TrkA Inhibitor Research Articles

Pharmacologic or genetic targeting of peripheral nerves prevents peri-articular traumatic heterotopic ossification

Heterotopic ossification (HO) is a pathological process that commonly arises following severe polytrauma, characterized by the anomalous differentiation of mesenchymal progenitor cells and resulting in the formation of ectopic bone in non-skeletal tissues. This abnormal bone growth contributes to pain and reduced mobility, especially when adjacent to a joint. Our prior observations suggested an essential role of NGF (Nerve Growth Factor)-responsive TrkA (Tropomyosin Receptor Kinase A)-expressing peripheral nerves in regulating abnormal osteochondral differentiation following tendon injury. Here, we utilized a recently developed mouse model of hip arthroplasty-induced HO to further validate the role of peripheral nerve regulation of traumatic HO. Nerve ingrowth was either modulated using a knockin transgenic animals with point mutation in TrkA, or local treatment with an FDA-approved formulation of long acting Bupivacaine which prevents peripheral nerve growth. Results demonstrate exuberant sensory and sympathetic nerve growth within the peri-articular HO site, and that both methods to reduce local innervation significantly reduced heterotopic bone formation. TrkA inhibition led to a 34% reduction in bone volume, while bupivacaine treatment resulted in a 50% decrease. Mechanistically, alterations in TGFβ and FGF signaling activation accompanied both methods of local denervation, and a shift in macrophages from M1 to M2 phenotypes was observed. In sum, these studies reinforce the observations that peripheral nerves play a role in the etiopathogenesis of HO, and that targeting local nerves represents a potential therapeutic approach for disease prevention.

Cisplatin induced alterations in nociceptor developmental trajectory elicits a TrkA dependent platinum-based chemotherapy induced neuropathic pain

Cisplatin-based chemotherapy is a common treatment for paediatric cancer. Unfortunately, cisplatin treatment causes neuropathic pain, a highly prevalent adverse health related complication in adult childhood cancer survivors. Due to minimal understanding of this condition, there are currently no condition tailored analgesics available. Here we investigated an alteration in nociceptor maturation that results in neuronal sensitisation and manifestation of cisplatin induced survivorship pain in a TrkA dependent manner. Cisplatin was administered (i.p. 0.1 mg/kg Postnatal day 14 and 16) to neonatal male and female Wistar rats and nociceptive behavioural assays were performed. In vitro studies utilised isolated neonatal dorsal root ganglia sensory neurons treated with cisplatin (5 μg/ml) to elucidate impact upon nociceptor activation and neurite growth, in combination with TrkA inhibition (GW441756 10 nM and 100 nM). Cisplatin treated male and female neonatal Wistar rats developed a delayed but lasting mechanical and heat hypersensitivity. Cisplatin administration led to increased TrkA expression in dorsal root ganglia sensory neurons. Nerve growth factor (NGF) induced TrkA activation led to sensory neuritogenesis and nociceptor sensitisation, which could be prevented through pharmacological TrkA inhibition (GW441756 either s.c. 100 nM or i.p. 2 mg/kg). Administration of TrkA antagonist suppressed cisplatin induced TRPV1 mediated nociceptor sensitisation and prevented cisplatin induced neuropathic pain. These studies provide greater understanding of the underlying mechanisms that cause cisplatin induced childhood cancer survivorship pain and allowing identification of potential therapeutic targets.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Dual inhibition of the TrkA and JAK2 pathways using entrectinib and pacritinib suppresses the growth and metastasis of HER2-positive and 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.

Nerve Growth Factor Signaling Modulates the Expression of Glutaminase in Dorsal Root Ganglion Neurons during Peripheral Inflammation.

Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain.

CCL19-Positive Lymph Node Stromal Cells Governthe Onset of Inflammatory Arthritis via Tropomyosin Receptor Kinase.

The study objective was to assess the role of CCL19+ lymph node stromal cells of the joint-draining popliteal lymph node (pLN) for the development of arthritis. CCL19+ lymph node stromal cells were spatiotemporally depleted for five days in the pLN before the onset of collagen-induced arthritis (CIA) using Ccl19-Cre × iDTR mice. In addition, therapeutic treatment with recombinant CCL19-immunoglobulin G (IgG), locally injected in the footpad, was used to confirm the results. RNA sequencing of lymph node stromal cells combined with T cell coculture assays using tropomyosin receptor kinase (Trk) family inhibitors together with in vivo local pLN small interfering RNA (siRNA) treatments were used to elucidate the pathway by which CCL19+ lymph node stromal cells initiate the onset of arthritis. Spatiotemporal depletion of CCL19+ lymph node stromal cells prevented disease onset in CIA mice. These inhibitory effects could be mimicked by local CCL19-IgG treatment. The messenger RNA sequencing analyses showed that CCL19+ lymph node stromal cells down-regulated the expression of the tropomyosin receptor kinase A (TrkA) just before disease onset. Blocking TrkA in lymph node stromal cells led to increased T cell proliferation in in vitro coculture assays. Similar effects were observed with the pan-Trk inhibitor larotrectinib in cocultures of lymph node stromal cells of patients with rheumatoid arthritis and T cells. Finally, local pLN treatment with TrkA inhibitor and TrkA siRNA led to exacerbated arthritis scores. CCL19+ lymph node stromal cells are crucially involved in the development of inflammatory arthritis. Therefore, targeting of CCL19+ lymph node stromal cells via TRK could provide a tool to prevent arthritis.

Nerve growth factor promote osteogenic differentiation of dental pulp stem cells through MEK/ERK signalling pathways.

Nerve growth factor (NGF) and its receptor, tropomyosin receptor kinase A (TrkA), are known to play important roles in the immune and nervous system. However, the effects of NGF on the osteogenic differentiation of dental pulp stem cells (DPSCs) remain unclear. This study aimed to investigate the role of NGF on the osteogenic differentiation of DPSCs in vitro and the underlying mechanisms. DPSCs were cultured in osteogenic differentiation medium containing NGF (50 ng/mL) for 7 days. Then osteogenic-related genes and protein markers were analysed using qRT-PCR and Western blot, respectively. Furthermore, addition of NGF inhibitor and small interfering RNA (siRNA) transfection experiments were used to elucidate the molecular signalling pathway responsible for the process. NGF increased osteogenic differentiation of DPSCs significantly compared with DPSCs cultured in an osteogenic-inducing medium. The NGF inhibitor Ro 08-2750 (10 μM) and siRNA-mediated gene silencing of NGF receptor, TrkA and ERK signalling pathways inhibitor U0126 (10 μM) suppressed osteogenic-related genes and protein markers on DPSCs. Furthermore, our data revealed that NGF-upregulated osteogenic differentiation of DPSCs may be associated with the activation of MEK/ERK signalling pathways via TrkA. Collectively, NGF was capable of promoting osteogenic differentiation of DPSCs through MEK/ERK signalling pathways, which may enhance the DPSCs-mediated bone tissue regeneration.

TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

BackgroundAs 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.MethodsLabel-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.ResultsAGPS 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.ConclusionBased 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.

ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells

BackgroundTriple-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.MethodsUsing 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.ResultsIn 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.ConclusionsThese 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.

Design, synthesis and anticancer evaluation of novel arylhydrazones of active methylene compounds

Nerve growth factor (NGF) and its receptor, tropomyosin kinase receptor kinase type A (TrkA) is emerging as an important target for Glioblastoma (GBM) treatment. TrkA is the cancer biomarker majorly involved in tumor invasion and migration into nearby normal tissue. However, currently, available Trk inhibitors exhibit many adverse effects in cancer patients, thus demanding a novel class of ligands to regulate Trk signaling. Here, we exploited the role of TrkA (NTRK1) expression from the 651 datasets of brain tumors. RNA sequence analysis identified overexpression of NTRK1 in GBM, recurrent GBM as well in Oligoastrocytoma patients. Also, TrkA expression tends to increase over the higher grades of GBM. TrkA protein targeting hydrazone derivatives, R48, R142, and R234, were designed and their mode of interaction was studied using molecular docking and dynamic simulation studies. Ligands' stability and binding assessment reveals R48, 2 2-(2-(2-hydroxy-4-nitrophenyl) hydrazineylidene)-1-phenylbutane-1,3-dione, as a potent ligand that interacts well with TrkA's hydrophobic residues, Ile, Phe, Leu, Ala, and Val. R48- TrkA exhibits stable binding potentials with an average RMSD value <0.8 nm. R48 obeyed Lipinski's rule of five and possessed the best oral bioavailability, suggesting R48 as a potential compound with drug-likeness properties. In-vitro analysis also revealed that R48 exhibited a higher cytotoxicity effect for U87 GBM cells than TMZ with the IC50 value of 68.99 μM. It showed the lowest percentage of cytotoxicity to the non-cancerous TrkA expressing MEF cells. However, further SiRNA analysis validates the non-specific binding of R48, necessitating structural alteration for the development of R48-based TrkA inhibitor for GBM therapeutics.

Abstract 12858: Myocardial Infarction Promotes Breast Cancer Growth Through the Nerve Growth Factor/Tropomyosin Receptor Kinase A Pathway

Background: Epidemiological studies have suggested that patients with heart failure due to myocardial infarction (MI) have a higher cancer incidence. Nerve growth factor (NGF) plays a pivotal role in sympathetic nerve remodeling after MI, but several studies have suggested that NGF also plays a significant role in cancer progression. However, the impact of NGF on cancer progression in the setting of MI remains to be elucidated. Methods and Results: MI was induced by permanent ligation of the left anterior descending artery in female BALB/c mice. 4T1 cells, mammary tumor cells, were injected into the right mammary fat pad 2 weeks after surgery. Levels of NGF expression in the myocardium 3 days after surgery (1.40±0.17 AU vs. 1.00±0.09 AU, P=0.049) and serum NGF at 2 weeks after surgery (279±34 pg/ml vs. 173±16 pg/ml, P=0.044) were higher in the MI group than in the sham group. In addition, the tumor volume was significantly larger in the MI group than in the sham group 3 weeks after 4T1 cell injection (465.3±32.6 mm 3 vs. 292.1±28.3 mm 3 , P&lt;0.001). The percentage of Ki67-positive cells in tumor tissue was higher in the MI group than in the sham group (37.1±5.7% vs. 16.5±3.6%, P=0.010). Moreover, phosphorylation activities of membranous tropomyosin receptor kinase A (TRKA, 1.54±0.04 AU vs. 1.00±0.09 AU, P&lt;0.001) and AKT (1.56±0.18 AU vs. 1.00±0.08 AU, P=0.046) were higher in the MI group than in the sham group. Inhibition of TRKA by a TRKA inhibitor, GW441756 (20 mg/kg/week, I.P.), suppressed tumor volume in MI mice compared to DMSO-treated MI mice (281.6±34.8 mm 3 vs. 443.9±50.8 mm 3 , P&lt;0.001). The percentage of Ki67-positive cells (31.9.1±2.8% vs. 42.2±2.0%, P=0.010), phosphorylation levels of TRKA (0.99±0.07 AU vs. 1.68±0.09 AU, P&lt;0.001) and AKT (0.90±0.01 AU vs. 1.19±0.05 AU, P=0.001) in tumor tissues were decreased in GW441756-treated MI mice than in DMSO-treated MI mice. Conclusion: The NGF/TRKA pathway is involved in accelerated tumor cell growth under MI-induced heart failure.

The effect of Banxia-houpo decoction on CUMS-induced depression by promoting M2 microglia polarization via TrkA/Akt signalling.

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.

Unlocking the potential of approved drugs for the allosteric inhibition of tropomyosin-receptor kinase A using molecular docking and molecular dynamics studies.

Tropomyosin-receptor kinase A (TrkA) is the primary isoform among the tropomyosin-receptor kinases that have been associated with human cancer development, contributing to approximately 7.4% of all cancer cases. TrkA represents an attractive target for cancer treatment; however, currently available TrkA inhibitors face limitations in terms of resistance development and potential toxicity. Hence, the objective of this study was to identify new allosteric-approved inhibitors of TrkA that can overcome these challenges and be employed in cancer therapy. To achieve this goal, a screening of 9,923 drugs from the ChEMBL database was conducted to assess their repurposing potential using molecular docking. The top 49 drug candidates, exhibiting the highest docking scores (-11.569 to -7.962kcal/mol), underwent MM-GBSA calculations to evaluate their binding energies. Delanzomib and tibalosin, the top two drugs with docking scores of -10.643 and -10.184kcal/mol, respectively, along with MM-GBSA dG bind values of -67.96 and -50.54kcal/mol, were subjected to 200ns molecular dynamic simulations, confirming their stable interactions with TrkA. Based on these findings, we recommend further experimental evaluation of delanzomib and tibalosin to determine their potential as allosteric inhibitors of TrkA. These drugs have the potential to provide more effective and less toxic therapeutic alternatives. The approach employed in this study, which involves repurposing drugs through molecular docking and molecular dynamics, serves as a valuable tool for identifying novel drug candidates with distinct therapeutic uses. This methodology can contribute to reducing the attrition rate and expediting the process of drug discovery.

Abstract 3630: TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignancy of the skeleton, and inordinately affects young adults. Despite improved outcomes with current multimodal care, the prognosis for patients with metastatic disease is grim. Peripheral neural regulation of cancer growth has long been suggested by clinical observations in pancreatic and prostate cancers. Prior reports suggest peripheral nerve sprouting in osteosarcoma, but the functional roles of sensory nerves in osteosarcoma are unknown. Here, the role of TrkA+ sensory nerves in OS disease progression was assayed. Methods: All experiments were conducted under IACUC approval at Johns Hopkins University. TrkAF592A mice which are homozygous for a phenylalanine-to-alanine point mutation in exon 12 of Ntrk1 gene were crossbred with NOD scid mice (TrkAF592A/Scid). TrkA inhibition was performed by 1NMPP1 treatment. In vivo tumor growth was examined using a periosteal xenograft model. 1 million human 143B-luc OS cells were injected. Analysis was performed using serial measurements of tumor size and IVIS imaging. Nerve distribution and macrophage polarization within the tumor microenvironment was documented using immunostaining for Beta III Tubulin (TUBB3), CD86, CD206 and F4/80. Results: Inhibition of TrkA led to a significant reduction in Tubb3+ nerves (88.4%) within and around the OS xenografts. In addition, tumors within TrkAF592A/Scid animals demonstrated slower growth, including a 51.5% reduction in tumor size and 60.0% reduction in IVIS luciferase activity at 4 wks. Histologic examination showed a reduction in macrophage infiltration and reprogramming of macrophages from an M2-like to M1-like phenotype in tumors with chemical denervation. Remarkably, this was associated with a reduction in Ki67 proliferative index (80.1%) and CD31 immunofluorescent staining (60.8%) within the tumors. Overall survival of mice with TrkA inhibition was significantly prolonged (p=0.0003). Retrograde tracing identified the sensory nerve fibers innervating the periosteum originated from L3-L5 lumbar DRGs and RNA seq identified OS-derived neurotrophins and axon guidance molecules. Discussion: Disruption of TrkA+ nerves significantly mitigates OS disease progression in a mouse xenograft model, including a significant improved overall survival. These findings are associated with a reduction of macrophage infiltration and macrophage re-polarization. Ongoing studies are examining the molecular mechanisms that mediate nerve-to-macrophage interaction in peripheral nerves and DRG neurons using a combination of sequencing approaches. Significance/Clinical Relevance: Sensory neurons positively regulate osteosarcoma growth and disease progression. Use of chemical or microsurgical approaches to denervate tumors may represent a future adjunctive therapy to both improve pain and prognosis in high grade bone sarcomas. Citation Format: Qizhi Qin, Sowmya Ramesh Ramesh, Thomas L. Clemens, Aaron W. James. TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3630.

Abstract 1721: Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers

Abstract Despite the current standard of care, metastatic triple-negative breast cancer (TNBC) and HER2-enriched breast cancer remain difficult to treat due to limited actionable targets, poor response to treatment, and intrinsic/acquired therapeutic resistance. Thus, more effective therapies are needed to reduce the high mortality rates associated with metastatic TNBC and HER2-enriched breast cancer. The Janus Kinase 2 (JAK2)-Signal Transducer and Activator of Transcription 3 (STAT3) pathway is dysregulated in multiple solid tumors, including breast cancer, and is being investigated as a therapeutic target. Similarly, Tropomyosin receptor tyrosine kinase A (TrkA) is an emerging target in metastatic breast cancers. The prevalence of aberrant JAK2 or TrkA signaling led to the development and FDA approval of orally active, selective inhibitors for JAK2 (pacritinib) and TrkA (entrectinib, larotrectinib). However, breast cancer patients receiving either JAK2 or TrkA inhibitors showed suboptimal clinical response, suggesting that oncogenic signaling events are sustained in the absence of either JAK2 or TrkA activity. In support of this paradigm, we previously reported a novel signaling crosstalk between the JAK2-STAT3 and TrkA pathways (Cancers, 10:2340-2360, 2021). We found that JAK2 and TrkA are preferentially co-activated in TNBC and HER2-enriched breast cancer, and both kinases converge to phosphorylate STAT3 to drive gene transcription and enhance breast cancer cell stemness. The observed significant functional crosstalk between JAK2 and TrkA pathways suggest that co-inhibition of these pathways would greatly benefit breast cancer patients with co-activation of these pathways. In this study, we aimed to determine the therapeutic utility of dual targeting JAK2 and TrkA in TNBC and HER2-enriched breast cancer using FDA-approved selective inhibitors. Our data showed that JAK2 and TrkA inhibitors synergize to reduce breast cancer cell viability in vitro. Our study further revealed that co-inhibition of JAK2 and TrkA potently inhibits breast cancer cell stemness since co-inhibition of these kinases significantly reduces the CD44high/CD24low cell population, mammosphere formation, and ALDH activity to a greater extent than vehicle or monotherapies. We also found that co-inhibition of JAK2 and TrkA more effectively induces apoptosis of breast cancer cells, as indicated by Annexin V flow cytometry, Western blot analysis for cleaved PARP, and Caspase 3/7 activity. Importantly, our in vivo studies showed that co-inhibition of JAK2 and TrkA significantly delays mammary tumor growth using cell line-derived and patient-derived xenograft breast cancer mouse models. Taken together, our data illustrates the therapeutic utility of dual targeting JAK2 and TrkA in metastatic TNBC and HER2-enriched breast cancers. Citation Format: Angelina T. Regua, Mariana K. Najjar, Grace L. Wong, Sara G. Manore, Austin Arrigo, Hui-Wen Lo. Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1721.

TrkA Co-Receptors: The Janus Face of TrkA?

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.

Auricular Vagal Nerve Stimulation Inhibited Central Nerve Growth Factor/Tropomyosin Receptor Kinase A/Phospholipase C-Gamma Signaling Pathway in Functional Dyspepsia Model Rats With Gastric Hypersensitivity

ObjectiveFunctional dyspepsia (FD), which has a complicated pathophysiologic process, is a common functional gastrointestinal disease. Gastric hypersensitivity is the key pathophysiological factor in patients with FD with chronic visceral pain. Auricular vagal nerve stimulation (AVNS) has the therapeutic effect of reducing gastric hypersensitivity by regulating the activity of the vagus nerve. However, the potential molecular mechanism is still unclear. Therefore, we investigated the effects of AVNS on the brain-gut axis through the central nerve growth factor (NGF)/ tropomyosin receptor kinase A (TrkA)/phospholipase C-gamma (PLC-γ) signaling pathway in FD model rats with gastric hypersensitivity. Materials and MethodsWe established the FD model rats with gastric hypersensitivity by means of colon administration of trinitrobenzenesulfonic acid on ten-day-old rat pups, whereas the control rats were given normal saline. AVNS, sham AVNS, K252a (an inhibitor of TrkA, intraperitoneally), and K252a + AVNS were performed on eight-week-old model rats for five consecutive days. The therapeutic effect of AVNS on gastric hypersensitivity was determined by the measurement of abdominal withdrawal reflex response to gastric distention. NGF in gastric fundus and NGF, TrkA, PLC-γ, and transient receptor potential vanilloid 1 (TRPV1) in the nucleus tractus solitaries (NTS) were detected separately by polymerase chain reaction, Western blot, and immunofluorescence tests. ResultsIt was found that a high level of NGF in gastric fundus and an upregulation of the NGF/TrkA/PLC-γ signaling pathway in NTS were manifested in model rats. Meanwhile, both AVNS treatment and the administration of K252a not only decreased NGF messenger ribonucleic acid (mRNA) and protein expressions in gastric fundus but also reduced the mRNA expressions of NGF, TrkA, PLC-γ, and TRPV1 and inhibited the protein levels and hyperactive phosphorylation of TrkA/PLC-γ in NTS. In addition, the expressions of NGF and TrkA proteins in NTS were decreased significantly after the immunofluorescence assay. The K252a + AVNS treatment exerted a more sensitive effect on regulating the molecular expressions of the signal pathway than did the K252a treatment. ConclusionAVNS can regulate the brain-gut axis effectively through the central NGF/TrkA/PLC-γ signaling pathway in the NTS, which suggests a potential molecular mechanism of AVNS in ameliorating visceral hypersensitivity in FD model rats.

