Neurotrophin Receptor Tyrosine Kinase Research Articles

Discovery of a pocket network on the domain 5 of the TrkB receptor - A potential new target in the quest for the new ligands.

The important role that the neurotrophin tyrosine kinase receptor - TrkB has in the pathogenesis of several neurodegenerative conditions such are Alzheimer's disease, Parkinson's disease, Huntington's disease, has been well described. This shouldn't be a surprise, since in the physiological conditions, once activated by brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), the TrkB receptor promotes neuronal survival, differentiation and synaptic function. Considering that the natural ligands for TrkB receptor are large proteins, it is a challenge to discover small molecule capable to mimic their effects. Even though, the surface of receptor that is interacting with BDNF or NT-4/5 is known, there was always a question which pocket and interaction is responsible for activation of it. In order to answer this challenging question, we have used molecular dynamic (MD) simulations and Pocketron algorithm which enabled us to detect, for the first time, a pocket network existing in the interacting domain (d5) of the receptor; to describe them and to see how they are communicating with each other. This new discovery gave us potential new areas on receptor that can be targeted and used for structure-based drug design approach in the development of the new ligands.

The Neurod1/4-Ntrk3-Src pathway regulates gonadotrope cell adhesion and motility

Pituitary gonadotrope cells are essential for the endocrine regulation of reproduction in vertebrates. These cells emerge early during embryogenesis, colonize the pituitary glands and organize in tridimensional networks, which are believed to be crucial to ensure proper regulation of fertility. However, the molecular mechanisms regulating the organization of gonadotrope cell population during embryogenesis remain poorly understood. In this work, we characterized the target genes of NEUROD1 and NEUROD4 transcription factors in the immature gonadotrope αT3-1 cell model by in silico functional genomic analyses. We demonstrated that NEUROD1/4 regulate genes belonging to the focal adhesion pathway. Using CRISPR/Cas9 knock-out approaches, we established a double NEUROD1/4 knock-out αT3-1 cell model and demonstrated that NEUROD1/4 regulate cell adhesion and cell motility. We then characterized, by immuno-fluorescence, focal adhesion number and signaling in the context of NEUROD1/4 insufficiency. We demonstrated that NEUROD1/4 knock-out leads to an increase in the number of focal adhesions associated with signaling abnormalities implicating the c-Src kinase. We further showed that the neurotrophin tyrosine kinase receptor 3 NTRK3, a target of NEUROD1/4, interacts physically with c-Src. Furthermore, using motility rescue experiments and time-lapse video microscopy, we demonstrated that NTRK3 is a major regulator of gonadotrope cell motility. Finally, using a Ntrk3 knock-out mouse model, we showed that NTRK3 regulates gonadotrope cells positioning in the developing pituitary, in vivo. Altogether our study demonstrates that the Neurod1/4-Ntrk3-cSrc pathway is a major actor of gonadotrope cell mobility, and thus provides new insights in the regulation of gonadotrope cell organization within the pituitary gland.

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

104P NTRK3 mutation affects the efficacy of immune checkpoint inhibitors in patients with advanced cancer

Immune checkpoint inhibitors (ICIs), as a revolutionary new immunotherapy therapy, can improve the survival of cancer patients. Nevertheless, ICIs are only effective for some patients. Therefore, there is an urgent need to explore biomarkers that can predict prognosis in cancer patients treated with ICIs. Neurotrophin tyrosine kinase receptor 3 (NTRK3) acts as a tumor suppressor or oncogene in the development of various cancers. However, the efficacy of NTRK3 mutation in immunotherapy for patients with cancer is unknown.

1693P Incidence of NTRK genes fusion in adult brain tumours: A prospective cohort of 140 patients with cerebral gliomas and brain metastases

Oncogenic fusion of neurotrophin receptor tyrosine kinase NTRK1, NTRK2, or NTRK3 genes have been found in different types of solid tumours. Nevertheless, the incidence of NTRK gene fusions in these cancers remains quite unexplored. The treatment of patients with TRK fusion cancer with a first-generation TRK inhibitor (such as larotrectinib or entrectinib) is associated with high response rates (>75%), regardless of tumour histology and presence of metastases, and acceptable toxicity profile. Due to the efficacy of TRK inhibitor therapy and the recent Food and Drug Administration and European Medicines Agency approval of larotrectinib and entrectinib, it is clinically important to identify patients accurately and efficiently with TRK fusion cancer.

Precision Medicine in Cholangiocarcinoma: Past, Present, and Future.

Cholangiocarcinoma (CCA), or biliary tract cancer, has a poor prognosis. The median survival time among patients with CCA is under 2 years from diagnosis, and the global 5-year survival rate is only 10%. First-line therapy with chemotherapeutic agents, gemcitabine plus cisplatin, has traditionally been used to treat unresectable advanced CCA. In recent years, precision medicine has become a mainstream cancer treatment due to innovative next-generation sequencing technology. Several genetic alterations, including mutations, gene fusions, and copy number variations, have been found in CCA. In this review, we summarized the current understanding of genetic profiling in CCA and targeted therapy in CCA. Owing to the high heterogeneity of CCA, tumor microenvironmental factors, and the complexity of tumor biology, only pemigatinib, infigratinib, ivosidenib, larotrbctinib, and entrectinib are currently approved for the treatment of CCA patients with fibroblast growth factor receptor 2 gene (FGFR2) fusion, isocitrate dehydrogenase gene (IDH1) mutation, and neurotrophin receptor tyrosine kinase gene (NRTK) fusion, respectively. Additional targeted therapies, including other FGFR2 inhibitors, PI3K/AKT/mTOR inhibitors, and BRAF-directed targeted therapy, have been discussed for the management of CCA, and immune checkpoint inhibitors, particularly pembrolizumab, can be administered to patients with high microsatellite instability tumors. There is a further need for improvement in precision medicine therapies in the treatment of CCA and discuss the approved and potential targeted therapies for CCA.

Characterization of NTRK alterations in metastatic colorectal cancer.

e15569 Background: Increased molecular profiling ability has resulted in the recognition of important actionable genes that can be used to direct targeted therapies in colorectal cancers (CRC). Neurotrophin receptor tyrosine kinase (NTRK) gene fusions are one of these molecular alterations that are potentially actionable with novel targeted therapeutics. However, other biologically pathogenic NTRK alterations, including point mutations, are not yet actionable in CRC. This study aimed to characterize NTRK alterations, including fusions in metastatic CRC patients. Methods: Molecular characteristics of 917 patients with CRC were collected from the Moffitt Cancer Center Clinical Genomics Action Committee database. Demographic, clinicopathological, and molecular data and treatment history were abstracted from electronic medical records. Mutations with potential oncogenic activity are considered “potentially pathogenic”, while alterations with proven oncogenic activity such as fusions are considered “pathogenic”. Results: There were 917 patients with CRC from November 2013 to December 2021, and 77 of them had NTRK alterations excluding synonymous mutations (8.3%, 77/917). Six patients had potentially pathogenic NTRK alterations, including one NTRK rearrangement (0.6%, 7/917); however, only 1 patient had an actionable pathogenic NTRK1 fusion (0.1%, 1/917), while the others (70) were a variant of unknown significance (VUS). The majority of NTRK alterations were missense mutations (92%; 71/77). The NTRK1 fusion partner was LMNA and occurred in the setting of an MSI-H tumor (3.7%; 1/27). Among patients with pathogenic and potentially pathogenic alterations (N = 7), there was only 1(14%) patient who had low tumor mutation burden (TMB) (< 10 mut/MB). Most patients were older than 50 years (70.1%, 54/77) and male (58.4%, 45/77). Thirty-six patients (46..7%) had right-sided tumor, while 41 patients (53.2%) left-sided. Across all NTRK alterations cohort, MSI-H was found in 10.3% (7/77), and POLE/POLD1 pathogenic mutations were seen in 6.4% (5/77) patients. Conclusions: NTRK gene fusion is a relatively rare event (< 1%) in CRC, including the MSI-H subtype. The majority of NTRK alterations in CRC are VUS, and they are not actionable, and they tend to be seen in the tumors with high TMB (TMB > 10). Hypermutator tumors cause frequent VUS alterations in the NTRK gene with unknown clinical relevance.

PD-L1, Mismatch Repair Protein, and NTRK Immunohistochemical Expression in Cervical Small Cell Neuroendocrine Carcinoma.

BackgroundCervical small cell neuroendocrine carcinoma (SCNC) is a rare and aggressive disease that lacks a standard treatment strategy or effective methods of targeted therapy. PD-L1 inhibitors for DNA mismatch repair system-deficient (dMMR) tumors and neurotrophin receptor tyrosine kinase (NTRK) inhibitors offer potential pan-cancer treatments.MethodsImmunohistochemistry was employed as the main detection method, and any NTRK positive cases, identified by immunohistochemistry, were further submitted for evaluation by fluorescence in situ hybridization (FISH) and real-time polymerase chain reaction (RT-PCR) methods.ResultsForty-six patients were enrolled. Positive PD-L1 expression was seen in 22 of the 43 patients (51.16%) with an average combined positive score of 6.82. PD-L1-positive patients were more likely to have a higher proliferation rate in the tumor, and they experienced less recurrence and death (p = 0.048 and 0.033, respectively) compared with the patients with negative PD-L1 expression. However, in the multivariate analysis, none of the clinical parameters was associated with the expression of PD-L1. There was no association between PD-L1 expression and disease recurrence or overall survival in the Kaplan-Meier analysis. All cases were found to be MMR-stable and lacked NTRK gene fusion. However, pan-Trk expressed in 14 (32.56%) of the 43 tested cases, but FISH and RT-PCR failed to confirm any positive fusion signals in IHC-positive cases.ConclusionsPD-L1 may be an effective therapeutic target for cervical SCNC. Cervical SCNC is a MMR-stable tumor and lacks NTRK gene fusion. IHC isn’t a reliable method in the detection of NTRK gene fusion in cervical SCNC.

A chemical-genetic investigation of BDNF-NtrkB signaling in mammalian sleep.

The neurotrophin brain-derived neurotrophic factor (BDNF) is hypothesized to be a molecular mediator of mammalian sleep homeostasis. This hypothesis is supported by correlational findings and results obtained from pharmacology. BDNF binds with high affinity to the membrane-bound receptor Neurotrophin Tyrosine Kinase Receptor B (NtrkB), which triggers several intracellular signaling cascades. It is therefore possible that BDNF's role in sleep homeostasis is mediated via NtrkB. We examined this hypothesis using a chemical-genetic technique that allows for rapid and selective inhibition of NtrkB in vivo. We used mutant mice bearing a point mutation in the NtrkB that allows for selective and reversible inactivation in the presence of a small binding molecule (1-NM-PP1). Using a crossover design, we determined the effects of NtrkB inhibition on baseline sleep architecture and sleep homeostasis. We find that NtrkB inhibition reduced rapid eye movement (REM) sleep time and changed state transitions but had no effect on sleep homeostasis. These findings suggest that BDNF-NtrkB receptor signaling has subtle roles in sleep architecture, but no role in sleep homeostasis.

Analgesic and Anti-Inflammatory Effects of the Synthetic Neurosteroid Analogue BNN27 during CFA-Induced Hyperalgesia.

Dehydroepiandrosterone (DHEA), an adrenal and neurosteroid hormone with strong neuroprotective and immunomodulatory properties, and ligand for all high-affinity neurotrophin tyrosine kinase receptors (Trk), also exerts important effects on hyperalgesia. Its synthetic, 17-spiro-epoxy analogue, BNN27, cannot be converted to estrogen or androgen as DHEA; it is a specific agonist of TrkA, the receptor of pain regulator Nerve Growth Factor (NGF), and it conserves the immunomodulatory properties of DHEA. Our study aimed to evaluate the anti-nociceptive and anti-inflammatory properties of BNN27 during Complete Freund’s Adjuvant (CFA)-induced inflammatory hyperalgesia in mice. Hyperalgesia was evaluated using the Hargreaves test. Inflammatory markers such as cytokines, NGF and opioids were measured, additionally to corticosterone and the protein kinase B (AKT) signaling pathway. We showed for the first time that treatment with BNN27 reversed hyperalgesia produced by CFA. The effect of BNN27 involved the inhibition of NGF in the dorsal root ganglia (DRG) and the increased synthesis of opioid peptides and their receptors in the inflamed paw. We also found alterations in the cytokine levels as well as in the phosphorylation of AKT2. Our findings strongly support that BNN27 represents a lead molecule for the development of analgesic and anti-inflammatory compounds with potential therapeutic applications in inflammatory hyperalgesia.

NTRK fusion positive colorectal cancer as a unique subset of CRC with high tumor mutation burden and microsatellite instability.

3544 Background: Neurotrophin receptor tyrosine kinase ( NTRK) gene fusions are rare but actionable oncogenic drivers that are present in a wide variety of solid tumors. This study aims to identify the frequency and the clinicopathologic and genetic features of NTRK-driven colorectal cancers (CRC). Methods: Colonic and rectal tumor DNA specimen from colorectal cancer patients submitted for molecular profiling at a CLIA-certified genomics laboratory in China that performed NTRK1/2/3 fusion detection by hybridization-based targeted next generation sequencing (NGS) were retrospectively reviewed. Patients’ demographic, clinical characteristics, and treatment history were retrieved from the database for further evaluation. Results: A total of 2,519 unique Chinese colorectal cancer cases were profiled from April 2016 to May 2020, and 17 NTRK+ fusion events were identified (0.7%, 17/2,519) consisting of 14 cases of NTRK1+ and 3 cases of NTRK3+ fusions. Furthermore, thirteen out of 17 NTRK+ CRC tumors (76%) were microsatellite instability-high (MSI-H) tumors, a much higher rate than that of the molecularly unselected CRC population (8%) or NTRK+ non-CRC tumors ( < 1%). NTRK+ CRC patients also had increased tumor mutation burden (median TMB = 65 mut/MB) compared to that of non- NTRK+ CRC (median TMB = 7.7 mut/MB) or NTRK+ non-CRC tumors (median TMB = 4 mut/MB). POLE/POLD1 mutations were also enriched in NTRK+ CRC (8/17, 47%) relative to molecularly unstratified CRC patients (8%) with over half carrying concurrent POLE and POLD1 mutations. TPM3 was the most common fusion partner of NTRK1 (78%, N = 14), followed by LMNA and TRP. Three NTRK3+ CRC were identified (ETV6-NTRK3, RUNX1-NTRK3, CSNK1G1-NTRK3). RNF43 (71%) was the most frequently mutated gene and the aberrations of RNF43 and ARID1 were significantly enriched in MSI-positive NTRK+ tumors as compared to the MSS NTRK+ subgroup. TP53 (53%) and APC (35%) aberrations frequently co-occurred with NTRK fusions, whereas the majority of the NTRK+ cohort were RAS/BRAF wildtype, except in one case that an oncogenic KRAS Q61R variant co-occurred with RUNX1-NTRK3. Conclusions: NTRK+ colorectal cancer is rare. In addition to the absence of canonical driver mutations, NTRK+ tumors demonstrated increased tumor mutation burden, higher frequency of microsatellite instability, and an enrichment of POLE/POLD1 mutations relative to molecularly unselected CRC population.

