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BACKGROUND: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare oncogenic drivers prevalent in 0.3% of solid tumors. They are most common in salivary gland cancer (2.6%), thyroid cancer (1.6%), and soft-tissue sarcoma (1.5%). Currently, there are 2 US Food and Drug Administration-approved targeted therapies for NTRK gene fusions: larotrectinib, approved in 2018, and entrectinib, approved in 2019. To date, the real-world uptake of tyrosine receptor kinase inhibitor (TRKi) use for NTRK-positive solid tumors in academic cancer centers remains largely unknown. OBJECTIVE: To describe the demographics, clinical and genomic characteristics, and testing and treatment patterns of patients with NTRK-positive solid tumors treated at US academic cancer centers. METHODS: This was a retrospective chart review study conducted in academic cancer centers in the United States. All patients diagnosed with an NTRK fusion-positive (NTRK1, NTRK2, NTRK3) solid tumor (any stage) and who received cancer treatment at participating sites between January 1, 2012, and July 1, 2023, were included in this study. Patient demographics, clinical characteristics, genomic characteristics, NTRK testing data, and treatment patterns were collected from electronic medical records and analyzed using descriptive statistics as appropriate. RESULTS: In total, 6 centers contributed data for 55 patients with NTRK-positive tumors. The mean age was 49.3 (SD = 20.5) years, 51% patients were female, and the majority were White (78%). The median duration of time from cancer diagnosis to NTRK testing was 85 days (IQR = 44-978). At the time of NTRK testing, 64% of patients had stage IV disease, compared with 33% at cancer diagnosis. Prevalent cancer types in the overall cohort included head and neck (15%), thyroid (15%), brain (13%), lung (13%), and colorectal (11%). NTRK1 fusions were most common (45%), followed by NTRK3 (40%) and NTRK2 (15%). Across all lines of therapy, 51% of patients (n = 28) received a TRKi. Among TRKi-treated patients, 71% had stage IV disease at TRKi initiation. The median time from positive NTRK test to initiation of TRKi was 48 days (IQR = 9-207). TRKis were commonly given as first-line (30%) or second-line (48%) therapies. Median duration of therapy was 610 (IQR = 182-764) days for TRKi use and 207.5 (IQR = 42-539) days for all other first-line therapies. CONCLUSIONS: This study reports on contemporary real-world NTRK testing patterns and use of TRKis in solid tumors, including time between NTRK testing and initiation of TRKi therapy and duration of TRKi therapy.

The alveolar type II epithelial cells (AEC2s) act as stem cells in the lung for alveolar epithelial maintenance and repair. Chemokine C-X-C motif chemokine 10 (CXCL10) is expressed in injured tissues, modulating multiple cellular functions. AEC2s, previously reported to release chemokines to recruit leukocytes, were found in our study to secrete CXCL10 after bleomycin injury. We found that Sftpc-Cxcl10 transgenic mice were protected from bleomycin injury. The transgenic mice showed an increase in the AEC2 population in the lung by flow cytometry analysis. Both endogenous and exogenous CXCL10 promoted the colony formation efficiency of AEC2s in a three-dimensional (3-D) organoid growth assay. We identified that the regenerative effect of CXCL10 was CXCR3 independent using Cxcr3-deficient mice, but it was related to the TrkA pathway. Binding experiments showed that CXCL10 interacted with TrkA directly and reversibly. This study demonstrates a previously unidentified AEC2 autocrine signaling of CXCL10 to promote their regeneration and proliferation, probably involving a CXCR3-independent TrkA pathway. NEW & NOTEWORTHY: CXCL10 may aid in lung injury recovery by promoting the proliferation of alveolar stem cells and using a distinct regulatory pathway from the classical one. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background As a novel necrosis manner, ferroptosis has been increasingly reported to play a role in tumor progression and treatment, however, the specific mechanisms underlying its development in prostate cancer remain unclear. Growing evidence showed that peroxisome plays a key role in ferroptosis. Herein, we identified a novel mechanism for the involvement of ferroptosis in prostate cancer progression, which may provide a new strategy for clinical treatment of prostate cancer. Methods Label-Free Mass spectrometry was used to screen and identify candidate proteins after ferroptosis inducer-ML210 treatment. Immunohistochemistry was undertaken to explore the protein expression of AGPS in prostate cancer tissues compared with normal tissues. Co-immunoprecipitation and GST pull-down were used to identify the directly binding of AGPS to MDM2 in vivo and in vitro. CCK8 assay and colony formation assay were used to illustrate the key role of AGPS in the progression of prostate cancer in vitro. The xenograft model was established to verify the key role of AGPS in the progression of prostate cancer in vivo. Results AGPS protein expression was downregulated in prostate cancer tissues compared with normal tissues from the first affiliated hospital of Zhengzhou University dataset. Lower expression was correlated with poorer overall survival of patients compared to those with high expression of AGPS. In addition, AGPS can promote ferroptosis by modulating the function of peroxisome-resulting in the lower survival of prostate cancer cells. Furthermore, it was shown that AGPS can be ubiquitinated and degraded by the E3 ligase-MDM2 through the proteasomal pathway. Meanwhile, kinase TrkA can promote the combination of AGPS and MDM2 by phosphorylating AGPS at Y451 site. It was verified that kinase TrkA inhibitor—Larotrectinib can increase the susceptibility of prostate cancer cells to ferroptosis, which leads to the inhibition of prostate cancer proliferation to a great extent in vitro and in vivo. Conclusion Based on these findings, we proposed the combination of ferroptosis inducer and TrkA inhibitor to synergistically exert anti-tumor effects, which may provide a new strategy for the clinical treatment of prostate cancer.

