A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreatic cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multi-drug therapies, providing a potential strategy for overcoming chemoresistance.

Divergent transcriptomic signatures from putative mesenchymal stimuli in glioblastoma cells.

In glioblastoma, a mesenchymal phenotype is associated with especially poor patient outcomes. Various glioblastoma microenvironmental factors and therapeutic interventions are purported drivers of the mesenchymal transition, but the degree to which these cues promote the same mesenchymal transitions and the uniformity of those transitions, as defined by molecular subtyping systems, is unknown. Here, we investigate this question by analyzing publicly available patient data, surveying commonly measured transcripts for mesenchymal transitions in glioma-initiating cells (GIC), and performing next-generation RNA sequencing of GICs. Analysis of patient tumor data reveals that TGFß, TNFα, and hypoxia signaling correlate with the mesenchymal subtype more than the proneural subtype. In cultured GICs, the microenvironment-relevant growth factors TGFß and TNFα and the chemotherapeutic temozolomide promote expression of commonly measured mesenchymal transcripts. However, next-generation RNA sequencing reveals that growth factors and temozolomide broadly promote expression of both mesenchymal and proneural transcripts, in some cases with equal frequency. These results suggest that glioblastoma mesenchymal transitions do not occur as distinctly as in epithelial-derived cancers, at least as determined using common subtyping ontologies and measuring response to growth factors or chemotherapeutics. Further understanding of these issues may identify improved methods for pharmacologically targeting the mesenchymal phenotype in glioblastoma.

Single-cell RNA sequencing reveals microenvironment context-specific routes for epithelial-mesenchymal transition in pancreas cancer cells.

In the PDAC tumor microenvironment, multiple factors initiate the epithelial-mesenchymal transition (EMT) that occurs heterogeneously among transformed ductal cells, but it is unclear if different drivers promote EMT through common or distinct signaling pathways. Here, we use single-cell RNA sequencing (scRNA-seq) to identify the transcriptional basis for EMT in pancreas cancer cells in response to hypoxia or EMT-inducing growth factors. Using clustering and gene set enrichment analysis, we find EMT gene expression patterns that are unique to the hypoxia or growth factor conditions or that are common between them. Among the inferences from the analysis, we find that the FAT1 cell adhesion protein is enriched in epithelial cells and suppresses EMT. Further, the receptor tyrosine kinase AXL is preferentially expressed in hypoxic mesenchymal cells in a manner correlating with YAP nuclear localization, which is suppressed by FAT1 expression. AXL inhibition prevents EMT in response to hypoxia but not growth factors. Relationships between FAT1 or AXL expression with EMT were confirmed through analysis of patient tumor scRNA-seq data. Further exploration of inferences from this unique dataset will reveal additional microenvironment context-specific signaling pathways for EMT that may represent novel drug targets for PDAC combination therapies.

An integrated mechanistic and data-driven computational model predicts cell responses to high- and low-affinity EGFR ligands.

The biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal transduction and may eventually be leveraged to understand receptor signaling system performance depends on cell context. One-sentence summary: Integrated kinetic and data-driven EGFR signaling model identifies the specific signaling mechanisms that dictate cell responses to EGFR activation by different ligands.

Correction: A computational modeling approach for predicting multicell spheroid patterns based on signaling-induced differential adhesion.

[This corrects the article DOI: 10.1371/journal.pcbi.1010701.].

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

A computational modeling approach for predicting multicell spheroid patterns based on signaling-induced differential adhesion.

