RESUMEN
Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.
Asunto(s)
Melanoma , Receptor de Factor de Crecimiento Nervioso , Humanos , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Tropomiosina , Melanoma/terapia , Receptor trkA/genética , Receptor trkA/metabolismo , Citoprotección , Inhibidores de Puntos de Control Inmunológico , Células T de Memoria , Terapia de Inmunosupresión , Inmunoterapia , Receptores de Antígenos de Linfocitos TRESUMEN
Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.
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Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Endosomas , Factores de Intercambio de Guanina Nucleótido , Factor de Crecimiento Nervioso , Proyección Neuronal , Receptor trkA , Animales , Ratones , Ratas , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Endosomas/metabolismo , Ganglios Espinales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Ratones Noqueados , Factor de Crecimiento Nervioso/metabolismo , Células PC12 , Transporte de Proteínas , Receptor trkA/metabolismoRESUMEN
The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.
Asunto(s)
Actinas/metabolismo , Endosomas/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptor trkA/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Ratones , Neurotrofina 3/metabolismo , Células PC12 , Transporte de Proteínas , Ratas , Transducción de Señal , Sistema Nervioso Simpático/citologíaRESUMEN
In neurons, many membrane proteins, synthesized in cell bodies, must be efficiently delivered to axons to influence neuronal connectivity, synaptic communication, and repair. Previously, we found that axonal targeting of TrkA neurotrophin receptors in sympathetic neurons occurs via an atypical transport mechanism called transcytosis, which relies on TrkA interactions with PTP1B, a protein tyrosine phosphatase. Here, we generated TrkAR685A mice, where TrkA receptor signaling is preserved, but its PTP1B-dependent transcytosis is disrupted to show that this mode of axonal transport is essential for sympathetic neuron development and autonomic function. TrkAR685A mice have decreased axonal TrkA levels in vivo, loss of sympathetic neurons, and reduced innervation of targets. The neuron loss and diminished target innervation phenotypes are specifically restricted to the developmental period when sympathetic neurons are known to rely on the TrkA ligand, nerve growth factor, for trophic support. Postnatal TrkAR685A mice exhibit reduced pupil size and eyelid ptosis, indicative of sympathetic dysfunction. Furthermore, we also observed a significant loss of TrkA-expressing nociceptive neurons in the dorsal root ganglia during development in TrkAR685A mice, suggesting that transcytosis might be a general mechanism for axonal targeting of TrkA receptors. Together, these findings establish the necessity of transcytosis in supplying TrkA receptors to axons, specifically during development, and highlight the physiological relevance of this axon targeting mechanism in the nervous system.
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Neuronas , Receptor trkA , Ratones , Animales , Receptor trkA/genética , Receptor trkA/metabolismo , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Axones/metabolismo , Transcitosis , Sistema Nervioso Simpático/metabolismoRESUMEN
NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.
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Neoplasias , Proteínas de Fusión Oncogénica , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/genética , Neoplasias/genética , Neoplasias/metabolismo , Fusión Génica , Receptor trkA/genética , Receptor trkA/metabolismo , Inhibidores de Proteínas QuinasasRESUMEN
A functional nerve growth factor NGF-Tropomyosin Receptor kinase A (TrkA) system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarifying the involvement of the NGF-TrkA system in pain sensation.
Asunto(s)
Modelos Animales de Enfermedad , Neuropatías Hereditarias Sensoriales y Autónomas , Receptor trkA , Humanos , Animales , Ratones , Mutación , Receptor trkA/genética , Técnicas de Sustitución del Gen , Factor de Crecimiento Nervioso/metabolismo , Fosforilación , Genes Letales , Dolor/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Piel/metabolismo , Piel/patología , Sistema Nervioso Simpático/metabolismo , Hipohidrosis/metabolismo , Conducta AnimalRESUMEN
Nerve growth factor (NGF) antagonism is on the verge of becoming a powerful analgesic treatment for numerous conditions, including osteoarthritis and lower back pain. This review summarizes the historical research, both fundamental and clinical, that led to our current understanding of NGF biology. We also discuss the surprising number of questions that remain about NGF expression patterns and NGF's various functions and interaction partners in relation to persistent pain and the potential side effects of anti-NGF therapy.