TrkA inhibition alleviates bladder overactivity in cyclophosphamide-induced cystitis by targeting hyperpolarization-activated cyclic nucleotide-gated channels.

To investigate the potential of Tropomyosin receptor kinase A (TrkA) for the treatment of interstitial cystitis/ bladder pain syndrome (IC/BPS). Sixty-four female rats were randomly assigned to the control and cyclophosphamide (CYP) groups. Quantitative reverse transcription polymerase chain reaction was utilized to detect the mRNA level of TrkA. Western blot analysis was used to measure the protein levels of TNF-α, IL-6, and TrkA. Immunostaining was used to detect the expression of TrkA in bladder sections. Contractility studies and urodynamic measurements were utilized to test the spontaneous contractions of detrusor muscle strips and the global bladder activity, respectively. Rat models of chronic cystitis were successfully established. The mRNA and protein levels of TrkA were significantly increased in the bladders of CYP-treated rats. Also, results of immunohistochemical staining and immunofluorescence staining showed that increased TrkA expression in the CYP group was mainly observed in the urothelium layer and bladder interstitial Cajal-like cells (ICC-LCs) but not in the detrusor smooth muscle cells. The specific inhibitor of TrkA, GW441756 (10 μM), significantly suppressed the robust spontaneous contractions of detrusor muscle strips in the CYP group and alleviated the overall bladder overactivity of CYP-treated rats. However, the inhibitory effects of GW441756 (10 μM) on the spontaneous contractions of detrusor muscle strips and the overall bladder activity were eliminated after pretreatments with the specific blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ZD7288 (50 μM). Our results suggested that increased TrkA expression during chronic cystitis promotes the development of bladder overactivity by targeting the HCN channels.

Rational design and crystallographic analysis of novel isoform-selective TRKA inhibitors for cancer therapy

Rational design and crystallographic analysis of novel isoform-selective TRKA inhibitors for cancer therapy

Design, synthesis, and biological evaluation of aminopyridine derivatives as novel tropomyosin receptor kinase inhibitors.

Tropomyosin receptor kinase (TRK) is a successful target for the treatment of various cancers caused by NTRK gene fusions. Herein, based on a rational drug design strategy, we designed and synthesized 35 aminopyrimidine derivatives that were shown to be TRKA inhibitors in the enzyme assay, among which compounds C3, C4, and C6 showed potent inhibitory activities against TRKA with IC50 values of 6.5, 5.0, and 7.0 nM, respectively. In vitro antiproliferative activity study showed that compound C3 significantly inhibited the proliferation of KM-12 cellsbut had weak inhibitory effect on MCF-7 cells and HUVEC cells. The preliminary druggability evaluation showed that compound C3 exhibited favorable liver microsomal and plasma stabilitiesand had weak or no inhibitory activity against cytochrome P450 isoforms at 10 µM. Compounds C3, C4, andC6 were also selected for ADME (absorption, distribution, metabolism, and elimination) properties prediction and molecular docking studies. Inhibition experiments showed that compound C3 was not selective for TRK subtypes. All results indicated that compound C3 was a useful candidate for the development of TRK inhibitors.