Trk Tyrosine Phosphorylation Research Articles

Abstract LB-118: Identification of TRKA and TRKB kinase domain mutations that induce resistance to a pan-TRK inhibitor

Abstract Background: Oncogene fusions involving members of the TRK family of receptor tyrosine kinases (encoded by NTRK1, NTRK2, and NTRK3) have been identified in multiple cancer types. The use of TRK inhibitors in vitro, in vivo and in patients has demonstrated activity against a number of different NTRK oncogene fusions in different tumor types. Thus, the use of a pan-TRK inhibitor presents a therapeutic opportunity for multiple patient populations. LOXO-101 is an inhibitor of the TRK kinase and is highly selective for the TRKA/B/C family of kinases. Kinase domain (KD) mutations are the most commonly identified mechanism of acquired drug resistance found in patients treated with kinase inhibitors. We therefore undertook an unbiased genetic approach to identify candidate resistance mutations in the TRK kinase domain. Methods: We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-MPRIP-NTRK1 and Ba/F3-TRIM24-NTRK2 cells to generate mutations permitting growth of Ba/F3 cells in the absence of IL-3 despite the presence of 100, 250, or 500 nM LOXO-101. Mutations identified by genomic DNA sequencing in the initial screen were validated by cloning the mutation-bearing cDNAs back into Ba/F3 cells to evaluate their sensitivity to LOXO-101 using both proliferation assays and TRK phosphorylation by immunoblot analyses. Modeling of the mutations was performed by mapping of the amino acid substitutions onto a drug-bound TRK kinase domain crystal structure. Results: We have identified 6 amino acid positions in the TRKA and 3 positions in the TRKB KD that induce resistance to LOXO-101. In the TRKA KD we identified V573M, and F589L/C, G595S, F600L, F646V, and G667S. In the TRKB KD, we identified Q596E/P, F617L/C/I, and G623S. The TRKA F589 and TRKB F617 position corresponds to the gatekeeper position, homologous to L1196 in ALK or T790 in EGFR. Several other TRKA and TRKB resistance mutations have homologous mutations in other kinases: TRKA G667 (ALK G1269), TRKA V573 (ALK V1180M), TRKA G595/TRKB G623 (ALK G1202 and ROS1 G2202), and TRKA F646 (ALK F1245). The TRKA F600 has not been previously identified as a resistance mutation in other kinases, but is adjacent to a homologous ALK S1206 resistance position. TRKB Q596 position has also not been previously but is adjacent to the homologous ALK F1174 resistance position. The TRK mutations identified here reduce target inhibition by LOXO-101 as measured by TRK tyrosine phosphorylation and resultant downstream signaling through the MAPK or other critical pathways. Conclusion: This genetic screen identified and validated several novel mutations in the TRKA and TRKB KD that may confer drug resistance to LOXO-101 in patients. Many, although not all, of the resistance mutations identified here are homologous to resistance mutations identified in other oncogenic kinases treated with kinase inhibitors. These data can be used to screen tumor samples from TRK+ patients who develop acquired resistance to TRK inhibitors and may assist in the design of next generation TRK inhibitors to potentially overcome acquired resistance in patients treated with LOXO-101 or structurally similar TRK inhibitors. Citation Format: Adriana Estrada-Bernal, Anh T. Le, Brian Tuch, Tatiana G. Kutateladze, Robert C. Doebele. Identification of TRKA and TRKB kinase domain mutations that induce resistance to a pan-TRK inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-118.

Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells

BackgroundTrk receptor tyrosine kinases regulate multiple important neuronal processes during the development and in the adulthood. Tyrosine phosphorylation of Trk serves as the initial step in the Trk signaling pathway and indicates receptor’ autocatalytic activity. However, methods allowing simple and large-scale Trk phosphorylation analyses in cultured cells are lacking. New methodWe describe an in situ phospho-Trk ELISA (enzyme-linked immunosorbent assay) method where cell culture, receptor stimulation and Trk phosphorylation analysis are all performed on the same multiwell plate. ResultsIn situ phospho-Trk ELISA readily and specifically detects neurotrophin-induced Trk phosphorylation in cultured cells. A proof-of-concept small molecule screening of a library composed of 2000 approved drugs and other bioactive compounds was carried out using this novel method. Comparison with existing methodsIn situ phospho-Trk ELISA utilizes the principles and advantages of conventional sandwich ELISA in an in situ context. ConclusionsWe describe a novel method that can be efficiently used to examine Trk receptor phosphorylation in cultured cells. Principally similar methods can be developed to examine the levels and signaling of any intracellular protein.

Distinct usages of phospholipase Cγ and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cγ (PLCγ) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCγ with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCγ-dependent increases in intracellular Ca 2+ levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCγ and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.

Gangliosides activate Trk receptors by inducing the release of neurotrophins

We used NIH‐3T3 fibroblasts expressing the different Trk receptors to examine whether GM1 ganglioside and its semisynthetic derivative LIGA20 activate various neurotrophin receptors. GM1 elicited a rapid activation of TrkC and TrkA, but not TrkB, tyrosine phosphorylation. In contrast, LIGA20 activated TrkB tyrosine phosphorylation only. These data suggest that gangliosides may act as ligands for specific Trk receptors similar to the neurotrophins. To test this hypothesis, we performed Scatchard analysis to determine if GM1 binds to the TrkC receptor. GM1 failed to displace neurotrophin‐3 (NT‐3) binding suggesting that gangliosides are not ligands for the Trk receptors. Moreover, GM1 did not activate a TNF/Trk chimeric receptor. We next determined whether gangliosides induce the release of neurotrophins. NIH‐3T3 cells and cerebellar granule neurons were exposed to GM1 or LIGA20 for 5 min and the levels of brain‐derived neurotrophic factor (BDNF) and NT‐3 measured in the medium. GM1 induced a significant increase in the amount of NT‐3, but not BDNF. In contrast, LIGA20 increased the secretion of BDNF, suggesting that gangliosides differentially affect the release of neurotrophins, which, in turn, activate Trk receptors. Therefore, Trk tyrosine phosphorylation was determined in the presence of Trk‐IgG receptor bodies. TrkB‐IgG, but not TrkC‐IgG, inhibited LIGA20‐mediated induction of TrkB tyrosine phosphorylation, while TrkC‐IgG, but not TrkB‐IgG, inhibited GM1‐mediated activation of TrkC, further suggesting that gangliosides activate Trk receptors by inducing the release of endogenous neurotrophins.

LIGA20 ganglioside prevents excitotoxicity through activation of TrkB receptor

Gangliosides protect cerebellar granule cells from excitotoxicity; however, their mechanism of action remains to be fully characterized. GM1 ganglioside has been shown to activate Trk, the tyrosine kinase receptor implicated in the neuroprotective properties of the neurotrophins. In these studies, we used primary cultures of cerebellar granule cells to determine whether gangliosides exert neuroprotective effect via the activation of Trk receptors. We first examined the relative potency of the neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin‐3 and nerve growth factor to prevent glutamate‐mediated apoptosis. BDNF was the only neurotrophin that elicited a complete neuronal protection against glutamate. GM1 and its semisynthetic compound LIGA20 also prevented glutamate toxicity, however, LIGA20 was more potent than GM1. Both LIGA20 and BDNF blocked glutamate‐mediated activation of caspase‐3 and consequently apoptosis; however, the anticaspase‐3 activity was seen only when these compounds were added to the cultures several hours before glutamate, suggesting that LIGA20 and BDNF share an identical molecular mechanism. To test this hypothesis, we compared the ability of LIGA20 and BDNF to activate TrkB. Both compounds elicited a similar time‐dependent increase in Trk tyrosine phosphorylation. Moreover, the neuroprotective effect of BDNF and LIGA20 was abolished in neurons exposed to the Trk tyrosine kinase inhibitor k252a, demonstrating a relationship between neuroprotection and activation of Trk receptors. Our data suggest that by activating the Trk neurotrophin receptors, gangliosides may be used as neuroprotective agents.

Gangliosides prevent excitotoxicity through activation of TrkB receptor.

Gangliosides protect cerebellar granule cells from excitotoxicity; however, their mechanism of action remains to be fully characterized. GM1 ganglioside has been shown to activate Trk, the tyrosine kinase receptor implicated in the neuroprotective properties of the neurotrophins. In these studies, we used primary cultures of cerebellar granule cells to determine whether gangliosides exert neuroprotective effect via the activation of Trk receptors. We first examined the relative potency of the neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin-3 and nerve growth factor to prevent glutamate-mediated apoptosis. BDNF was the only neurotrophin that elicited a complete neuronal protection against glutamate. GM1 and its semisynthetic compound LIGA20 also prevented glutamate toxicity, however, LIGA20 was more potent than GM1. Both LIGA20 and BDNF blocked glutamate-mediated activation of caspase-3 and consequently apoptosis; however, the anticaspase-3 activity was seen only when these compounds were added to the cultures several hours before glutamate, suggesting that LIGA20 and BDNF share an identical molecular mechanism. To test this hypothesis, we compared the ability of LIGA20 and BDNF to activate TrkB. Both compounds elicited a similar time-dependent increase in Trk tyrosine phosphorylation. Moreover, the neuroprotective effect of BDNF and LIGA20 was abolished in neurons exposed to the Trk tyrosine kinase inhibitor k252a, demonstrating a relationship between neuroprotection and activation of Trk receptors. Our data suggest that by activating the Trk neurotrophin receptors, gangliosides may be used as neuroprotective agents.