Asunto(s)
Factor de Crecimiento Nervioso/metabolismo , Dolor/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Animales , Dolor Crónico/metabolismo , HumanosRESUMEN
Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Resistencia a Antineoplásicos , Reordenamiento Génico , Lamina Tipo A , Mutación , Neurofibromina 2 , Inhibidores de Proteínas Quinasas , Receptor trkA , Sarcoma , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Resistencia a Antineoplásicos/genética , Receptor trkA/genética , Receptor trkA/antagonistas & inhibidores , Receptor trkA/metabolismo , Sarcoma/genética , Sarcoma/tratamiento farmacológico , Sarcoma/patología , Sarcoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Piridonas/farmacología , Benzamidas/farmacología , Pirimidinonas/farmacología , Sirolimus/farmacología , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/patología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Transducción de Señal/efectos de los fármacos , Sinergismo Farmacológico , IndazolesRESUMEN
In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas , Humanos , Resistencia a Antineoplásicos/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Neurofibromina 2/genética , Proteínas de Fusión Oncogénica/genética , Benzamidas/uso terapéutico , Benzamidas/farmacología , Receptor trkA/genética , Receptor trkA/metabolismo , Transducción de Señal/genética , Indazoles/uso terapéutico , Indazoles/farmacología , Mutación , Sarcoma/genética , Sarcoma/tratamiento farmacológico , Sarcoma/patología , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Fibrosarcoma , Neoplasias de los Tejidos Conjuntivo y Blando , Neoplasias de los Tejidos Blandos , Adulto , Humanos , Niño , Receptor trkA/genética , Proteínas Proto-Oncogénicas B-raf/genética , Recurrencia Local de Neoplasia/genética , Fibrosarcoma/genética , Fibrosarcoma/patología , Neoplasias de los Tejidos Blandos/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Proteínas de Fusión Oncogénica/genéticaRESUMEN
The nervous system consists of an ensemble of billions of neurons interconnected in a highly specific pattern that allows proper propagation and integration of neural activities. The organization of these specific connections emerges from sequential developmental events including axon guidance, target selection, and synapse formation. These events critically rely on cell-cell recognition and communication mediated by cell-surface ligands and receptors. Recent studies have uncovered central roles for leucine-rich repeat (LRR) domain-containing proteins, not only in organizing neural connectivity from axon guidance to target selection to synapse formation, but also in various nervous system disorders. Their versatile LRR domains, in particular, serve as key sites for interactions with a wide diversity of binding partners. Here, we focus on a few exquisite examples of secreted or membrane-associated LRR proteins in Drosophila and mammals and review the mechanisms by which they regulate diverse aspects of nervous system development and function.
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Red Nerviosa/embriología , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Proteínas/metabolismo , Animales , Axones/metabolismo , Movimiento Celular/fisiología , Dendritas/metabolismo , Humanos , Proteínas Repetidas Ricas en Leucina , Trastornos Mentales/patología , Trastornos Mentales/fisiopatología , Modelos Moleculares , Vaina de Mielina/metabolismo , Red Nerviosa/anatomía & histología , Vías Nerviosas/anatomía & histología , Vías Nerviosas/embriología , Vías Nerviosas/crecimiento & desarrollo , Neuronas/citología , Neuronas/fisiología , Conformación Proteica , Proteínas/química , Proteínas/genética , Receptor trkA/genética , Receptor trkA/metabolismo , Sinapsis/fisiologíaRESUMEN
We recently identified pathogenic KIF1Bß mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bß in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bß is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bß mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bß-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bß. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bß independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bß loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bß mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.