Background: As a novel necrosis manner, ferroptosis has been increasingly reported to play a role in tumor progression and treatment, however, the specific mechanisms underlying its development in prostate cancer remain unclear. Growing evidence showed that peroxisome plays a key role in ferroptosis. Herein, we identified a novel mechanism for the involvement of ferroptosis in prostate cancer progression, which may provide a new strategy for clinical treatment of prostate cancer. Methods: Label-Free Mass spectrometry was used to screen and identify candidate proteins after ferroptosis inducer-ML210 treatment. Immunohistochemistry was undertaken to explore the protein expression of AGPS in prostate cancer tissues compared with normal tissues. Co-immunoprecipitation and GST pull-down were used to identify the directly binding of AGPS to MDM2 in vivo and in vitro. CCK8 assay and colony formation assay were used to illustrate the key role of AGPS in the progression of prostate cancer in vitro. The xenograft model was established to verify the key role of AGPS in the progression of prostate cancer in vivo. Results: AGPS protein expression was downregulated in prostate cancer tissues compared with normal tissues from the first affiliated hospital of Zhengzhou University dataset. Lower expression was correlated with poorer overall survival of patients compared to those with high expression of AGPS. In addition, AGPS can promote ferroptosis by modulating the function of peroxisome-resulting in the lower survival of prostate cancer cells. Furthermore, it was shown that AGPS can be ubiquitinated and degraded by the E3 ligase-MDM2 through the proteasomal pathway. Meanwhile, kinase TrkA can promote the combination of AGPS and MDM2 by phosphorylating AGPS at Y451 site. It was verified that kinase TrkA inhibitor—Larotrectinib can increase the susceptibility of prostate cancer cells to ferroptosis, which leads to the inhibition of prostate cancer proliferation to a great extent in vitro and in vivo. Conclusion: Based on these findings, we proposed the combination of ferroptosis inducer and TrkA inhibitor to synergistically exert anti-tumor effects, which may provide a new strategy for the clinical treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300–345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization. [ABSTRACT FROM AUTHOR]

Abstract Background Triple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood. Methods Using a human blood–brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors. Results In this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model. Conclusions These disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

Background: Triple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood. Methods: Using a human blood–brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors. Results: In this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model. Conclusions: These disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Introduction: Axonal transport of pro nerve growth factor (proNGF) is impaired in aged basal forebrain cholinergic neurons (BFCNs), which is associated with their degeneration. ProNGF is neurotrophic in the presence of its receptor tropomyosin-related kinase A (TrkA) but induces apoptosis via the pan-neurotrophin receptor (p75NTR) when TrkA is absent. It is well established that TrkA is lost while p75NTR is maintained in aged BFCNs, but whether aging differentially affects transport of proNGF via each receptor is unknown. Nitrative stress increases during aging, but whether age-induced nitrative stress differentially affects proNGF transport via TrkA versus p75NTR has not yet been studied. Answering these questions is essential for developing an accurate understanding of the mechanisms contributing to age-induced loss of proNGF transport and BFCN degeneration. Methods: In this study, fluorescence microscopy was used to analyze axonal transport of quantum dot labeled proNGF in rat BFCNs in vitro. Receptor specific effects were studied with proNGF mutants that selectively bind to either TrkA (proNGF-KKE) or p75NTR (proNGF-Δ9-13). Signaling factor activity was quantified via immunostaining. Results: Young BFCNs transported proNGF-KKE but not proNGF-Δ9-13, and proNGF transport was not different in p75NTR knockout BFCNs compared to wildtype BFCNs. These results indicate that young BFCNs transport proNGF via TrkA. In vitro aging increased transport of proNGF-Δ9-13 but decreased transport of proNGF-KKE. Treatment with the nitric oxide synthase inhibitor L-NAME reduced retrograde transport of proNGF-Δ9-13 in aged BFCNs while increasing retrograde transport of proNGF-KKE but did not affect TrkA or p75NTR levels. ProNGF-Δ9-13 induced greater pro-apoptotic signaling and neurodegeneration and less pro-survival signaling relative to proNGF-KKE. Discussion: Together, these results indicate that age-induced nitrative stress decreases proNGF transport via TrkA while increasing proNGF transport via p75NTR. These transport deficits are associated with decreased survival signaling, increased apoptotic signaling, and neurodegeneration. Our findings elucidate the receptor specificity of age-and nitrative stress-induced proNGF transport deficits. These results may help to rescue the neurotrophic signaling of proNGF in aging to reduce age-induced loss of BFCN function and cognitive decline. [ABSTRACT FROM AUTHOR]

It has been reported that Banxia‐houpo decoction (BXHPD) serves as the anti‐depressant treatment for a mild and severe depressive disease with limited side effects. The present study was performed to evaluate the protective effect of BXHPD on chronic unpredicted mild stress (CUMS)‐induced depression and explore its effect on TrkA/Akt‐mediated microglia polarization. The CUMS procedure was carried out, and the mice were intragastrically treated with BXHPD once daily. The selective TrkA inhibitor GW441756 was applied to further investigate the role of TrkA in BXHPD‐mediated microglia polarization. The behaviour test including open field test (OFT), sucrose preference test (SPT), novelty‐suppressed feeding test (NSFT), tail suspension test (TST) and forced swim test (FST) was performed. The concentrations of pro‐inflammatory cytokines IL‐6, TNF‐α, IL‐1β, IL‐12 and anti‐inflammatory cytokines IL‐4, IL‐10 were determined using Enzyme‐linked immunosorbent assay. The population of Iba1+ cells and the length of microglia processes were observed under the fluorescence microscope. The mRNA expressions of Arg1, Ym1 and Fizzl1 were measured by PCR. The protein expressions of TrkA, p‐Tyr490‐TrkA, p‐Ser473‐Akt, p‐Ser473‐Akt1, p‐Ser474‐Akt2, p‐CREB and Jmjd3 were detected by western blot. Our results showed that BXHPD attenuated CUMS‐induced depressive‐like behaviour, promoted anti‐inflammatory cytokines, inhibited pro‐inflammatory cytokines, suppressed microglia activation, promoted M2 phenotype‐specific indices and upregulated the expressions of TrkA, p‐Tyr490‐TrkA, p‐Ser473‐Akt, p‐Ser473‐Akt1, p‐Ser474‐Akt2, p‐CREB and Jmjd3. The above beneficial effect of BXHPD can be blocked by TrkA inhibitor GW441756. This work demonstrated that BXHPD exerted an anti‐depressant effect by promoting M2 phenotype microglia polarization via TrkA/Akt pathway. [ABSTRACT FROM AUTHOR]

Introduction: The neurotrophin system plays a pivotal role in the development, morphology, and survival of the nervous system, and its dysregulation has been manifested in numerous neurodegenerative and neuroinflammatory diseases. Neurotrophins NGF and BDNF are major growth factors that prevent neuronal death and synaptic loss through binding with high affinity to their specific tropomyosin-related kinase receptors namely, TrkA and TrkB, respectively. The poor pharmacokinetic properties prohibit the use of neurotrophins as therapeutic agents. Our group has previously synthesized BNN27, a prototype small molecule based on dehydroepiandrosterone, mimicking NGF through the activation of the TrkA receptor. Methods: To obtain a better understanding of the stereo-electronic requirements for selective activation of TrkA and TrkB receptors, 27 new dehydroepiandrosterone derivatives bearing a C17-spiro-dihydropyran or cyclobutyl moiety were synthesized. The new compounds were evaluated for their ability (a) to selectively activate the TrkA receptor and its downstream signaling kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death, and (b) to induce phosphorylation of TrkB and to promote cell survival under serum deprivation conditions in NIH3T3 cells stable transfected with the TrkB receptor and primary cortical astrocytes. In addition the metabolic stability and CYP-mediated reaction was assessed. Results: Among the novel derivatives, six were able to selectively protect PC12 cells through interaction with the TrkA receptor and five more to selectively protect TrkB-expressing cells via interaction with the TrkB receptor. In particular, compound ENT-A025 strongly induces TrkA and Erk1/2 phosphorylation, comparable to NGF, and can protect PC12 cells against serum deprivation- induced cell death. Furthermore, ENT-A065, ENT-A066, ENT-A068, ENT-A069, and ENT-A070 showed promising pro-survival effects in the PC12 cell line. Concerning TrkB agonists, ENT-A009 and ENT-A055 were able to induce phosphorylation of TrkB and reduce cell death levels in NIH3T3-TrkB cells. In addition, ENT- A076, ENT-A087, and ENT-A088 possessed antiapoptotic activity in NIH-3T3-TrkB cells exclusively mediated through the TrkB receptor. The metabolic stability and CYP-mediated reaction phenotyping of the potent analogs did not reveal any major liabilities. Discussion: We have identified small molecule selective agonists of TrkA and TrkB receptors as promising lead neurotrophin mimetics for the development of potential therapeutics against neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

Basal forebrain cholinergic neurons (BFCNs) modulate cognitive functions such as attention, learning and memory. The NGF/TrkA pathway plays an important role in the development and function of BFCNs, although two mouse models conditionally deleting TrkA expression in the central nervous system (CNS) have shown contradictory results. To shed light into this discrepancy, we used a mouse model with a gain-of-function in TrkA receptor signaling. Our results indicate that enhanced TrkA signaling did not alter hippocampal cholinergic innervation, general locomotion or anxiety-related behaviors, but it increases ChAT expression, the number of cholinergic neurons at early postnatal stages and, mutant mice showed impaired motor learning and memory functions. These data demonstrate that proper functioning of the cholinergic system in CNS requires a balanced NGF/TrkA signaling. [ABSTRACT FROM AUTHOR]

The tropomyosin receptor kinase (TRK) family of proteins is encoded by neurotrophic tyrosine receptor kinase (NTRK) genes and has a role in the development and normal functioning of the nervous system. NTRK gene fusions have been identified as oncogenic drivers in a wide range of tumors in both adult and pediatric patients. There has recently been a paradigm shift in cancer treatment toward biomarker-based targeted therapies, as an increasing number of actionable targets are being identified across different tumors and/or tumor histologies. These targeted agents offer greater comparative effectiveness and safety vs historical nontargeted standard therapies. The development of drugs that specifically target oncogenic drivers of cancer has led to the emergence of screening technologies to identify the patients most likely to benefit from targeted therapy. This review describes the role of NTRK gene fusions in cancer and outlines the epidemiology of NTRK gene fusions, the therapeutic benefits of targeting TRK fusions with small molecule inhibitors, and recommendations for NTRK gene fusion testing in adult and pediatric patients with cancer, in order to guide treatment decisions.

Transductional targeting of herpes simplex virus (HSV)-based gene therapy vectors offers the potential for improved tissue-specific delivery and can be achieved by modification of the viral entry machinery to incorporate ligands that bind the desired cell surface proteins. The interaction of nerve growth factor (NGF) with tropomyosin receptor kinase A (TrkA) is essential for survival of sensory neurons during development and is involved in chronic pain signaling. We targeted HSV infection to TrkA-bearing cells by replacing the signal peptide and HVEM binding domain of glycoprotein D (gD) with pre-pro-NGF. This TrkA-targeted virus (KNGF) infected cells via both nectin-1 and TrkA. However, infection through TrkA was inefficient, prompting a genetic search for KNGF mutants showing enhanced infection following repeat passage on TrkA-expressing cells. These studies revealed unique point mutations in envelope glycoprotein gH and in UL24, a factor absent from mature particles. Together these mutations rescued efficient infection of TrkA-expressing cells, including neurons, and facilitated the production of a completely retargeted KNGF derivative. These studies provide insight into HSV vector improvements that will allow production of replication-defective TrkA-targeted HSV for delivery to the peripheral nervous system and may be applied to other retargeted vector studies in the central nervous system.

IntroductionAxonal transport of pro nerve growth factor (proNGF) is impaired in aged basal forebrain cholinergic neurons (BFCNs), which is associated with their degeneration. ProNGF is neurotrophic in the presence of its receptor tropomyosin-related kinase A (TrkA) but induces apoptosis via the pan-neurotrophin receptor (p75NTR) when TrkA is absent. It is well established that TrkA is lost while p75NTR is maintained in aged BFCNs, but whether aging differentially affects transport of proNGF via each receptor is unknown. Nitrative stress increases during aging, but whether age-induced nitrative stress differentially affects proNGF transport via TrkA versus p75NTR has not yet been studied. Answering these questions is essential for developing an accurate understanding of the mechanisms contributing to age-induced loss of proNGF transport and BFCN degeneration.MethodsIn this study, fluorescence microscopy was used to analyze axonal transport of quantum dot labeled proNGF in rat BFCNs in vitro. Receptor specific effects were studied with proNGF mutants that selectively bind to either TrkA (proNGF-KKE) or p75NTR (proNGF-Δ9-13). Signaling factor activity was quantified via immunostaining.ResultsYoung BFCNs transported proNGF-KKE but not proNGF-Δ9-13, and proNGF transport was not different in p75NTR knockout BFCNs compared to wildtype BFCNs. These results indicate that young BFCNs transport proNGF via TrkA. In vitro aging increased transport of proNGF-Δ9-13 but decreased transport of proNGF-KKE. Treatment with the nitric oxide synthase inhibitor L-NAME reduced retrograde transport of proNGF-Δ9-13 in aged BFCNs while increasing retrograde transport of proNGF-KKE but did not affect TrkA or p75NTR levels. ProNGF-Δ9-13 induced greater pro-apoptotic signaling and neurodegeneration and less pro-survival signaling relative to proNGF-KKE.DiscussionTogether, these results indicate that age-induced nitrative stress decreases proNGF transport via TrkA while increasing proNGF transport via p75NTR. These transport deficits are associated with decreased survival signaling, increased apoptotic signaling, and neurodegeneration. Our findings elucidate the receptor specificity of age-and nitrative stress-induced proNGF transport deficits. These results may help to rescue the neurotrophic signaling of proNGF in aging to reduce age-induced loss of BFCN function and cognitive decline.

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot–Marie–Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease. [ABSTRACT FROM AUTHOR]

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

IntroductionThe neurotrophin system plays a pivotal role in the development, morphology, and survival of the nervous system, and its dysregulation has been manifested in numerous neurodegenerative and neuroinflammatory diseases. Neurotrophins NGF and BDNF are major growth factors that prevent neuronal death and synaptic loss through binding with high affinity to their specific tropomyosin-related kinase receptors namely, TrkA and TrkB, respectively. The poor pharmacokinetic properties prohibit the use of neurotrophins as therapeutic agents. Our group has previously synthesized BNN27, a prototype small molecule based on dehydroepiandrosterone, mimicking NGF through the activation of the TrkA receptor.MethodsTo obtain a better understanding of the stereo-electronic requirements for selective activation of TrkA and TrkB receptors, 27 new dehydroepiandrosterone derivatives bearing a C17-spiro-dihydropyran or cyclobutyl moiety were synthesized. The new compounds were evaluated for their ability (a) to selectively activate the TrkA receptor and its downstream signaling kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death, and (b) to induce phosphorylation of TrkB and to promote cell survival under serum deprivation conditions in NIH3T3 cells stable transfected with the TrkB receptor and primary cortical astrocytes. In addition the metabolic stability and CYP-mediated reaction was assessed.ResultsAmong the novel derivatives, six were able to selectively protect PC12 cells through interaction with the TrkA receptor and five more to selectively protect TrkB-expressing cells via interaction with the TrkB receptor. In particular, compound ENT-A025 strongly induces TrkA and Erk1/2 phosphorylation, comparable to NGF, and can protect PC12 cells against serum deprivation-induced cell death. Furthermore, ENT-A065, ENT-A066, ENT-A068, ENT-A069, and ENT-A070 showed promising pro-survival effects in the PC12 cell line. Concerning TrkB agonists, ENT-A009 and ENT-A055 were able to induce phosphorylation of TrkB and reduce cell death levels in NIH3T3-TrkB cells. In addition, ENT-A076, ENT-A087, and ENT-A088 possessed antiapoptotic activity in NIH-3T3-TrkB cells exclusively mediated through the TrkB receptor. The metabolic stability and CYP-mediated reaction phenotyping of the potent analogs did not reveal any major liabilities.DiscussionWe have identified small molecule selective agonists of TrkA and TrkB receptors as promising lead neurotrophin mimetics for the development of potential therapeutics against neurodegenerative conditions.