Subcellular localization of epitope-tagged neurotrophins in neuroendocrine cells.

A growing body of evidence suggests that neurotrophins (NTs) play a critical role in synaptic plasticity and other activity dependent processes in the CNS. Release of these growth factors by neurons and neuroendocrine cells was recently shown to occur via the regulated secretory pathway, representing a possible mechanism for preferentially supplying NTs locally to active synapses. However, the identity and characteristics of the intracellular storage compartment for NTs undergoing stimulus-coupled secretion remains controversial. As a step towards addressing these issues we have investigated the subcellular localization of epitope-tagged nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in neuroendocrine cells. Placement of the myc-epitope tag at the neurotrophin carboxy terminus did not affect essential properties of the NTs such as their ability to induce Trk tyrosine phosphorylation or their sorting into the regulated secretory pathway in PC12 and AtT-20 neuroendocrine cells. Epitope-tagged NTs colocalize with dense core vesicle (DCV)-markers at the light microscopic level in both cell lines investigated. Furthermore, at an EM level immunoreactivity (IR) for myc-tagged NGF was found over dense core granules (DCGs) in PC12 cells. These data provide evidence that NTs can be stored in DCVs in neuronal model cell lines and, potentially, in neurons as well.

Protective effect of neurotrophin against glutamate neurotoxicity in cortical cultures

This study was performed to investigate the effects of neurotrophins on glutamate cytotoxicity by using cultured cortical neurons. Primary cultures obtained from the cerebral cortex of fetal rats (17-19 days gestation) were used for experiments. NGF did not elicit tyrosine phosphorylation of Trks whereas BDNF induced Trk tyrosine phosphorylation within 10 min, followed by time-dependent decrease. Brief glutamate exposure to the cell induced delayed cytotoxicity. Similar cytotoxicity was observed with the brief application of a calcium ionophore, ionomycin, and nitric oxide (NO) generating agents, S-nitrosocysteine (SNOC) and SIN-1. Exposure of the cultures to NGF and BDNF for 1 or 24 hr prior to glutamate exposure reduced glutamate-induced cytotoxicity. In contrast, simultaneous addition of NGF and BDNF with glutamate did not affect glutamate-induced cytotoxicity. Ionomycin-induced cytotoxicity was prevented by exposing cultures to NGF and BDNF for 24-hr. Moreover, NGF and BDNF ameliorated cytotoxicity induced by SNOC and SIN-1. These results suggest that neurotrophins prevent NO mediated glutamate cytotoxicity.

Overexpression of N-Acetylglucosaminyltransferase III Disrupts the Tyrosine Phosphorylation of Trk with Resultant Signaling Dysfunction in PC12 Cells Treated with Nerve Growth Factor

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) is a pivotal glycosyltransferase which participates in branch formation by catalysis of the synthesis of a bisecting GlcNAc structure in N-glycans. These structures are thought to be one of the unique features of the N-glycans of neural tissues. To examine the intracellullar role of GnT-III expression and its product in neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells which normally express a low level of GnT-III. In the GnT-III gene-transfected cells, lectin blot analysis showed that some glycoproteins showed increased levels of bisecting GlcNAc structures. Following treatment with nerve growth factor (NGF) the control cells showed neurite outgrowth for differentiation whereas the transfectants showed no morphological response or change in the rate of cell growth. Transient tyrosine phosphorylation of the Trk/NGF receptor was detected at 5-15 min after NGF treatment in control cells, but not detected in the GnT-III gene-transfected cells despite the intact binding of NGF to the cells. Moreover the dimerization of Trk with NGF treatment was not induced in the GnT-III transfectant as compared with the dimerization seen in control cells. These results indicate that overexpression of GnT-III gene in PC12 cells affects some functions of glycoprotein receptors such as Trk by alteration of N-glycan structures, and results in changes in the intracellular signaling pathway of tyrosine phosphorylation modified by NGF.

Intraparenchymal NGF Injections in Adult and Aged Rats Induce Long-Lasting Trk Tyrosine Phosphorylation

Neurotrophic factors, particularly the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and related molecules are proposed for the experimental treatment of neurodegenerative disease. Earlier observations had suggested down-regulation of the neurotrophin receptor response with chronic stimulation. We therefore tested for effects of acute and chronic NGF treatmentin vivoon the tyrosine phosphorylation response of Trk-type neurotrophin receptors in adult and aged rats. Rats were treated for 1 week with daily injections of NGF directly into the striatum. Surprisingly, this chronic neurotrophin treatment induced long-lasting tyrosine phosphorylation of Trk type receptors beyond the last injection. A similar result was obtained with 1 week of daily injections of BDNF into the hippocampus. Persistent Trk tyrosine phosphorylation was also observed after single neurotrophin injections. With 1 μg of NGF injected, Trk-type receptors were maximally stimulated from immediately after the injection until 3 days after the treatment. Maintaining Trk tyrosine phosphorylation required maintained energy levels in the tissue. Incubation of microslices of brain tissue from NGF-injected animals in glucose-free buffer completely abolished all Trk tyrosine phosphorylation signals. Recovery of the tissue in presence of glucose restored the signals in microslices derived from NGF-injected animals, in absence of acute NGF treatment. This result, together with dose–response comparisons after 2-h and 2-day survival times suggest that Trk protein remains tyrosine phosphorylated due to trophic protein which is only slowly being cleared out of the tissue during several days after the injection. Experiments with aged rats indicated similar extent and duration of Trk receptor activation after NGF administration in young adult and in aged brain.

Regulated neurotrophin receptor responsiveness during neuronal migrationand early differentiation

The response of brain tissue to neurotrophins during rat development was examined using a novel in vitro assay for Trk/neurotrophin receptor activity. In this assay, brain tissues were exposed to neutrophins and ligand-induced Trk tyrosine phosphorylation was measured. During the perinatal period, Trk tyrosine phsphorylation in all brain area was induced very similarly by the TrkB and TrkC ligands brain-derived neurotrophic factor (BNDF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT-4/5). In the adult brain, minimal signals were observed after treatment with these three factors, despite the continued presence of full length and truncated TrikB protein. In contrast, responsiveness to the TrkA ligand NGF was absent in the ebmryo and increased during the first 2 weeks after birth in various brain areas, particularly in striatum, basal forebrain, and hippocampus. Our results, showing maximal responsiveness of brain tissue to BDNF, NT-3, and NT-4/5 during early neuronal differentiation and migration, suggest involvement of TrkB in these events. The lack of a significant response to these neurotrophins in the adult brain indicates effective posttranslational mechanisms that control the response of Trk family receptors. Our findings further demonstrate that neurons of the striatum and basal forebrain remain NGF responsive in the adult, confirming at the molecular level results obtained earlier at the cellular level for the basal forebrain cholinergic neurons.

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.

K-252b selectively potentiates cellular actions and trk tyrosine phosphorylation mediated by neurotrophin-3.

K-252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K-252b was found to prevent trophic actions of two other neurotrophins, brain-derived neurotrophic factor, and neurotrophin-3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC12 pheochromocytoma cells, when used at greater than 2 microM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1-100 nM, K-252b selectively enhanced the trophic action of neurotrophin-3 on central cholinergic neurons, peripheral sensory neurons, and PC12 cells. In PC12 cells, K-252b potentiated the neurotrophin-3-induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K-252a, K-252b, and staurosporine, only the first two also mediated neurotrophin-3 potentiation. These findings indicate that K-252b generally and selectively potentiates the neurotrophic action of neurotrophin-3 and suggest that this action involves trk-type neurotrophin receptors.

Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF

Nerve growth factor (NGF) is a neurotrophic factor responsible for the differentiation and survival of sympathetic and sensory neurons as well as selective populations of cholinergic neurons. NGF binds to specific cell-surface receptors but the mechanism for transduction of the neurotrophic signal is unknown. Several experiments using the NGF-responsive pheochromocytoma cell line, PC12, have implicated tyrosine phosphorylation in NGF-mediated responses, although no NGF-specific tyrosine kinases have been identified. Here we show that NGF induces tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product, a tyrosine kinase receptor whose expression is restricted in vivo to neurons of the sensory spinal and cranial ganglia of neural crest origin. Tyrosine phosphorylation of trk by NGF is rapid, specific and occurs with picomolar quantities of factor, indicating that the response is mediated by physiological amounts of NGF. Activation of the trk tyrosine kinase receptor provides a possible mechanism for signal transduction by NGF.