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.].

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.

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.

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.

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.

EGF augments TGFß-induced epithelial-mesenchymal transition by promoting SHP2 binding to GAB1.

In many epithelial cells, epidermal growth factor (EGF) augments the epithelial-mesenchymal transition (EMT) that occurs when cells are treated with transforming growth factor ß (TGFß). We demonstrate that this augmentation requires activation of SH2 domain-containing phosphatase-2 (SHP2; also known as PTPN11), a proto-oncogene. In lung and pancreatic cancer cell lines, reductions in E-cadherin expression, increases in vimentin expression and increases in cell scatter rates were larger when cells were treated with TGFß and EGF versus TGFß or EGF alone. SHP2 knockdown promoted epithelial characteristics basally and antagonized EMT in response to TGFß alone or in combination with EGF. Whereas EGF promoted SHP2 binding to tyrosine phosphorylated GAB1, which promotes SHP2 activity, TGFß did not induce SHP2 association with phosphotyrosine-containing proteins. Knockdown of endogenous SHP2 and reconstitution with an SHP2 mutant with impaired phosphotyrosine binding ability eliminated the EGF-mediated EMT augmentation that was otherwise restored with wild-type SHP2 reconstitution. These results demonstrate roles for basal and ligand-induced SHP2 activity in EMT and further motivate efforts to identify specific ways to inhibit SHP2, given the role of EMT in tumor dissemination and chemoresistance.

p53Ψ is a transcriptionally inactive p53 isoform able to reprogram cells toward a metastatic-like state.

Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3' splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.

Multivariate signaling regulation by SHP2 differentially controls proliferation and therapeutic response in glioma cells.

Information from multiple signaling axes is integrated in the determination of cellular phenotypes. Here, we demonstrate this aspect of cellular decision making in glioblastoma multiforme (GBM) cells by investigating the multivariate signaling regulatory functions of the protein tyrosine phosphatase SHP2 (also known as PTPN11). Specifically, we demonstrate that the ability of SHP2 to simultaneously drive ERK1/2 and antagonize STAT3 pathway activities produces qualitatively different effects on the phenotypes of proliferation and resistance to EGFR and c-MET co-inhibition. Whereas the ERK1/2 and STAT3 pathways independently promote proliferation and resistance to EGFR and c-MET co-inhibition, SHP2-driven ERK1/2 activity is dominant in driving cellular proliferation and SHP2-mediated antagonism of STAT3 phosphorylation prevails in the promotion of GBM cell death in response to EGFR and c-MET co-inhibition. Interestingly, the extent of these SHP2 signaling regulatory functions is diminished in glioblastoma cells that express sufficiently high levels of the EGFR variant III (EGFRvIII) mutant, which is commonly expressed in GBM. In cells and tumors that express EGFRvIII, SHP2 also antagonizes the phosphorylation of EGFRvIII and c-MET and drives expression of HIF-1α and HIF-2α, adding complexity to the evolving understanding of the regulatory functions of SHP2 in GBM.

Regulation of EGFR trafficking and cell signaling by Sprouty2 and MIG6 in lung cancer cells.

The duration and specificity of epidermal growth factor receptor (EGFR) activation and signaling are determinants of cellular decision processes and are tightly regulated by receptor dephosphorylation, internalization and degradation. In addition, regulatory proteins that are upregulated or activated post-transcriptionally upon receptor activation may initiate feedback loops that play crucial roles in spatiotemporal regulation of signaling. We examined the roles of Sprouty2 (SPRY2) and mitogen-inducible gene 6 (MIG6), two feedback regulators of EGFR trafficking and signaling, in lung cancer cells with or without EGFR-activating mutations. These mutations are of interest because they confer unusual cellular sensitivity to EGFR inhibition through a mechanism involving an impairment of EGFR endocytosis. We found that the endocytosis of wild-type and mutant EGFR was promoted by SPRY2 knockdown and antagonized by MIG6 knockdown. SPRY2 knockdown also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation, EGFR expression, and EGFR recycling. In a cell line expressing mutant EGFR, this effect on ERK led to a marked increase in cell death response to EGFR inhibition. The effects of SPRY2 knockdown on EGFR endocytosis and recycling were primarily the result of the concomitant change in EGFR expression, but this was not true for the observed changes in ERK phosphorylation. Thus, our study demonstrates that SPRY2 and MIG6 are important regulators of wild-type and mutant EGFR trafficking and points to an EGFR expression-independent function of SPRY2 in the regulation of ERK activity that may impact cellular sensitivity to EGFR inhibitors, especially in the context of EGFR mutation.

Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.

Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.

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.

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 that hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multidrug therapies, providing a potential strategy for overcoming chemoresistance. SIGNIFICANCE: Integrated regulation of histone methylation and MAPK signaling by the low-oxygen environment of pancreatic cancer drives long-lasting EMT that promotes chemoresistance and shortens patient survival and that can be pharmacologically inhibited. See related commentary by Wirth and Schneider, p. 1739.

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.

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.

Computational analysis of the regulation of EGFR by protein tyrosine phosphatases.

The tyrosine phosphorylated epidermal growth factor receptor (EGFR) initiates numerous cell signaling pathways. Although EGFR phosphorylation levels are ultimately determined by the balance of receptor kinase and protein tyrosine phosphatase (PTP) activities, the kinetics of EGFR dephosphorylation are not well understood. Previous models of EGFR signaling have generally neglected PTP activity or computed PTP activity by considering data that do not fully reveal the kinetics and compartmentalization of EGFR dephosphorylation. We developed a compartmentalized, mechanistic model to elucidate the kinetics of EGFR dephosphorylation and the coupling of this process to phosphorylation-dependent EGFR endocytosis. Model regression against data from HeLa cells for EGFR phosphorylation response to EGFR activation, PTP inhibition, and EGFR kinase inhibition led to the conclusion that EGFR dephosphorylation occurs at the plasma membrane and in the cell interior with a timescale that is smaller than that for ligand-mediated EGFR endocytosis. The model further predicted that sufficiently rapid dephosphorylation of EGFR at the plasma membrane could potentially impede EGFR endocytosis, consistent with recent experimental findings. Overall, our results suggest that PTPs regulate multiple receptor-level phenomena via their action at the plasma membrane and cell interior and point to new possibilities for targeting PTPs for modulation of EGFR dynamics.

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.

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.