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Diferenciación Celular/genética , Cinesinas/genética , Cinesinas/metabolismo , Neuroblastoma/genética , Neuronas/citología , Receptor trkA/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Mutación , Neuroblastoma/fisiopatología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Células PC12 , Ratas , Transducción de Señal/genética , Sistema Nervioso Simpático/citología , Proteínas ras/genéticaRESUMEN
Mesenchymal spindle cell tumors with kinase fusions, often presenting in superficial or deep soft tissue locations, may rarely occur in bone. Herein, we describe the clinicopathologic and molecular data of eight bone tumors characterized by various kinase fusions from our files and incorporate the findings with the previously reported seven cases, mainly as single case reports. In the current series all but one of the patients were young children or teenagers, with an age range from newborn to 59 years (mean 19 years). Most tumors (n = 5) presented in the head and neck area (skull base, mastoid, maxilla, and mandible), and remaining three in the tibia, pelvic bone, and chest wall. The fusions included NTRK1 (n = 3), RET (n = 2), NTRK3 (n = 2), and BRAF (n = 1). In the combined series (n = 15), most tumors (73%) occurred in children and young adults (<30 years) and showed a predilection for jaw and skull bones (40%), followed by long and small tubular bones (33%). The fusions spanned a large spectrum of kinase genes, including in descending order NTRK3 (n = 6), NTRK1 (n = 4), RET (n = 2), BRAF (n = 2), and RAF1 (n = 1). All fusions confirmed by targeted RNA sequencing were in-frame and retained the kinase domain within the fusion oncoprotein. Similar to the soft tissue counterparts, most NTRK3-positive bone tumors in this series showed high-grade morphology (5/6), whereas the majority of NTRK1 tumors were low-grade (3/4). Notably, all four tumors presenting in the elderly were high-grade spindle cell sarcomas, with adult fibrosarcoma (FS)-like, malignant peripheral nerve sheath tumor (MPNST)-like and MPNST phenotypes. Overall, 10 tumors had high-grade morphology, ranging from infantile and adult-types FS, MPNST-like, and MPNST, whereas five showed benign/low-grade histology (MPNST-like and myxoma-like). Immunohistochemically (IHC), S100 and CD34 positivity was noted in 57% and 50%, respectively, while co-expression of S100 and CD34 in 43% of cases. One-third of tumors (4 high grade and the myxoma-like) were negative for both S100 and CD34. IHC for Pan-TRK was positive in all eight NTRK-fusion positive tumors tested and negative in two tumors with other kinase fusions. Clinical follow-up was too limited to allow general conclusions.
Asunto(s)
Neoplasias Óseas , Fibrosarcoma , Mixoma , Neurofibrosarcoma , Neoplasias de los Tejidos Blandos , Niño , Recién Nacido , Adolescente , Adulto Joven , Humanos , Preescolar , Anciano , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de los Tejidos Blandos/genética , Fibrosarcoma/genética , Proteínas Tirosina Quinasas Receptoras , Neoplasias Óseas/genética , Proteínas de Fusión Oncogénica/genética , Biomarcadores de Tumor/genética , Receptor trkA/genéticaRESUMEN
The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.
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Diferenciación Celular , Quinasa 5 Dependiente de la Ciclina , Metformina , Factor de Crecimiento Nervioso , Neuronas , Receptor trkA , Animales , Metformina/farmacología , Ratas , Células PC12 , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Receptor trkA/metabolismo , Receptor trkA/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Diferenciación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , FosfotransferasasRESUMEN
BACKGROUND: Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. RESULTS: The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. CONCLUSIONS: The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.
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Prostatitis , Receptor trkA , Vejiga Urinaria Hiperactiva , Animales , Masculino , Ratones , Ratas , Administración Oral , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Factor de Crecimiento Nervioso/antagonistas & inhibidores , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Próstata/efectos de los fármacos , Próstata/patología , Próstata/metabolismo , Prostatitis/tratamiento farmacológico , Prostatitis/patología , Prostatitis/metabolismo , Ratas Sprague-Dawley , Receptor trkA/antagonistas & inhibidores , Receptor trkA/metabolismo , Receptor trkB/antagonistas & inhibidores , Receptor trkB/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/patología , Vejiga Urinaria/metabolismo , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Vejiga Urinaria Hiperactiva/etiologíaRESUMEN
BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.
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Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas , Receptor trkA , Receptor trkB , Receptor trkC , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Receptor trkA/genética , Receptor trkA/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Receptor trkB/antagonistas & inhibidores , Receptor trkB/genética , Receptor trkC/genética , Receptor trkC/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Ratas , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Pirazoles/farmacología , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Pirimidinas/farmacología , Mutación , Femenino , Glicoproteínas de MembranaRESUMEN
Herein, we present a misleading case of advanced papillary thyroid carcinoma with lung, node, and pleural metastases, initially diagnosed as metastatic lung adenocarcinoma with papillary features, based on the histological and immunohistochemical analysis of a pleural biopsy. Between August 2019 and August 2020, the patient received 2 ineffective lines of systemic therapy, including a first line of chemotherapy with cisplatin and pemetrexed, and a second line of immunotherapy with atezolizumab. Comprehensive genomic profiling by next-generation sequencing on the archival pleural biopsy revealed an NTRK1-TMP3 fusion and comutation of the TERT promoter, commonly found in papillary thyroid carcinoma. After palliative partial thyroidectomy that confirmed the diagnosis of papillary thyroid carcinoma, in February 2021, the patient was enrolled in the STARTRK-2 GO40782 basket trial and received entrectinib, an oral pan-TRK inhibitor specifically targeting NTRK-rearranged tumors. After initially experiencing drug-related grade 2 anorexia, dysgeusia, and neurotoxicity and grade 3 asthenia, the dose was reduced, and an excellent and durable objective response was observed.
