RESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Animales , Neoplasias Encefálicas/enzimología , Línea Celular Tumoral , Reparación del ADN/fisiología , Ciencia de los Datos , Dimetilsulfóxido/uso terapéutico , Resistencia a Antineoplásicos , Femenino , Gefitinib/uso terapéutico , Glioblastoma/enzimología , Xenoinjertos , Humanos , Indoles/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Análisis de los Mínimos Cuadrados , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Modelos Biológicos , Trasplante de Neoplasias , Fosfohidrolasa PTEN/metabolismo , Fenotipo , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Transducción de Señal , Sulfonas/uso terapéutico , Temozolomida/uso terapéutico , Factor de Transcripción AP-1/metabolismo , Dominios Homologos srcRESUMEN
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.
Asunto(s)
Citosina Desaminasa/genética , Genes Transgénicos Suicidas/genética , Terapia Genética , Neoplasias/terapia , Timidina Quinasa/genética , Animales , Citosina Desaminasa/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Vectores Genéticos/genética , Xenoinjertos , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Simplexvirus/enzimología , Timidina Quinasa/farmacología , Células Tumorales Cultivadas , Proteínas ras/genéticaRESUMEN
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.
Asunto(s)
Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ligandos , Ratones Desnudos , Modelos Biológicos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Línea Celular Tumoral , Humanos , Immunoblotting , Fosforilación/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal/fisiología , Espectrometría de FluorescenciaRESUMEN
Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis, but aberrant protein phosphorylation regulation leads to various diseases. The study of signaling regulation by protein phosphorylation is complicated in part by the sheer scope of the kinome and phosphoproteome, dependence of signaling protein functionality on cellular localization, and the complex multivariate relationships that exist between protein phosphorylation dynamics and the cellular phenotypes they control. Additional complexities arise from the ability of microenvironmental factors to influence phosphorylation-dependent signaling and from the tendency for some signaling processes to occur heterogeneously among cells. These considerations should be taken into account when measuring cell signaling regulation by protein phosphorylation.
Asunto(s)
Fenómenos Fisiológicos Celulares , Fosfoproteínas/metabolismo , Transducción de Señal , Animales , Humanos , FosforilaciónRESUMEN
An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aß peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aß residues 33-42 in CDR3. Interestingly, co-selection for improved Aß binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aß binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments.