RESUMEN
Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106-mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. SIGNIFICANCE: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirimidinas/farmacología , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Inmunidad Adaptativa , Animales , Apoptosis , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Neoplasias del Colon/patología , Femenino , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.
Asunto(s)
Encéfalo/metabolismo , Microglía/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/patología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Modelos Animales de Enfermedad , Femenino , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Masculino , Ratones , Neurogénesis , Enfermedad de Parkinson/metabolismo , Fagocitosis , Proteína S/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Tirosina Quinasas Receptoras/deficiencia , Transducción de Señal , Nicho de Células Madre , Regulación hacia Arriba , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
The TAM receptors Tyro3, Axl and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and downregulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection. Unexpectedly, mice lacking Mertk and/or Axl, but not Tyro3, exhibited greater vulnerability to infection with neuroinvasive West Nile and La Crosse encephalitis viruses. This phenotype was associated with increased blood-brain barrier permeability, which enhanced virus entry into and infection of the brain. Activation of Mertk synergized with interferon-ß to tighten cell junctions and prevent virus transit across brain microvascular endothelial cells. Because TAM receptors restrict pathogenesis of neuroinvasive viruses, these findings have implications for TAM antagonists that are currently in clinical development.
Asunto(s)
Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/virología , Encefalitis de California/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Fiebre del Nilo Occidental/enzimología , Inmunidad Adaptativa , Animales , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/patología , Quimiocinas/sangre , Encefalitis de California/patología , Encefalitis de California/virología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Interferón beta/metabolismo , Virus La Crosse/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Permeabilidad , Sustancias Protectoras , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/deficiencia , Tolerancia a Radiación , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/deficiencia , Transducción de Señal , Análisis de Supervivencia , Carga Viral , Fiebre del Nilo Occidental/patología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/fisiología , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
The receptor tyrosine kinase ALK (anaplastic lymphoma kinase), as its name suggests, is aberrantly activated, mutated, or abundant in multiple cancers. Although widely studied in the context of cancer, the ligand that normally binds to and activates ALK in vertebrates has remained elusive. In this issue of Science Signaling, Murray and colleagues identify heparin as an ALK ligand.
Asunto(s)
Activación Enzimática/fisiología , Heparina/metabolismo , Ligandos , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , HumanosRESUMEN
The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.
Asunto(s)
Células Dendríticas/inmunología , Macrófagos/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Apoptosis/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Immunoblotting , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Rastreo , Fagocitosis/inmunología , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosfatidilserinas/metabolismo , Proteína S/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Línea Celular , Embrión de Mamíferos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/genética , Fosfatidilserinas/farmacología , Cultivo Primario de Células , Proteína S/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
Upon activation by the ligands Gas6 and Protein S, Tyro3/Axl/Mer (TAM) receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection. Upon virus challenge, TAM-deficient DCs display type I IFN responses that are elevated in comparison to wild-type cells. As a consequence, TAM-deficient DCs are relatively resistant to infection by flaviviruses and pseudotyped retroviruses, but infection can be restored with neutralizing type I IFN antibodies. Correspondingly, a TAM kinase inhibitor antagonizes the infection of wild-type DCs. Thus, TAM receptors are engaged by viruses in order to attenuate type I IFN signaling and represent potential therapeutic targets.