Basal forebrain cholinergic neurons (BFCNs) modulate cognitive functions such as attention, learning and memory. The NGF/TrkA pathway plays an important role in the development and function of BFCNs, although two mouse models conditionally deleting TrkA expression in the central nervous system (CNS) have shown contradictory results. To shed light into this discrepancy, we used a mouse model with a gain-of-function in TrkA receptor signaling. Our results indicate that enhanced TrkA signaling did not alter hippocampal cholinergic innervation, general locomotion or anxiety-related behaviors, but it increases ChAT expression, the number of cholinergic neurons at early postnatal stages and, mutant mice showed impaired motor learning and memory functions. These data demonstrate that proper functioning of the cholinergic system in CNS requires a balanced NGF/TrkA signaling.

Prion protein peptide (PrP) has demonstrated neurotoxicity in brain cells, resulting in the progression of prion diseases with spongiform degenerative, amyloidogenic, and aggregative properties. Thymosin beta 4 (Tβ4) plays a role in the nervous system and may be related to motility, axonal enlargement, differentiation, neurite outgrowth, and proliferation. However, no studies about the effects of Tβ4 on prion disease have been performed yet. In the present study, we investigated the protective effect of Tβ4 against synthetic PrP (106–126) and considered possible mechanisms. Hippocampal neuronal HT22 cells were treated with Tβ4 and PrP (106–126) for 24 h. Tβ4 significantly reversed cell viability and reactive oxidative species (ROS) affected by PrP (106–126). Apoptotic proteins induced by PrP (106–126) were reduced by Tβ4. Interestingly, a balance of neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) and receptors (nerve growth factor receptor p75, tropomyosin related kinase A and B) were competitively maintained by Tβ4 through receptors reacting to PrP (106–126). Our results demonstrate that Tβ4 protects neuronal cells against PrP (106–126) neurotoxicity via the interaction of neurotrophic factors/receptors. [ABSTRACT FROM AUTHOR]

Simple Summary: NGF was the first growth factor discovered by Rita Levi Montalcini in 1950. TrkA, its high affinity receptor, is an oncogene that is overexpressed in many cancers. However, targeted therapies against TrkA, in particular kinase inhibitors, have not yet demonstrated efficacy in the context of overexpression. In this review, after describing the state-of-the-art TrkA-targeted therapies, we will elicit the failures of these therapies by focusing on non-genomic resistance. Larotrectinib and Entrectinib are specific pan-Trk tyrosine kinase inhibitors (TKIs) approved by the Food and Drug Administration (FDA) in 2018 for cancers with an NTRK fusion. Despite initial enthusiasm for these compounds, the French agency (HAS) recently reported their lack of efficacy. In addition, primary and secondary resistance to these TKIs has been observed in the absence of other mutations in cancers with an NTRK fusion. Furthermore, when TrkA is overexpressed, it promotes ligand-independent activation, bypassing the TKI. All of these clinical and experimental observations show that genetics does not explain all therapeutic failures. It is therefore necessary to explore new hypotheses to explain these failures. This review summarizes the current status of therapeutic strategies with TrkA inhibitors, focusing on the mechanisms potentially involved in these failures and more specifically on the role of TrkA. [ABSTRACT FROM AUTHOR]

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol–water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol–water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains—transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) regulates many aspects of neuronal biology by retrogradely propagating signals along axons to the targets of those axons. How this occurs when axons contain a plethora of proteins that can silence those signals has long perplexed the neurotrophin field. In this issue of the JCI, Li et al. suggest an answer to this vexing problem, while exploring why the Elp1 gene that is mutated in familial dysautonomia (FD) causes peripheral neuropathy. They describe a distinctive function of Elp1 as a protein that is required to sustain NGF signaling by blocking the activity of its phosphatase that shuts off those signals. This finding helps explain the innervation deficits prominent in FD and reveals a unique role for Elp1 in the regulation of NGF-dependent TrkA activity.

BackgroundEntrectinib is a potent inhibitor of tropomyosin receptor kinase (TRK) A, B, and C, which has been shown to have anti-tumour activity against NTRK gene fusion-positive solid tumours, including CNS activity due to its ability to penetrate the blood-brain barrier. We present an integrated efficacy and safety analysis of patients with metastatic or locally advanced solid tumours harbouring oncogenic NTRK1, NTRK2, and NTRK3 gene fusions treated in three ongoing, early-phase trials.MethodsAn integrated database comprised the pivotal datasets of three, ongoing phase 1 or 2 clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2), which enrolled patients aged 18 years or older with metastatic or locally advanced NTRK fusion-positive solid tumours who received entrectinib orally at a dose of at least 600 mg once per day in a capsule. All patients had an Eastern Cooperative Oncology Group performance status of 0-2 and could have received previous anti-cancer therapy (except previous TRK inhibitors). The primary endpoints, the proportion of patients with an objective response and median duration of response, were evaluated by blinded independent central review in the efficacy-evaluable population (ie, patients with NTRK fusion-positive solid tumours who were TRK inhibitor-naive and had received at least one dose of entrectinib). Overall safety evaluable population included patients from STARTRK-1, STARTRK-2, ALKA-372-001, and STARTRK-NG (NCT02650401; treating young adult and paediatric patients [aged ≤21 years]), who received at least one dose of entrectinib, regardless of tumour type or gene rearrangement. NTRK fusion-positive safety evaluable population comprised all patients who have received at least one dose of entrectinib regardless of dose or follow-up. These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001).FindingsPatients were enrolled in ALKA-372-001 from Oct 26, 2012, to March 27, 2018; in STARTRK-1 from Aug 7, 2014, to May 10, 2018; and in STARTRK-2 from Nov 19, 2015 (enrolment is ongoing). At the data cutoff date for this analysis (May 31, 2018) the efficacy-evaluable population comprised 54 adults with advanced or metastatic NTRK fusion-positive solid tumours comprising ten different tumour types and 19 different histologies. Median follow-up was 12.9 months (IQR 8·77-18·76). 31 (57%; 95% CI 43·2-70·8) of 54 patients had an objective response, of which four (7%) were complete responses and 27 (50%) partial reponses. Median duration of response was 10 months (95% CI 7·1 to not estimable). The most common grade 3 or 4 treatment-related adverse events in both safety populations were increased weight (seven [10%] of 68 patients in the NTRK fusion-positive safety population and in 18 [5%] of 355 patients in the overall safety-evaluable population) and anaemia (8 [12%] and 16 [5%]). The most common serious treatment-related adverse events were nervous system disorders (three [4%] of 68 patients and ten [3%] of 355 patients). No treatment-related deaths occurred.InterpretationEntrectinib induced durable and clinically meaningful responses in patients with NTRK fusion-positive solid tumours, and was well tolerated with a manageable safety profile. These results show that entrectinib is a safe and active treatment option for patients with NTRK fusion-positive solid tumours. These data highlight the need to routinely test for NTRK fusions to broaden the therapeutic options available for patients with NTRK fusion-positive solid tumours.FundingIgnyta/F Hoffmann-La Roche.