Design, synthesis and biological activity of bicyclic carboxamide derivatives as TRK inhibitors

‘precision medicine’ is characterized by the selection of targeted drugs based on genetic characteristics of tumor from patients, and no longer selected basis on the type of cancer tissue. Among them, clinical trials on neurotrophin receptor tyrosine kinase genes (NTRK) have proven that great anti-cancer effects can be achieved in different cancer patients. In this paper, a novel total of twenty compounds in two categories have been designed and synthesized. Results of Kinase activity tests showed that I-9 (TRKA IC50 = 1.3 nM, TRKAG595R IC50 = 6.1 nM), and I-10 (TRKA IC50 = 1.1 nM, TRKAG595R IC50 = 5.3 nM) have significant inhibitory activity, and results of cell viability tests showed that I-9 and I-10 can maintain a great inhibitory effect in the Ba/F3-LMNA-NTRK1 cell line(IC50 = 81.1 nM and 41.7 nM, respectively), and in Ba/F3-LMNA-NTRK1-G595R cell line, I-9 and I-10 have better cell activity (IC50 was 495.3 nM, 336.6 nM, respectively) compared with the positive control drug LOXO-101. These results indicate that I-9 and I-10 are potential TRK inhibitors that can overcome drug resistance for further investigation.

Non-small cell lung cancer targetable mutations: present and future

Rapid advances in the utility of molecular testing to identify common and uncommon driver mutations as well as the expeditious rate of drug development have intensified the need to obtain multiplex next generation sequencing from tissue and/or blood at the time of diagnosis in order to determine the most appropriate treatment for patients with advanced lung cancers. In this review, we discuss the currently available investigational targeted therapies for the treatment of sensitizing epidermal growth factor receptor ( EGFR ) mutations, anaplastic lymphoma kinase ( ALK ) fusions, ROS proto-oncogene 1 ( ROS1 ) fusions, B-Raf proto-oncogene ( BRAF ) V600E mutations, and neurotrophin receptor tyrosine kinase ( NTRK ) fusions. In addition, we have highlighted promising investigational therapies aimed at high level mesenchymal-to- epithelial transition factor ( MET ) amplifications or exon 14 skipping mutations, EGFR or human epidermal growth factor receptor 2 ( HER2 ) exon 20 insertion mutations, rearranged during transfection ( RET ) fusions, and Kirsten rat sarcoma viral oncogene homolog ( KRAS ) G12C mutations that lend support towards routine testing for these driver mutations as well.

The Evolving Diagnostic and Treatment Landscape of NTRK-Fusion-Driven Pediatric Cancers.

The neurotrophin receptor tyrosine kinase (NTRK1-3) genes have been identified as key fusion partners in a range of pediatric cancers. In childhood cancers, ETV6-NTRK3 fusions are found in the majority of infantile fibrosarcomas and congenital mesoblastic nephromas. NTRK fusions are also found in mammary analog secretory carcinomas (MASC), secretory breast carcinomas, and with modest frequency in high-grade gliomas in very young children. While there are a range of multi-receptor tyrosine kinase inhibitors that show efficacy against TRK kinases, there are now multiple highly selective TRK inhibitors in clinical evaluation. Entrectinib and larotrectinib have been evaluated in early-phase clinical trials for children and demonstrated high response rates with good durability of response. Both agents are now approved in the United States in an age and histology agnostic manner for children (age > 12years for entrectinib; all ages for larotrectinib) for the treatment of solid tumors harboring NTRK fusions without an option for complete surgical resection, with relapsed disease, or without a viable alternative systemic option. More recently, two second-generation TRK inhibitors, selitrectinib and repotrectinib, have been developed and are currently being evaluated in pediatric early phase trials. The Children's Oncology Group has also launched a phase II trial of larotrectinib as a neoadjuvant agent for patients with newly diagnosed infantile fibrosarcoma. While the clinical use of these agents has developed rapidly, many questions remain in terms of duration of therapy, treatment of CNS disease, and long-term toxicities. Further development of this class of agents will continue to require multi-center trials for these rare tumors. Tumor sequencing and potentially sequencing of circulating tumor DNA will improve our understanding of patterns of resistance and the most effective treatment strategies for these patients.