Asunto(s)
Células Dendríticas/inmunología , Flavivirus/inmunología , Tolerancia Inmunológica , Inmunidad Innata , Retroviridae/inmunología , Animales , Línea Celular , Humanos , Interferón Tipo I/biosíntesis , Ratones , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
Although TAM receptor tyrosine kinases play key roles in immune regulation, cancer metastasis, and viral infection, the relative importance of the two TAM ligands-Gas6 and Protein S-has yet to be resolved in any setting in vivo. We have now performed a genetic dissection of ligand function in the retina, where the TAM receptor Mer is required for the circadian phagocytosis of photoreceptor outer segments by retinal pigment epithelial cells. This process is severely attenuated in Mer mutant mice, which leads to photoreceptor death. We find that retinal deletion of either Gas6 or Protein S alone yields retinae with a normal number of photoreceptors. However, concerted deletion of both ligands fully reproduces the photoreceptor death seen in Mer mutants. These results demonstrate that Protein S and Gas6 function as independent, bona fide Mer ligands, and are, to a first approximation, interchangeable with respect to Mer-driven phagocytosis in the retina.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fagocitosis/fisiología , Proteína S/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Epitelio Pigmentado de la Retina/citología , Retinitis Pigmentosa/fisiopatología , Animales , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Ligandos , Ratones , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/fisiología , Segmento Externo de las Células Fotorreceptoras Retinianas/fisiología , Epitelio Pigmentado de la Retina/fisiología , Tirosina Quinasa c-MerRESUMEN
Autophosphorylation of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) is mediated by a sequential and precisely ordered three-stage autophosphorylation reaction. First-stage autophosphorylation of an activation loop tyrosine leads to 50- to 100-fold stimulation of kinase activity and is followed by second-stage phosphorylation of three additional tyrosine residues, which are binding sites for signaling molecules. Finally, third-stage phosphorylation of a second activation loop tyrosine leads to an additional 10-fold stimulation of FGFR1 catalytic activity. In this report, we show that sequential autophosphorylation of five tyrosines in the FGFR1 kinase domain is under kinetic control, mediated by both the amino acid sequence surrounding the tyrosines and their locations within the kinase structure, and, moreover, that phosphoryl transfer is the rate-limiting step. Furthermore, the strict order of autophosphorylation is disrupted by a glioblastoma-derived, oncogenic FGFR1 point mutation in the kinase domain. We propose that disrupted stepwise activation of tyrosine autophosphorylation caused by oncogenic and other activating FGFR mutations may lead to aberrant activation of and assembly of signaling molecules by the activated receptor.
Asunto(s)
Mutación Puntual , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Sitios de Unión , Glioblastoma/genética , Humanos , Cinética , Fosforilación , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Tirosina/metabolismoRESUMEN
Mutations in fibroblast growth factor receptor 2 (FGFR2) and its ligand, FGF10, are known to cause lacrimo-auriculo-dento-digital (LADD) syndrome. Multiple gain-of-function mutations in FGF receptors have been implicated in a variety of severe skeletal disorders and in many cancers. We aimed to elucidate the mechanism by which a missense mutation in the tyrosine kinase domain of FGFR2, described in the sporadic case of LADD syndrome, leads to reduced tyrosine kinase activity. In this report, we describe the crystal structure of a FGFR2 A628T LADD mutant in complex with a nucleotide analog. We demonstrate that the A628T LADD mutation alters the configuration of key residues in the catalytic pocket that are essential for substrate coordination, resulting in reduced tyrosine kinase activity. Further comparison of the structures of WT FGFR2 and WT FGFR1 kinases revealed that FGFR2 uses a less stringent mode of autoinhibition than FGFR1, which was also manifested in faster in vitro autophosphorylation kinetics. Moreover, the nearly identical conformation of WT FGFR2 kinase and the A628T LADD mutant to either the phosphorylated FGFR2 or FGFR2 harboring pathological activating mutations in the kinase hinge region suggests that FGFR autoinhibition and activation are better explained by changes in the conformational dynamics of the kinase rather than by static crystallographic snapshots of minor structural variations.
Asunto(s)
Anomalías Múltiples , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Adenosina Trifosfato/metabolismo , Alanina/genética , Alanina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación/genética , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Homología Estructural de Proteína , Especificidad por Sustrato , SíndromeRESUMEN
Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.
Asunto(s)
Anomalías Múltiples , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Aparato Lagrimal/anomalías , Deformidades Congénitas de las Extremidades , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Glándulas Salivales/anomalías , Transducción de Señal/fisiología , Anomalías Dentarias , Línea Celular , Factor 10 de Crecimiento de Fibroblastos/genética , Humanos , Modelos Moleculares , Conformación Proteica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , SíndromeRESUMEN
Tyrosine phosphorylation of cellular proteins induced by extracellular cues serves as a critical mediator in the control of a great variety of cellular processes. Here, we describe an integrated experimental approach including rapid quench methodology and ESI-LC-MS/MS as well as time-resolved ESI-MS to demonstrate that tyrosine autophosphorylation of the catalytic tyrosine kinase domain of FGF-receptor-1 (FGFR1) is mediated by a sequential and precisely ordered reaction. We also demonstrate that the rate of catalysis of two FGFR substrates is enhanced by 50- to 100-fold after autophosphorylation of Y653 in the activation loop, whereas autophosphorylation of the second site in the activation loop (Y654) results in 500- to 1,000-fold increase in the rate of substrate phosphorylation. We propose that FGFR1 is activated by a two-step mechanism mediated by strictly ordered and regulated autophosphorylation, suggesting that distinct phosphorylation states may provide both temporal and spatial resolution to receptor signaling.
Asunto(s)
Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Cromatografía Liquida , Humanos , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.