Previously, we reported a family in which bipolar disorder (BD) co-segregates with a Mendelian kidney disorder linked to 1q22. The causative renal gene was later identified as MUC1. Genome-wide linkage analysis of BD in the family yielded a peak at 1q22 that encompassed the NTRK1 and MUC1 genes. NTRK1 codes for TrkA (Tropomyosin-related kinase A) which is essential for development of the cholinergic nervous system. Whole genome sequencing of the proband identified a damaging missense mutation, E492K, in NTRK1. Induced pluripotent stem cells were generated from family members, and then differentiated to neural stem cells (NSCs). E492K NSCs had reduced neurite outgrowth. A conditional knock-in mouse line, harboring the point mutation in the brain, showed depression-like behavior in the tail suspension test following challenge by physostigmine, a cholinesterase inhibitor. These results are consistent with the cholinergic hypothesis of depression. They imply that the NTRK1 E492K mutation, impairs cholinergic neurotransmission, and may convey susceptibility to bipolar disorder.

ObjectiveTo investigate the TrkA inhibitor, ASP7962, for treatment of painful knee osteoarthritis.DesignPhase 2a, double-blind, placebo- and naproxen-controlled, double-dummy, parallel-group study. Adults with knee osteoarthritis were randomized (2:2:1) to ASP7962 (100 mg), placebo, or naproxen (500 mg) twice daily (BID) for 4 weeks. Primary endpoint: change from baseline to Week 4 in Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain subscale score. Secondary endpoints: change from baseline to Weeks 1, 2, and End of Treatment (EoT) in WOMAC pain subscale score; change from baseline to Weeks 1, 2, 4, and EoT in WOMAC physical function and stiffness subscales, walking pain and WOMAC total scores; and change from baseline in daily average pain score.Results215 participants were randomized (ASP7962 100 mg BID, n = 85; placebo, n = 87; naproxen 500 mg BID, n = 43). No significant difference was observed between ASP7962 and placebo in change from baseline to Week 4 in WOMAC pain subscale score (-0.14; 90% 2-sided CI: -0.62, 0.34; P = 0.316); a significant difference was observed between naproxen and placebo (-0.67; 80% 2-sided CI: -1.12, -0.23; P = 0.027). No differences were observed between ASP7962 and placebo in change from baseline in any WOMAC subscale score; statistically significant changes were observed between naproxen and placebo (P ≤ 0.01, all time points for all WOMAC endpoints). ASP7962 was safe and well-tolerated.ConclusionsFour-week treatment with ASP7962 (100 mg BID) did not improve pain or physical function in individuals with painful knee osteoarthritis. ClinicalTrials.gov, NCT02611466; EudraCT Number, 2014-004996-22.

Nerve growth factor (NGF) plays a key role in controlling growth, maintenance and survival of neurons as well as nociceptor development. Many of its critical actions are believed to be mediated by activation of corresponding transcription factors. Here I attempt to reveal signaling cascades that relay NGF stimulation to NFAT, a family of Ca2+/calcineurin-dependent transcription factors that are implicated in playing a pivotal role in neuronal development and survival, and pain sensation. With a luciferase gene reporter assay, it was confirmed that NGF can trigger NFAT-dependent gene expression in PC12 cells. Subsequent research with pharmacological tools uncovers that this effect was mediated by the TrkA-MEK1/2 pathway. Furthermore, it was determined that NFATc3 is the predominant isoform by calculating an absolute copy number of each NFAT isoform with quantitative PCR. Taken together, it is proposed that elucidating cellular connections linking NGF and NFAT may help reveal novel therapeutic targets, which can be exploited to devise innovative remedies for the treatment of NGF and NFAT-associated clinical disorders such as anomalous neuronal development and hyperalgesia.

