Cell-specific models reveal conformation-specific RAF inhibitor combinations that synergistically inhibit ERK signaling in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges for targeted clinical interventions due to prevalent KRAS mutations, rendering PDAC resistant to RAF and MEK inhibitors (RAFi and MEKi). In addition, responses to targeted therapies vary between patients. Here, we explored the differential sensitivities of PDAC cell lines to RAFi and MEKi and developed an isogenic pair comprising the most sensitive and resistant PDAC cells. To simulate patient- or tumor-specific variations, we constructed cell-line-specific mechanistic models based on protein expression profiling and differential properties of KRAS mutants. These models predicted synergy between two RAFi with different conformation specificity (type I½ and type II RAFi) in inhibiting phospho-ERK (ppERK) and reducing PDAC cell viability. This synergy was experimentally validated across all four studied PDAC cell lines. Our findings underscore the need for combination approaches to inhibit the ERK pathway in PDAC.

Cell State Transition Models Stratify Breast Cancer Cell Phenotypes and Reveal New Therapeutic Targets.

Understanding signaling patterns of transformation and controlling cell phenotypes is a challenge of current biology. Here we applied a cell State Transition Assessment and Regulation (cSTAR) approach to a perturbation dataset of single cell phosphoproteomic patterns of multiple breast cancer (BC) and normal breast tissue-derived cell lines. Following a separation of luminal, basal, and normal cell states, we identified signaling nodes within core control networks, delineated causal connections, and determined the primary drivers underlying oncogenic transformation and transitions across distinct BC subtypes. Whereas cell lines within the same BC subtype have different mutational and expression profiles, the architecture of the core network was similar for all luminal BC cells, and mTOR was a main oncogenic driver. In contrast, core networks of basal BC were heterogeneous and segregated into roughly four major subclasses with distinct oncogenic and BC subtype drivers. Likewise, normal breast tissue cells were separated into two different subclasses. Based on the data and quantified network topologies, we derived mechanistic cSTAR models that serve as digital cell twins and allow the deliberate control of cell movements within a Waddington landscape across different cell states. These cSTAR models suggested strategies of normalizing phosphorylation networks of BC cell lines using small molecule inhibitors.

Lipid Peroxidation and Type I Interferon Coupling Fuels Pathogenic Macrophage Activation Causing Tuberculosis Susceptibility.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

A key feature of arteriogenesis is capillary-to-arterial endothelial cell fate transition. Although a number of studies in the past two decades suggested this process is driven by VEGF activation of Notch signaling, how arteriogenesis is regulated remains poorly understood. Here we report that arterial specification is mediated by fluid shear stress (FSS) independent of VEGFR2 signaling and that a decline in VEGFR2 signaling is required for arteriogenesis to fully take place. VEGF does not induce arterial fate in capillary ECs and, instead, counteracts FSS-driven capillary-to-arterial cell fate transition. Mechanistically, FSS-driven arterial program involves both Notch-dependent and Notch-independent events. Sox17 is the key mediator of the FSS-induced arterial specification and a target of VEGF-FSS competition. These findings suggest a new paradigm of VEGF-FSS crosstalk coordinating angiogenesis, arteriogenesis and capillary maintenance.

cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling.

Fluid shear stress (FSS) from blood flow is sensed by vascular endothelial cells (ECs) to determine vessel stability, remodeling and susceptibility to atherosclerosis and other inflammatory diseases but the regulatory networks that govern these behaviors are only partially understood. We used cSTAR, a powerful new computational method, to define EC transcriptomic states under low shear stress (LSS) that triggers vessel inward remodeling, physiological shear stress (PSS) that stabilizes vessels, high shear stress (HSS) that triggers outward remodeling, and oscillatory shear stress (OSS) that confers disease susceptibility, all in comparison to cells under static conditions (STAT). We combined these results with the LINCS database where EC transcriptomic responses to drug treatments to define a preliminary regulatory network in which the cyclin-dependent kinases CDK1/2 play a central role in promoting vessel stability. Experimental analysis showed that PSS induced a strong late G1 cell cycle arrest in which CDK2 was activated. EC deletion of CDK2 in mice resulted in inward artery remodeling and both pulmonary and systemic hypertension. These results validate use of cSTAR to determine EC state and in vivo vessel behavior, reveal unexpected features of EC phenotype under different FSS conditions, and identify CDK2 as a key element within the EC regulatory network that governs artery remodeling.

Cell state transition analysis identifies interventions that improve control of Mycobacterium tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.

A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression.

Cancer cells often adapt to targeted therapies, yet the molecular mechanisms underlying adaptive resistance remain only partially understood. Here, we explore a mechanism of RAS/RAF/MEK/ERK (MAPK) pathway reactivation through the upregulation of RAF isoform (RAFs) abundance. Using computational modeling and in vitro experiments, we show that the upregulation of RAFs changes the concentration range of paradoxical pathway activation upon treatment with conformation-specific RAF inhibitors. Additionally, our data indicate that the signaling output upon loss or downregulation of one RAF isoform can be compensated by overexpression of other RAF isoforms. We furthermore demonstrate that, while single RAF inhibitors cannot efficiently inhibit ERK reactivation caused by RAF overexpression, a combination of two structurally distinct RAF inhibitors synergizes to robustly suppress pathway reactivation.

Reversing pathological cell states: the road less travelled can extend the therapeutic horizon.

Acquisition of omics data advances at a formidable pace. Yet, our ability to utilize these data to control cell phenotypes and design interventions that reverse pathological states lags behind. Here, we posit that cell states are determined by core networks that control cell-wide networks. To steer cell fate decisions, core networks connecting genotype to phenotype must be reconstructed and understood. A recent method, cell state transition assessment and regulation (cSTAR), applies perturbation biology to quantify causal connections and mechanistically models how core networks influence cell phenotypes. cSTAR models are akin to digital cell twins enabling us to purposefully convert pathological states back to physiologically normal states. While this capability has a range of applications, here we discuss reverting oncogenic transformation.

Control of cell state transitions.

Understanding cell state transitions and purposefully controlling them is a longstanding challenge in biology. Here we present cell state transition assessment and regulation (cSTAR), an approach for mapping cell states, modelling transitions between them and predicting targeted interventions to convert cell fate decisions. cSTAR uses omics data as input, classifies cell states, and develops a workflow that transforms the input data into mechanistic models that identify a core signalling network, which controls cell fate transitions by influencing whole-cell networks. By integrating signalling and phenotypic data, cSTAR models how cells manoeuvre in Waddington's landscape1 and make decisions about which cell fate to adopt. Notably, cSTAR devises interventions to control the movement of cells in Waddington's landscape. Testing cSTAR in a cellular model of differentiation and proliferation shows a high correlation between quantitative predictions and experimental data. Applying cSTAR to different types of perturbation and omics datasets, including single-cell data, demonstrates its flexibility and scalability and provides new biological insights. The ability of cSTAR to identify targeted perturbations that interconvert cell fates will enable designer approaches for manipulating cellular development pathways and mechanistically underpinned therapeutic interventions.

Relationship Between Dimensionality and Convergence of Optimization Algorithms: A Comparison Between Data-Driven Normalization and Scaling Factor-Based Methods Using PEPSSBI.

Ordinary differential equation models are used to represent intracellular signaling pathways in silico, aiding and guiding biological experiments to elucidate intracellular regulation. To construct such quantitative and predictive models of intracellular interactions, unknown parameters need to be estimated. Most of biological data are expressed in relative or arbitrary units, raising the question of how to compare model simulations with data. It has recently been shown that for models with large number of unknown parameters, fitting algorithms using a data-driven normalization of the simulations (DNS) performs best in terms of the convergence time and parameter identifiability. DNS approach compares model simulations and corresponding data both normalized by the same normalization procedure, without requiring additional parameters to be estimated, as necessary for widely used scaling factor-based methods. However, currently there is no parameter estimation software that directly supports DNS. In this chapter, we show how to apply DNS to dynamic models of systems and synthetic biology using PEPSSBI (Parameter Estimation Pipeline for Systems and Synthetic Biology). PEPSSBI is the first software that supports DNS, through algorithmically supported data normalization and objective function construction. PEPSSBI also supports model import using SBML and repeated parameter estimation runs executed in parallel either on a personal computer or a multi-CPU cluster.

Can Systems Biology Advance Clinical Precision Oncology?

Precision oncology is perceived as a way forward to treat individual cancer patients. However, knowing particular cancer mutations is not enough for optimal therapeutic treatment, because cancer genotype-phenotype relationships are nonlinear and dynamic. Systems biology studies the biological processes at the systems' level, using an array of techniques, ranging from statistical methods to network reconstruction and analysis, to mathematical modeling. Its goal is to reconstruct the complex and often counterintuitive dynamic behavior of biological systems and quantitatively predict their responses to environmental perturbations. In this paper, we review the impact of systems biology on precision oncology. We show examples of how the analysis of signal transduction networks allows to dissect resistance to targeted therapies and inform the choice of combinations of targeted drugs based on tumor molecular alterations. Patient-specific biomarkers based on dynamical models of signaling networks can have a greater prognostic value than conventional biomarkers. These examples support systems biology models as valuable tools to advance clinical and translational oncological research.

Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.

Modeling the Nonlinear Dynamics of Intracellular Signaling Networks.

