model of SARS-CoV-2 Infection Informs Drug Discovery, Clinical Dose, and Regimen Selection.

Quantitative systems pharmacology (QSP) has been an important tool to project safety and efficacy of novel or repurposed therapies for the SARS-CoV-2 virus. Here, we present a QSP modeling framework to predict response to antiviral therapeutics with three mechanisms of action (MoA): cell entry inhibitors, anti-replicatives, and neutralizing biologics. We parameterized three distinct model structures describing virus-host interaction by fitting to published viral kinetics data of untreated COVID-19 patients. The models were used to test theoretical behaviors and map therapeutic design criteria of the different MoAs, identifying the most rapid and robust antiviral activity from neutralizing biologic and anti-replicative MoAs. We found good agreement between model predictions and clinical viral load reduction observed with anti-replicative nirmatrelvir/ritonavir (Paxlovid®) and neutralizing biologics bamlanivimab and casirivimab/imdevimab (REGEN-COV®), building confidence in the modeling framework to inform a dose selection. Finally, the model was applied to predict antiviral response with ensovibep, a novel DARPin therapeutic designed as a neutralizing biologic. We developed a new in silico measure of antiviral activity, area under the curve (AUC) of free spike protein concentration, as a metric with larger dynamic range than viral load reduction. By benchmarking to bamlanivimab predictions, we justified dose levels of 75, 225, and 600 mg ensovibep to be administered intravenously in a Phase 2 clinical investigation. Upon trial completion, we found model predictions to be in good agreement with the observed patient data. These results demonstrate the utility of this modeling framework to guide the development of novel antiviral therapeutics.

TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes.

Cell-to-cell heterogeneity is a feature of the tumor necrosis factor (TNF)-stimulated inflammatory response mediated by the transcription factor NF-κB, motivating an exploration of the underlying sources of this noise. Here, we combined single-transcript measurements with computational models to study transcriptional noise at six NF-κB-regulated inflammatory genes. In the basal state, NF-κB-target genes displayed an inverse correlation between mean and noise characteristic of transcriptional bursting. By analyzing transcript distributions with a bursting model, we found that TNF primarily activated transcription by increasing burst size while maintaining burst frequency for gene promoters with relatively high basal histone 3 acetylation (AcH3) that marks open chromatin environments. For promoters with lower basal AcH3 or when AcH3 was decreased with a small molecule drug, the contribution of burst frequency to TNF activation increased. Finally, we used a mathematical model to show that TNF positive feedback amplified gene expression noise resulting from burst size-mediated transcription, leading to a subset of cells with high TNF protein expression. Our results reveal potential sources of noise underlying intercellular heterogeneity in the TNF-mediated inflammatory response.

Considering Abundance, Affinity, and Binding Site Availability in the NF-κB Target Selection Puzzle.

The NF-κB transcription regulation system governs a diverse set of responses to various cytokine stimuli. With tools from in vitro biochemical characterizations, to omics-based whole genome investigations, great strides have been made in understanding how NF-κB transcription factors control the expression of specific sets of genes. Nonetheless, these efforts have also revealed a very large number of potential binding sites for NF-κB in the human genome, and a puzzle emerges when trying to explain how NF-κB selects from these many binding sites to direct cell-type- and stimulus-specific gene expression patterns. In this review, we surmise that target gene transcription can broadly be thought of as a function of the nuclear abundance of the various NF-κB dimers, the affinity of NF-κB dimers for the regulatory sequence and the availability of this regulatory site. We use this framework to place quantitative information that has been gathered about the NF-κB transcription regulation system into context and thus consider questions it answers, and questions it raises. We end with a brief discussion of some of the future prospects that new approaches could bring to our understanding of how NF-κB transcription factors orchestrate diverse responses in different biological contexts.

Fold-Change Detection of NF-κB at Target Genes with Different Transcript Outputs.

The transcription factor nuclear factor (NF)-κB promotes inflammatory and stress-responsive gene transcription across a range of cell types in response to the cytokine tumor necrosis factor (TNF). Although NF-κB signaling exhibits significant variability across single cells, some target genes supporting high levels of TNF-inducible transcription exhibit fold-change detection of NF-κB, which may buffer against stochastic variation in signaling molecules. It is unknown whether fold-change detection is maintained at NF-κB target genes with low levels of TNF-inducible transcription, for which stochastic promoter events may be more pronounced. Here, we used a microfluidic cell-trapping device to measure how TNF-induced activation of NF-κB controls transcription in single Jurkat T cells at the promoters of integrated HIV and the endogenous cytokine gene IL6, which produce only a few transcripts per cell. We tracked TNF-stimulated NF-κB RelA nuclear translocation by live-cell imaging and then quantified transcript number by RNA FISH in the same cell. We found that TNF-induced transcript abundance at 2 h for low- and high-abundance target genes correlates with similar strength with the fold change in nuclear NF-κB. A computational model of TNF-NF-κB signaling, which implements fold-change detection from competition for binding to κB motifs, could reproduce fold-change detection across the experimentally measured range of transcript outputs. However, multiple model parameters affecting transcription had to be simultaneously varied across promoters to maintain fold-change detection while also matching other trends in the single-cell data for low-abundance transcripts. Our results suggest that cells use multiple biological mechanisms to tune transcriptional output while maintaining robustness of NF-κB fold-change detection.