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Receptor trkA , Neoplasias de la Tiroides , Humanos , Receptor trkA/genética , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genéticaRESUMEN
Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.
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Neoplasias , Humanos , Inmunohistoquímica , Canadá , Anticuerpos Monoclonales , Receptor trkA/genética , Proteínas de Fusión Oncogénica/genética , Biomarcadores de Tumor/genéticaRESUMEN
Protein kinases play crucial roles in many cellular signaling processes, making them become important targets for drug discovery. But drug resistance mediated by mutation puts a barrier to the therapeutic effect of kinase inhibitors. Fragment-based drug discovery has been successfully applied to overcome such resistance. However, the complicate kinase-inhibitor fragment interaction and fragment-to-lead process seriously limit the efficiency of kinase inhibitor discovery against resistance caused by mutation. Here, we constructed a comprehensive web platform KinaFrag for the fragment-based kinase inhibitor discovery to overcome resistance. The kinase-inhibitor fragment space was investigated from 7783 crystal kinase-inhibitor fragment complexes, and the structural requirements of kinase subpockets were analyzed. The core fragment-based virtual screening workflow towards specific subpockets was developed to generate new kinase inhibitors. A series of tropomyosin receptor kinase (TRK) inhibitors were designed, and the most potent compound YT9 exhibits up to 70-fold activity improvement than marketed drugs larotrectinib and selitrectinib against G595R, G667C and F589L mutations of TRKA. YT9 shows promising antiproliferative against tumor cells in vitro and effectively inhibits tumor growth in vivo for wild type TRK and TRK mutants. Our results illustrate the great potential of KinaFrag in the kinase inhibitor discovery to combat resistance mediated by mutation. KinaFrag is freely available at http://chemyang.ccnu.edu.cn/ccb/database/KinaFrag/.
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Antineoplásicos , Neoplasias , Antineoplásicos/uso terapéutico , Humanos , Mutación , Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor trkA/genética , Receptor trkA/metabolismoRESUMEN
Influenza A virus (IAV) infection causes acute respiratory disease with potential severe and deadly complications. Viral pathogenesis is not only due to the direct cytopathic effect of viral infections but also to the exacerbated host inflammatory responses. Influenza viral infection can activate various host signaling pathways that function to activate or inhibit viral replication. Our previous studies have shown that a receptor tyrosine kinase TrkA plays an important role in the replication of influenza viruses in vitro, but its biological roles and functional mechanisms in influenza viral infection have not been characterized. Here we show that IAV infection strongly activates TrkA in vitro and in vivo. Using a chemical-genetic approach to specifically control TrkA kinase activity through a small molecule compound 1NMPP1 in a TrkA knock-in (TrkA KI) mouse model, we show that 1NMPP1-mediated TrkA inhibition completely protected mice from a lethal IAV infection by significantly reducing viral loads and lung inflammation. Using primary lung cells isolated from the TrkA KI mice, we show that specific TrkA inhibition reduced IAV viral RNA synthesis in airway epithelial cells (AECs) but not in alveolar macrophages (AMs). Transcriptomic analysis confirmed the cell-type-specific role of TrkA in viral RNA synthesis, and identified distinct gene expression patterns under the TrkA regulation in IAV-infected AECs and AMs. Among the TrkA-activated targets are various proinflammatory cytokines and chemokines such as IL6, IL-1ß, IFNs, CCL-5, and CXCL9, supporting the role of TrkA in mediating lung inflammation. Indeed, while TrkA inhibitor 1NMPP1 administered after the peak of IAV replication had no effect on viral load, it was able to decrease lung inflammation and provided partial protection in mice. Taken together, our results have demonstrated for the first time an important biological role of TrkA signaling in IAV infection, identified its cell-type-specific contribution to viral replication, and revealed its functional mechanism in virus-induced lung inflammation. This study suggests TrkA as a novel host target for therapeutic development against influenza viral disease.