Breast cancer represents the most common type of cancer and is the leading cause of death due to cancer among women. Thus, the prevention and early diagnosis of breast cancer is of primary urgency, as well as the development of new treatments able to improve its prognosis. Nerve Growth Factor (NGF) is a neurotrophic factor involved in the regulation of neuronal functions through the binding of the Tropomyosin receptor kinase A (TrkA) and the Nerve Growth Factor receptor or Pan-Neurotrophin Receptor 75 (NGFR/p75NTR). In addition, its precursor (pro-NGF) can extert biological activity by forming a trimeric complex with NGFR/p75NTR and sortilin, or by binding to TrkA receptors with low affinity. Several examples of in vitro and in vivo evidence show that NGF is both synthesized and released by breast cancer cells, and has mitogen, antiapoptotic and angiogenic effects on these cells through the activation of different signaling cascades that involve TrkA and NGFR/p75NTR receptors. Conversely, pro-NGF signaling has been related to breast cancer invasion and metastasis. Other studies suggested that NGF and its receptors could represent a good diagnostic and prognostic tool, as well as promising therapeutic targets for breast cancer. In this paper, we comprehensively summarize and systematically review the current experimental evidence on this topic. INPLASY ID: INPLASY2022100017.

Abstract Background Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation 11C and 18F-labeled Trk inhibitors, e.g., [18F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [18F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe. Methods In vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [18F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2 tm1Bbd /J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples. Results Uptake of [18F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [18F]TRACK uptake in vitro (IC50 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [18F]TRACK showed uptake into KM12 tumors (SUVmean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [18F]TRACK uptake reached SUVmean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUVmean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue. Conclusion The present data show that radiotracer [18F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.

Supplemental Digital Content is Available in the Text. Sánchez-Sánchez et al. demonstrated that ARMS/Kidins220 protein plays a seminal role in the thermal, inflammatory, and capsaicin nociception that is dependent on brain-derived neurotrophic factor secretion and that ARMS levels are reduced in vitro and in vivo in response to capsaicin. Pain is an alarm mechanism to prevent body damage in response to noxious stimuli. The nerve growth factor (NGF)/TrkA axis plays an essential role as pain mediator, and several clinical trials using antibodies against NGF have yielded promising results, but side effects have precluded their clinical approval. A better understanding of the mechanism of NGF/TrkA–mediated nociception is needed. Here, we find that ARMS/Kidins220, a scaffold protein for Trk receptors, is a modulator of nociception. Male mice, with ARMS/Kidins220 reduction exclusively in TrkA-expressing cells, displayed hyperalgesia to heat, inflammatory, and capsaicin stimuli, but not to cold or mechanical stimuli. Simultaneous deletion of brain-derived neurotrophic factor (BDNF) reversed the effects of ARMS/Kidins220 knock down alone. Mechanistically, ARMS/Kidins220 levels are reduced in vitro and in vivo in response to capsaicin through calpains, and this reduction leads to enhanced regulated BDNF secretion from dorsal root ganglion. Altogether, these data indicate that ARMS/Kidins220 protein levels have a role as a pain modulator in the NGF/TrkA axis regulating BDNF secretion. [ABSTRACT FROM AUTHOR]

TrkA and TrkB knockout HT-22 cell lines were used to prove that the neuroprotective properties of GK-2 [bis(N-monosuccinyl-L-glutamyl-L-lysine hexamethylenediamide], a low-molecular-weight dipeptide mimetic of the NGF 4th loop, are definitely mediated by selective interaction with TrkA receptor upon oxidative stress-induced cellular damage in vitro. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) is a neurotrophin that has been implicated in pain signaling, apoptosis, inflammation and proliferation. The resultant effects depend on interaction with two different receptors; tyrosine kinase A (TrkA) and p75NTR. NGF increases in synovial fluid from osteoarthritic joints, and monoclonal antibody therapy is trialed to treat osteoarthritis (OA)‐related pain. Investigation of the complex and somewhat contradictory signaling pathways of NGF is conducted in neural research, but has not followed through to orthopaedic studies. The objectives of this study were to compare the expression of NGF receptors and the downstream regulator BAX in synovial membranes from joints in various stages of OA. The horse was used as a model. Synovial membranes were harvested from five healthy horses postmortem and from clinical cases with spontaneous OA undergoing arthroscopic surgery for lameness. Four horses with synovitis without gross cartilage changes, four horses with synovitis and cartilage damage, and four horses with synovitis and intracarpal fractures were included. Samples were investigated by immunohistochemistry and results showed that nuclear staining of TrkA, p75NTR and BAX increases in OA‐associated synovitis. TrkA expression increased in early disease stages whereas increases in p75NTR were most prominent in later disease stages with cartilage damage and fibrosis. Clinical significance: Suppression of NGF may result in varied effects depending on different stages of the osteoarthritic disease process. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) was the first neurotrophin described. This neurotrophin contributes to organogenesis by promoting sensory innervation and angiogenesis in the endocrine and immune systems. Neuronal and non-neuronal cells produce and secrete NGF, and several cell types throughout the body express the high-affinity neurotrophin receptor TrkA and the low-affinity receptor p75NTR. NGF is essential for glucose-stimulated insulin secretion and the complete development of pancreatic islets. Plus, this factor is involved in regulating lipolysis and thermogenesis in adipose tissue. Immune cells produce and respond to NGF, modulating their inflammatory phenotype and the secretion of cytokines, contributing to insulin resistance and metabolic homeostasis. This neurotrophin regulates the synthesis of gonadal steroid hormones, which ultimately participate in the metabolic homeostasis of other tissues. Therefore, we propose that this neurotrophin's imbalance in concentrations and signaling during metabolic syndrome contribute to its pathophysiology. In the present work, we describe the multiple roles of NGF in immunoendocrine organs that are important in metabolic homeostasis and related to the pathophysiology of metabolic syndrome. [ABSTRACT FROM AUTHOR]

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM. [ABSTRACT FROM AUTHOR]

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC’s interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC’s in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300–345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

Using TrkA or TrkB receptor gene knockout HT-22 cells, the selectivity of the interaction of the low-molecular-weight dipeptide BDNF mimetic GSB-106 (hexamethylenediamide bis(N-monosuccinyl-L-seryl-L-lysine)) with TrkB receptors was shown. [ABSTRACT FROM AUTHOR]

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

BackgroundThe highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection.MethodsA total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively.ResultsA total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (>98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues.ConclusionsChildren with locally advanced TRK fusion sarcomas may proceed to surgical resection after treatment with the selective TRK inhibitor larotrectinib, thereby sparing them the potentially significant morbidity noted with current approaches. These results support the evaluation of larotrectinib as presurgical therapy in children with newly diagnosed TRK fusion sarcomas.

The binding of nerve growth factor (NGF) to the tropomyosin–related kinase A (TrkA) and p75NTR receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA‐p75NTR interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma‐derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75NTR, or both. We found that TrkA‐null cells become unresponsive to NGF. Conversely, the absence of p75NTR enhances the phosphorylation of TrkA and its effectors. Using a patch‐clamp, we demonstrated that the individual activation of TrkA and p75NTR by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75NTR in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing. [ABSTRACT FROM AUTHOR]

Breast cancer represents the most common type of cancer and is the leading cause of death due to cancer among women. Thus, the prevention and early diagnosis of breast cancer is of primary urgency, as well as the development of new treatments able to improve its prognosis. Nerve Growth Factor (NGF) is a neurotrophic factor involved in the regulation of neuronal functions through the binding of the Tropomyosin receptor kinase A (TrkA) and the Nerve Growth Factor receptor or Pan-Neurotrophin Receptor 75 (NGFR/p75NTR). In addition, its precursor (pro-NGF) can extert biological activity by forming a trimeric complex with NGFR/p75NTR and sortilin, or by binding to TrkA receptors with low affinity. Several examples of in vitro and in vivo evidence show that NGF is both synthesized and released by breast cancer cells, and has mitogen, antiapoptotic and angiogenic effects on these cells through the activation of different signaling cascades that involve TrkA and NGFR/p75NTR receptors. Conversely, pro-NGF signaling has been related to breast cancer invasion and metastasis. Other studies suggested that NGF and its receptors could represent a good diagnostic and prognostic tool, as well as promising therapeutic targets for breast cancer. In this paper, we comprehensively summarize and systematically review the current experimental evidence on this topic. INPLASY ID: INPLASY2022100017. [ABSTRACT FROM AUTHOR]

Among the factors involved in diabetic retinopathy (DR), nerve growth factor (NGF) and vascular endothelial growth factor A (VEGFA) have been shown to affect both neuronal survival and vascular function, suggesting that their crosstalk might influence DR outcomes. To address this question, the administration of eye drops containing NGF (ed-NGF) to adult Sprague Dawley rats receiving streptozotocin (STZ) intraperitoneal injection was used as an experimental paradigm to investigate NGF modulation of VEGFA and its receptor VEGFR2 expression. We show that ed-NGF treatment prevents the histological and vascular alterations in STZ retina, VEGFR2 expression decreased in GCL and INL, and preserved the co-expression of VEGFR2 and NGF-tropomyosin-related kinase A (TrkA) receptor in retinal ganglion cells (RGCs). The WB analysis confirmed the NGF effect on VEGFR2 expression and activation, and showed a recovery of VEGF isoform dysregulation by suppressing STZ-induced VEGFA121 expression. Reduction in inflammatory and pro-apoptotic intracellular signals were also found in STZ+NGF retina. These findings suggest that ed-NGF administration might favor neuroretina protection, and in turn counteract the vascular impairment by regulating VEGFR2 and/or VEGFA isoform expression during the early stages of the disease. The possibility that an increase in the NGF availability might contribute to the switch from the proangiogenic/apoptotic to the neuroprotective action of VEGF is discussed. [ABSTRACT FROM AUTHOR]

Abstract Background CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. Methods After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. Results We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. Conclusion Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

HER2-positive and triple-negative breast cancers (TNBC) are difficult to treat and associated with poor prognosis. Despite showing initial response, HER2-positive breast cancers often acquire resistance to HER2-targeted therapies, and TNBC lack effective therapies. To overcome these clinical challenges, we evaluated the therapeutic utility of co-targeting TrkA and JAK2/STAT3 pathways in these breast cancer subtypes. Here, we report the novel combination of FDA-approved TrkA inhibitors (Entrectinib or Larotrectinib) and JAK2 inhibitors (Pacritinib or Ruxolitinib) synergistically inhibited in vitro growth of HER2-positive breast cancer cells and TNBC cells. The Entrectinib-Pacritinib combination inhibited the breast cancer stem cell subpopulation, reduced expression of stemness genes, SOX2 and MYC , and induced apoptosis. The Entrectinib-Pacritinib combination suppressed orthotopic growth of HER2-positive Trastuzumab-refractory breast cancer xenografts and basal patient-derived xenograft (PDXs), reduced tumoral SOX2 and MYC, and induced apoptosis in both mouse models. The Entrectinib-Pacritinib combination inhibited overall metastatic burden, and brain and bone metastases of intracardially inoculated TNBC cells without toxicity. Together, our results demonstrate for the first time that co-inhibition of TrkA and JAK2 synergistically suppresses breast cancer growth and metastasis, thereby providing preclinical evidence that supports future clinical evaluations. • TrkA and JAK2 inhibitors synergistically reduce breast cancer cell viability. • Dual targeting of TrkA and JAK2 impeded growth of breast cancer xenografts in vivo. • Co-inhibition of TrkA and JAK2 reduced metastasis burden of TNBC cells. [ABSTRACT FROM AUTHOR]