NF-κB-Chromatin Interactions Drive Diverse Phenotypes by Modulating Transcriptional Noise.

Noisy gene expression generates diverse phenotypes, but little is known about mechanisms that modulate noise. Combining experiments and modeling, we studied how tumor necrosis factor (TNF) initiates noisy expression of latent HIV via the transcription factor nuclear factor κB (NF-κB) and how the HIV genomic integration site modulates noise to generate divergent (low-versus-high) phenotypes of viral activation. We show that TNF-induced transcriptional noise varies more than mean transcript number and that amplification of this noise explains low-versus-high viral activation. For a given integration site, live-cell imaging shows that NF-κB activation correlates with viral activation, but across integration sites, NF-κB activation cannot account for differences in transcriptional noise and phenotypes. Instead, differences in transcriptional noise are associated with differences in chromatin state and RNA polymerase II regulation. We conclude that, whereas NF-κB regulates transcript abundance in each cell, the chromatin environment modulates noise in the population to support diverse HIV activation in response to TNF.

Finding the Optimal Tradeoffs.

Computational analyses of a half-million circuit topologies provide a rationale for why certain fold-change detection topologies are more prevalent in nature.

NF-κB signalling and cell fate decisions in response to a short pulse of tumour necrosis factor.

In tissues and tumours, cell behaviours are regulated by multiple time-varying signals. While in the laboratory cells are often exposed to a stimulus for the duration of the experiment, in vivo exposures may be much shorter. In this study, we monitored NF-κB and caspase signalling in human cancer cells treated with a short pulse of Tumour Necrosis Factor (TNF). TNF is an inflammatory cytokine that can induce both the pro-survival NF-κB-driven gene transcription pathway and the pro-apoptotic caspase pathway. We find that a few seconds of exposure to TNF is sufficient to activate the NF-κB pathway in HeLa cells and induce apoptotic cell death in both HeLa and Kym-1 cells. Strikingly, a 1-min pulse of TNF can be more effective at killing than a 1-hour pulse, indicating that in addition to TNF concentration, duration of exposure also coordinates cell fate decisions.

Redefining Signaling Pathways with an Expanding Single-Cell Toolbox.

Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways.

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors.

The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage.

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.

Fold change of nuclear NF-κB determines TNF-induced transcription in single cells.

In response to tumor necrosis factor (TNF), NF-κB enters the nucleus and promotes inflammatory and stress-responsive gene transcription. Because NF-κB deregulation is associated with disease, one might expect strict control of NF-κB localization. However, nuclear NF-κB levels exhibit considerable cell-to-cell variability, even in unstimulated cells. To resolve this paradox and determine how transcription-inducing signals are encoded, we quantified single-cell NF-κB translocation dynamics and transcription in the same cells. We show that TNF-induced transcription correlates best with fold change in nuclear NF-κB, not absolute nuclear NF-κB abundance. Using computational modeling, we find that an incoherent feedforward loop, from competition for binding to κB motifs, could provide memory of the preligand state necessary for fold-change detection. Experimentally, we observed three gene-specific transcriptional patterns that our model recapitulates by modulating competition strength alone. Fold-change detection buffers against stochastic variation in signaling molecules and explains how cells tolerate variability in NF-κB abundance and localization.

Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit.

Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)-related kinases Tank-binding kinase-1 (TBK1) and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.

Exploring the contextual sensitivity of factors that determine cell-to-cell variability in receptor-mediated apoptosis.

Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand) variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions.

The MEK2-binding tumor suppressor hDlg is recruited by E-cadherin to the midbody ring.

BACKGROUND: The human homologue of the Drosophila Discs-large tumor suppressor protein, hDlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. At both synaptic junctions and epithelia cell-cell junctions, hDlg is known to recruit several signaling proteins into macromolecular complexes. hDlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear. RESULTS: Here we describe the interaction of hDlg with the activated form of MEK2 of the canonical RAF/MEK/ERK pathway, a protein that is found at the midbody during cytokinesis. We show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the PDZ domains of hDlg and the C-terminal portion of MEK2 is dependent on the phosphorylation of MEK2. Finally, we found that E-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hDlg, but not activated MEK2, to that structure. CONCLUSION: Our results suggest that like at other cell-cell junction sites, hDlg is part of a macromolecular complex of structural and signaling proteins at the midbody.