Physiological and pathological processes including embryogenesis and tumorigenesis rely on the ability of individual cells to work collectively to form multicell patterns. In these heterogeneous multicell systems, cell-cell signaling induces differential adhesion between cells that leads to tissue-level patterning. However, the sensitivity of pattern formation to changes in the strengths of signaling or cell adhesion processes is not well understood. Prior work has explored these issues using synthetically engineered heterogeneous multicell spheroid systems, in which cell subpopulations engage in bidirectional intercellular signaling to regulate the expression of different cadherins. While engineered cell systems provide excellent experimental tools to observe pattern formation in cell populations, computational models of these systems may be leveraged to explore more systematically how specific combinations of signaling and adhesion parameters can drive the emergence of unique patterns. We developed and validated two- and three-dimensional agent-based models (ABMs) of spheroid patterning for previously described cells engineered with a bidirectional signaling circuit that regulates N- and P-cadherin expression. Systematic exploration of model predictions, some of which were experimentally validated, revealed how cell seeding parameters, the order of signaling events, probabilities of induced cadherin expression, and homotypic adhesion strengths affect pattern formation. Unsupervised clustering was also used to map combinations of signaling and adhesion parameters to these unique spheroid patterns predicted by the ABM. Finally, we demonstrated how the model may be deployed to design new synthetic cell signaling circuits based on a desired final multicell pattern.

Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes tumor growth and progression in triple-negative breast cancer.

Triple-Negative Breast Cancers (TNBCs) constitute roughly 10-20% of breast cancers and are associated with poor clinical outcomes. Previous work from our laboratory and others has determined that the cytoplasmic adaptor protein Breast Cancer Antiestrogen Resistance 3 (BCAR3) is an important promoter of cell motility and invasion of breast cancer cells. In this study, we use both in vivo and in vitro approaches to extend our understanding of BCAR3 function in TNBC. We show that BCAR3 is upregulated in ductal carcinoma in situ (DCIS) and invasive carcinomas compared to normal mammary tissue, and that survival of TNBC patients whose tumors contained elevated BCAR3 mRNA is reduced relative to individuals whose tumors had less BCAR3 mRNA. Using mouse orthotopic tumor models, we further show that BCAR3 is required for efficient TNBC tumor growth. Analysis of publicly available RNA expression databases revealed that MET receptor signaling is strongly correlated with BCAR3 mRNA expression. A functional role for BCAR3-MET coupling is supported by data showing that both proteins participate in a single pathway to control proliferation and migration of TNBC cells. Interestingly, the mechanism through which this functional interaction operates appears to differ in different genetic backgrounds of TNBC, stemming in one case from potential differences in the strength of downstream signaling by the MET receptor and in another from BCAR3-dependent activation of an autocrine loop involving the production of HGF mRNA. Together, these data open the possibility for new approaches to personalized therapy for individuals with TNBCs.

CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of 'STimulator of Interferon Genes' (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP-AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS-STING-TBK1-IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair.

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

Mathematical Model Predicts that Acceleration of Diabetic Wound Healing is Dependent on Spatial Distribution of VEGF-A mRNA (AZD8601).

INTRODUCTION: Pharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing. METHODS: We modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing. RESULTS: When simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1-2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue. CONCLUSIONS: These findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00678-9.

Data-Driven Computational Modeling Identifies Determinants of Glioblastoma Response to SHP2 Inhibition.

Oncogenic protein tyrosine phosphatases have long been viewed as drug targets of interest, and recently developed allosteric inhibitors of SH2 domain-containing phosphatase-2 (SHP2) have entered clinical trials. However, the ability of phosphatases to regulate many targets directly or indirectly and to both promote and antagonize oncogenic signaling may make the efficacy of phosphatase inhibition challenging to predict. Here we explore the consequences of antagonizing SHP2 in glioblastoma, a recalcitrant cancer where SHP2 has been proposed as a useful drug target. Measuring protein phosphorylation and expression in glioblastoma cells across 40 signaling pathway nodes in response to different drugs and for different oxygen tensions revealed that SHP2 antagonism has network-level, context-dependent signaling consequences that affect cell phenotypes (e.g., cell death) in unanticipated ways. To map specific signaling consequences of SHP2 antagonism to phenotypes of interest, a data-driven computational model was constructed based on the paired signaling and phenotype data. Model predictions aided in identifying three signaling processes with implications for treating glioblastoma with SHP2 inhibitors. These included PTEN-dependent DNA damage repair in response to SHP2 inhibition, AKT-mediated bypass resistance in response to chronic SHP2 inhibition, and SHP2 control of hypoxia-inducible factor expression through multiple MAPKs. Model-generated hypotheses were validated in multiple glioblastoma cell lines, in mouse tumor xenografts, and through analysis of The Cancer Genome Atlas data. Collectively, these results suggest that in glioblastoma, SHP2 inhibitors antagonize some signaling processes more effectively than existing kinase inhibitors but can also limit the efficacy of other drugs when used in combination. SIGNIFICANCE: These findings demonstrate that allosteric SHP2 inhibitors have multivariate and context-dependent effects in glioblastoma that may make them useful components of some combination therapies, but not others.

