Mutations in NTRK3 suggest a novel signaling pathway in human congenital heart disease.

Congenital heart defects (CHDs) are the most common major birth defects and the leading cause of death from congenital malformations. The etiology remains largely unknown, though genetic variants clearly contribute. In a previous study, we identified a large copy-number variant (CNV) that deleted 46 genes in a patient with a malalignment type ventricular septal defect (VSD). The CNV included the gene NTRK3 encoding neurotrophic tyrosine kinase receptor C (TrkC), which is essential for normal cardiogenesis in animal models. To evaluate the role of NTRK3 in human CHDs, we studied 467 patients with related heart defects for NTRK3 mutations. We identified four missense mutations in four patients with VSDs that were not found in ethnically matched controls and were predicted to be functionally deleterious. Functional analysis using neuroblastoma cell lines expressing mutant TrkC demonstrated that one of the mutations (c.278C>T, p.T93M) significantly reduced autophosphorylation of TrkC in response to ligand binding, subsequently decreasing phosphorylation of downstream target proteins. In addition, compared with wild type, three of the four cell lines expressing mutant TrkC showed altered cell growth in low-serum conditions without supplemental neurotrophin 3. These findings suggest a novel pathophysiological mechanism involving NTRK3 in the development of VSDs.

AZ64 inhibits TrkB and enhances the efficacy of chemotherapy and local radiation in neuroblastoma xenografts.

Neuroblastoma is a common pediatric tumor characterized by clinical heterogeneity. Because it is derived from sympathetic neuroblasts, the NTRK family of neurotrophin receptors plays an integral role in neuroblastoma cell survival, growth, and differentiation. Indeed, high expression of NTRK1 is associated with favorable clinical features and outcome, whereas expression of NTRK2 and its ligand, brain-derived neurotrophic factor (BDNF), are associated with unfavorable features and outcome. AZ64 (Astra Zeneca) is a potent and selective inhibitor of the NTRK tyrosine kinases that blocks phosphorylation at nanomolar concentrations. To determine the preclinical activity of AZ64, we performed intervention trials in a xenograft model with NTRK2-overexpressing neuroblastomas. AZ64 alone significantly inhibited tumor growth compared to vehicle-treated animals (p = 0.0006 for tumor size). Furthermore, the combination of AZ64 with conventional chemotherapeutic agents, irinotecan and temozolomide (irino-temo), showed significantly enhanced anti-tumor efficacy compared to irino-temo alone [(p < 0.0001 for tumor size, p < 0.0005 for event-free survival (EFS)]. We also assessed the combination of AZ64 and local radiation therapy (RT) on a neuroblastoma hindlimb xenograft model, and the efficacy of local RT was significantly increased when animals were treated simultaneously with AZ64 (p < 0.0001 for tumor size, p = 0.0006 for EFS). We conclude that AZ64 can inhibit growth of NTRK-expressing neuroblastomas both in vitro and in vivo. More importantly, it can significantly enhance the efficacy of conventional chemotherapy as well as local RT, presumably by inhibition of the NTRK2/BDNF autocrine survival pathway.

Clinical significance of NTRK family gene expression in neuroblastomas.

BACKGROUND: Neuroblastomas (NBs) are characterized by clinical heterogeneity, from spontaneous regression to relentless progression. The pattern of NTRK family gene expression contributes to these disparate behaviors. TrkA/NTRK1 is expressed in favorable NBs that regress or differentiate, whereas TrkB/NTRK2 and its ligand brain-derived neurotrophic factor (BDNF) are co-expressed in unfavorable NBs, representing an autocrine survival pathway. We determined the significance of NTRK family gene expression in a large, representative set of primary NBs. PATIENTS AND METHODS: We analyzed the expression of the following genes in 814 NBs using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR): NTRK1, NTRK2, NTRK3, P75/NGFR, nerve growth factor (NGF), BDNF, IGFR1, and EGFR. Expression (high vs. low) was dichotomized by median expression value and compared to clinical and biological variables as well as outcome. RESULTS: High NTRK1 expression was strongly correlated with favorable age, stage, MYCN status, histology, ploidy, risk group, and outcome (P < 0.0001 for all). However, it did not add significantly to the panel of prognostic variables currently used for cooperative group trials. NTRK2 expression was associated with risk factors but not with outcome. High NGF expression was also associated with most risk factors and weakly with unfavorable outcome. CONCLUSIONS: High expression of NTRK1 is strongly associated with favorable risk factors and outcome in a large, representative population of NB patients. It did not add significantly to the current risk prediction algorithm, but it may contribute to future expression classifiers. Indeed, prospective assessment of NTRK1 and NTRK2 expression will identify tumors that would be candidates for NTRK-targeted therapy, either alone or in combination with conventional agents.

The effect of P75 on Trk receptors in neuroblastomas.

Neuroblastomas (NBs) with favorable outcome usually express TrkA, whereas unfavorable NBs frequently express TrkB and its cognate ligand BDNF. P75 (p75(LNTR), NGFR, TNFRSF16) binds NGF-related neurotrophins with low affinity and usually is coexpressed with Trk receptors in NBs. Here, we investigated the importance of p75 coexpression with Trk receptors in NBs. We transfected p75 into two Trk-null NB cell lines, SH-SY5Y and NLF that were also engineered to stably express TrkA or TrkB. Cell numbers were compared between single (Trk alone) and double (Trk+p75) transfectants, and proliferation was assessed by flow cytometry. P75 coexpression had little effect on cell growth in Trk NB cells in the absence of ligand, but it increased sensitivity and greatly enhanced the effect of cognate ligand. Exogenous NGF induced greater phosphorylation of TrkA and AKT. This was associated with increased cell number in TrkA/p75 cells compared to TrkA cells (p<0.01), which was due to increased proliferation in TrkA/p75 cells (p<0.05), followed by differentiation. Exogenous BDNF also increased cell number in TrkB/p75 compared to TrkB cells (p<0.01), due to an increase in proliferation, but without differentiation. Coexpression of p75 also increased specificity of Trk-expressing cells to ligand. NT3-induced phosphorylation of TrkA and AKT was reduced in TrkA/p75 cells. NT3-induced phosphorylation of TrkB (as well as AKT and MAPK) was also reduced with p75 coexpression. Our results suggest that p75 plays an important role in enhancing both the sensitivity of Trk receptors to low levels of ligand, as well as increasing the specificity of Trks to their cognate ligands. It also enhances ligand-induced differentiation in TrkA/p75 but not TrkB/p75 cells.

Trk receptor expression and inhibition in neuroblastomas.

Neuroblastoma, the most common and deadly solid tumor in children, exhibits heterogeneous clinical behavior, from spontaneous regression to relentless progression. Current evidence suggests that the TRK family of neurotrophin receptors plays a critical role in these diverse behaviors. Neuroblastomas expressing TrkA are biologically favorable and prone to spontaneous regression or differentiation, depending on the absence or presence of its ligand (NGF) in the microenvironment. In contrast, TrkB-expressing tumors frequently have MYCN amplification and are very aggressive and often fatal tumors. These tumors also express the TrkB ligand (BDNF), resulting in an autocrine or paracrine survival pathway. Exposure to BDNF promotes survival, drug resistance, and angiogenesis of TrkB-expressing tumors. Here we review the role of Trks in normal development, the different functions of Trk isoforms, and the major Trk signaling pathways. We also review the roles these receptors play in the heterogeneous biological and clinical behavior of neuroblastomas, and the activation of Trk receptors in other cancers. Finally we address the progress that has been made in developing targeted therapy with Trk-selective inhibitors to treat neuroblastomas and other tumors with activated Trk expression.