Lyapunov exponents and phase diagrams reveal multi-factorial control over TRAIL-induced apoptosis.

Receptor-mediated apoptosis proceeds via two pathways: one requiring only a cascade of initiator and effector caspases (type I behavior) and the second requiring an initiator-effector caspase cascade and mitochondrial outer membrane permeabilization (type II behavior). Here, we investigate factors controlling type I versus II phenotypes by performing Lyapunov exponent analysis of an ODE-based model of cell death. The resulting phase diagrams predict that the ratio of XIAP to pro-caspase-3 concentrations plays a key regulatory role: type I behavior predominates when the ratio is low and type II behavior when the ratio is high. Cell-to-cell variability in phenotype is observed when the ratio is close to the type I versus II boundary. By positioning multiple tumor cell lines on the phase diagram we confirm these predictions. We also extend phase space analysis to mutations affecting the rate of caspase-3 ubiquitylation by XIAP, predicting and showing that such mutations abolish all-or-none control over activation of effector caspases. Thus, phase diagrams derived from Lyapunov exponent analysis represent a means to study multi-factorial control over a complex biochemical pathway.

Systematic calibration of a cell signaling network model.

BACKGROUND: Mathematical modeling is being applied to increasingly complex biological systems and datasets; however, the process of analyzing and calibrating against experimental data is often challenging and a rate limiting step in model development. To address this problem, we developed a systematic methodology for calibrating quantitative models of dynamic biological processes and illustrate its utility by validating a model of TRAIL (Tumor necrosis factor Related Apoptosis-Inducing Ligand)-induced cell death. RESULTS: We propose a serial framework integrating analysis and calibration modules and we compare various methods for global sensitivity analysis and global parameter estimation. First, adequacy of the network structure is checked by global sensitivity analysis to changes in concentrations of molecular species, validating that the model can reproduce qualitative features of the system behavior derived from experiments or literature surveys. Second, rate parameters are ranked by importance using gradient-based and variance-based sensitivity indices, and we systematically determine the optimal number of parameters to include in model calibration. Third, deterministic, stochastic and hybrid algorithms for global optimization are applied to estimate the values of the most important parameters by fitting to time series data. We compare the performance of these three optimization algorithms. CONCLUSIONS: Our proposed framework covers the entire process from validating a proto-model to establishing a realistic model for in silico experiments and thereby provides a generalized workflow for the construction of predictive models of complex network systems.

Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis.

In microorganisms, noise in gene expression gives rise to cell-to-cell variability in protein concentrations. In mammalian cells, protein levels also vary and individual cells differ widely in their responsiveness to uniform physiological stimuli. In the case of apoptosis mediated by TRAIL (tumour necrosis factor (TNF)-related apoptosis-inducing ligand) it is common for some cells in a clonal population to die while others survive-a striking divergence in cell fate. Among cells that die, the time between TRAIL exposure and caspase activation is highly variable. Here we image sister cells expressing reporters of caspase activation and mitochondrial outer membrane permeabilization after exposure to TRAIL. We show that naturally occurring differences in the levels or states of proteins regulating receptor-mediated apoptosis are the primary causes of cell-to-cell variability in the timing and probability of death in human cell lines. Protein state is transmitted from mother to daughter, giving rise to transient heritability in fate, but protein synthesis promotes rapid divergence so that sister cells soon become no more similar to each other than pairs of cells chosen at random. Our results have implications for understanding 'fractional killing' of tumour cells after exposure to chemotherapy, and for variability in mammalian signal transduction in general.

Collecting and organizing systematic sets of protein data.

Systems biology, particularly of mammalian cells, is data starved. However, technologies are now in place to obtain rich data, in a form suitable for model construction and validation, that describes the activities, states and locations of cell-signalling molecules. The key is to use several measurement technologies simultaneously and, recognizing each of their limits, to assemble a self-consistent compendium of systematic data.

The response of human epithelial cells to TNF involves an inducible autocrine cascade.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that induces conflicting pro- and antiapoptotic signals whose relative strengths determine the extent of cell death. TNF receptor (TNFR) has been studied in considerable detail, but it is not known how crosstalk among antagonistic pro- and antiapoptotic signals is achieved. Here we report an experimental and computational analysis of crosstalk between prodeath TNF and prosurvival growth factors in human epithelial cells. By applying classifier-based regression to a cytokine-signaling compendium of approximately 8000 intracellular protein measurements, we demonstrate that cells respond to TNF both directly, via activated TNF receptor, and indirectly, via the sequential release of transforming growth factor-alpha (TGF-alpha), interleukin-1alpha (IL-1alpha), and IL-1 receptor antagonist (IL-1ra). We refer to the contingent and time-varying series of extracellular signals induced by TNF as an "autocrine cascade." Time-dependent crosstalk of synergistic and antagonistic autocrine circuits may serve to link cellular responses to the local environment.