MECHANISTIC AND DATA-DRIVEN MODELS OF CELL SIGNALING: TOOLS FOR FUNDAMENTAL DISCOVERY AND RATIONAL DESIGN OF THERAPY.

A full understanding of cell signaling processes requires knowledge of protein structure/function relationships, protein-protein interactions, and the abilities of pathways to control phenotypes. Computational models offer a valuable framework for integrating that knowledge to predict the effects of system perturbations and interventions in health and disease. Whereas mechanistic models are well suited for understanding the biophysical basis for signal transduction and principles of therapeutic design, data-driven models are particularly suited to distill complex signaling relationships among samples and between multivariate signaling changes and phenotypes. Both approaches have limitations and provide incomplete representations of signaling biology, but their careful implementation and integration can provide new understanding for how manipulating system variables impacts cellular decisions.

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers.

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here.

Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex.

Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.

Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling.

SPRY2 is a purported tumor suppressor in certain cancers that promotes tumor growth and resistance to receptor tyrosine kinase inhibitors in glioblastoma. Here, we identify a SPRY2-dependent bypass signaling mechanism in glioblastoma that drives resistance to EGFR and MET inhibition. In glioblastoma cells treated with EGFR and MET inhibitors, SPRY2 expression is initially suppressed but eventually rebounds due to NF-κB pathway activation, resultant autocrine FGFR activation, and reactivation of ERK, which controls SPRY2 transcription. In cells where FGFR autocrine signaling does not occur and ERK does not reactivate, or in which ERK reactivates but SPRY2 cannot be expressed, EGFR and MET inhibitors are more effective at promoting death. The same mechanism also drives acquired resistance to EGFR and MET inhibition. Furthermore, tumor xenografts expressing an ERK-dependent bioluminescent reporter engineered for these studies reveal that this bypass resistance mechanism plays out in vivo but can be overcome through simultaneous FGFR inhibition.

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

BACKGROUND: Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear. METHODS: We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. RESULTS: OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. CONCLUSIONS: Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

Disrupting the transmembrane domain-mediated oligomerization of protein tyrosine phosphatase receptor J inhibits EGFR-driven cancer cell phenotypes.

Receptor protein tyrosine phosphatases (RPTPs) play critical regulatory roles in mammalian signal transduction. However, the structural basis for the regulation of their catalytic activity is not fully understood, and RPTPs are generally not therapeutically targetable. This knowledge gap is partially due to the lack of known natural ligands or selective agonists of RPTPs. Contrary to what is known from structure-function studies of receptor tyrosine kinases (RTKs), RPTP activities have been reported to be suppressed by dimerization, which may prevent RPTPs from accessing their RTK substrates. We report here that homodimerization of protein tyrosine phosphatase receptor J (PTPRJ, also known as DEP-1) is regulated by specific transmembrane (TM) residues. We found that disrupting these interactions destabilizes homodimerization of full-length PTPRJ in cells, reduces the phosphorylation of the known PTPRJ substrate epidermal growth factor receptor (EGFR) and of other downstream signaling effectors, antagonizes EGFR-driven cell phenotypes, and promotes substrate access. We demonstrate these observations in human cancer cells using mutational studies and identified a peptide that binds to the PTPRJ TM domain and represents the first example of an allosteric agonist of RPTPs. The results of our study provide fundamental structural and functional insights into how PTPRJ activity is tuned by TM interactions in cells. Our findings also open up opportunities for developing peptide-based agents that could be used as tools to probe RPTPs' signaling mechanisms or to manage cancers driven by RTK signaling.

Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells.

Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.