This protocol illustrates a pipeline for modeling the nonlinear behavior of intracellular signaling pathways. At fixed spatial points, nonlinear signaling dynamics are described by ordinary differential equations (ODEs). At constant parameters, these ODEs may have multiple attractors, such as multiple steady states or limit cycles. Standard optimization procedures fine-tune the parameters for the system trajectories localized within the basin of attraction of only one attractor, usually a stable steady state. The suggested protocol samples the parameter space and captures the overall dynamic behavior by analyzing the number and stability of steady states and the shapes of the assembly of nullclines, which are determined as projections of quasi-steady-state trajectories into different 2D spaces of system variables. Our pipeline allows identifying main qualitative features of the model behavior, perform bifurcation analysis, and determine the borders separating the different dynamical regimes within the assembly of 2D parametric planes. Partial differential equation (PDE) systems describing the nonlinear spatiotemporal behavior are derived by coupling fixed point dynamics with species diffusion.

A systematic analysis of signaling reactivation and drug resistance.

Increasing evidence suggests that the reactivation of initially inhibited signaling pathways causes drug resistance. Here, we analyze how network topologies affect signaling responses to drug treatment. Network-dependent drug resistance is commonly attributed to negative and positive feedback loops. However, feedback loops by themselves cannot completely reactivate steady-state signaling. Newly synthesized negative feedback regulators can induce a transient overshoot but cannot fully restore output signaling. Complete signaling reactivation can only occur when at least two routes, an activating and inhibitory, connect an inhibited upstream protein to a downstream output. Irrespective of the network topology, drug-induced overexpression or increase in target dimerization can restore or even paradoxically increase downstream pathway activity. Kinase dimerization cooperates with inhibitor-mediated alleviation of negative feedback. Our findings inform drug development by considering network context and optimizing the design drug combinations. As an example, we predict and experimentally confirm specific combinations of RAF inhibitors that block mutant NRAS signaling.

Systems biology approaches to macromolecules: the role of dynamic protein assemblies in information processing.

Macromolecular protein assemblies govern many cellular processes and are disturbed in many diseases including cancer. Often seen as static molecular machines, protein complexes involved in signal transduction networks exhibit intricate dynamics that are critical for their function. Using the RAS-RAF-MEK-ERK pathway as example we discuss recent progress in our understanding of protein complex dynamics achieved through mathematical modelling, computational simulations and structural studies. The emerging picture highlights that both spatial and temporal dynamics cooperate to enable correct signal processing and the fine tuning of timing, duration and strengths of signalling. These dynamic processes are subverted by oncogenic mutations and contribute to tumorigenesis and drug resistance.

Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration.

Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear, whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

An Integrative Computational Approach for a Prioritization of Key Transcription Regulators Associated With Nanomaterial-Induced Toxicity.

A rapid increase of new nanomaterial (NM) products poses new challenges for their risk assessment. Current traditional methods for estimating potential adverse health effect of NMs are complex, time consuming, and expensive. In order to develop new prediction tests for nanotoxicity evaluation, a systems biology approach, and data from high-throughput omics experiments can be used. We present a computational approach that combines reverse engineering techniques, network analysis and pathway enrichment analysis for inferring the transcriptional regulation landscape and its functional interpretation. To illustrate this approach, we used published transcriptomic data derived from mice lung tissue exposed to carbon nanotubes (NM-401 and NRCWE-26). Because fibrosis is the most common adverse effect of these NMs, we included in our analysis the data for bleomycin (BLM) treatment, which is a well-known fibrosis inducer. We inferred gene regulatory networks for each NM and BLM to capture functional hierarchical regulatory structures between genes and their regulators. Despite the different nature of the lung injury caused by nanoparticles and BLM, we identified several conserved core regulators for all agents. We reason that these regulators can be considered as early predictors of toxic responses after NMs exposure. This integrative approach, which refines traditional methods of transcriptomic analysis, can be useful for prioritization of potential core regulators and generation of new hypothesis about mechanisms of nanoparticles toxicity.

Mapping connections in signaling networks with ambiguous modularity.

Modular Response Analysis (MRA) is a suite of methods that under certain assumptions permits the precise reconstruction of both the directions and strengths of connections between network modules from network responses to perturbations. Standard MRA assumes that modules are insulated, thereby neglecting the existence of inter-modular protein complexes. Such complexes sequester proteins from different modules and propagate perturbations to the protein abundance of a downstream module retroactively to an upstream module. MRA-based network reconstruction detects retroactive, sequestration-induced connections when an enzyme from one module is substantially sequestered by its substrate that belongs to a different module. Moreover, inferred networks may surprisingly depend on the choice of protein abundances that are experimentally perturbed, and also some inferred connections might be false. Here, we extend MRA by introducing a combined computational and experimental approach, which allows for a computational restoration of modular insulation, unmistakable network reconstruction and discrimination between solely regulatory and sequestration-induced connections for a range of signaling pathways. Although not universal, our approach extends MRA methods to signaling networks with retroactive interactions between modules arising from enzyme sequestration effects.