Neurotrophin receptor tyrosine kinases regulated with near-infrared light

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

TRK Fusion Cancers in Children: A Clinical Review and Recommendations for Screening.

Chromosomal translocations involving the NTRK1, NTRK2, and NTRK3 genes (TRK fusions), which encode the neurotrophin tyrosine kinase receptors TRKA, TRKB, and TRKC, can result in constitutive activation and aberrant expression of TRK kinase. Certain cancers almost universally harbor TRK fusions, including infantile fibrosarcoma, cellular congenital mesoblastic nephroma, secretory breast cancer, and mammary analog secretory carcinoma of the salivary gland. TRK fusions have also been identified at lower frequencies across a broad range of other pediatric cancers, including undifferentiated sarcomas, gliomas, papillary thyroid cancers, spitzoid neoplasms, inflammatory myofibroblastic tumors, and acute leukemias. Here we review the prevalence and diseases associated with TRK fusions and methods of detection of these fusions in light of the recent development of selective TRK inhibitors, such as larotrectinib, which demonstrated a 75% response rate across children and adults with TRK fusion cancers. We provide recommendations for screening pediatric tumors for the presence of TRK fusions, including the use of immunohistochemistry or fluorescence in situ hybridization for patients with tumors likely to harbor TRK fusions. Further, we recommend next-generation sequencing for tumors that have a relatively low prevalence of TRK fusions, both to identify patients who may benefit from TRK inhibition and to identify other targetable oncogenic drivers that exist in the same tumor types.

Abstract 1084: TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis

Abstract Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs play an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remains incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, a RING domain E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin receptor tyrosine kinase, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and genetic ablation of TrkA abolished TRAF4-dependent cell invasion. TRAF4 promoted atypical K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream effector proteins. TRAF4 knockdown also suppressed nerve growth factor-stimulated TrkA downstream p38 MAPK activation and subsequent invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated receptor tyrosine kinase activation in cancer cells. Citation Format: Ramesh Singh, Dileep Karri, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Bert W. O'Malley, Ping Yi. TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1084.

TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis.

Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of WT RTKs plays an important role in driving cancer progression. However, the mechanisms underlying how RTKs drive prostate cancer remain incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, an E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK-mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin RTK, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and found that inhibition of TrkA activity abolished TRAF4-dependent cell invasion. TRAF4 promoted K27- and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine autophosphorylation and its interaction with downstream proteins. TRAF4 knockdown also suppressed nerve growth factor (NGF) stimulated TrkA downstream p38 MAPK activation and invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating that this signaling axis is significantly activated during oncogenesis. Our results revealed a posttranslational modification mechanism contributing to aberrant non-mutated RTK activation in cancer cells.

A comprehensive analysis of neurotrophins and neurotrophin tyrosine kinase receptors expression during development of zebrafish.

Neurotrophins (NTF) are a family of secreted nerve growth factors with affinity for tyrosine kinase (Ntrk) and p75 receptors. To fully understand the variety of developmental roles played by NTFs, it is critical to know when and where genes encoding individual ligands and receptors are transcribed. Identification of ntf and ntrk transcripts in zebrafish development remains to be fully characterized for further uncovering the potential function(s) of the NTF signal transduction pathway. Here, we conducted a systematic analysis of the expression profiles of four ntf and five ntrk genes during zebrafish development using whole-mount in situ hybridization. Our study unveils new expression domains in the developing embryo, confirms those previously known, and shows that ntf and ntrk genes have different degrees of cell- and tissue-type specificity. The unique and overlapping expression patterns here depicted indicate the coordination of the redundant and divergent functions of NTFs and represent valuable tools for deciphering the molecular pathways involved in the specification and function of embryonic cell types.

LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate.

The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesion molecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesion ligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTP was capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network.