Trka, a Novel Binding Partner of NPM-ALK Oncogenic Tyrosine Kinase, Facilitates the Survival of T-Cell Anaplastic Large-Cell Lymphoma
Authors: Shi Wenyu , George Suraj Konnath , Curry Choladda V. , Alkan Serhan , Amin Hesham M. 
Published: November 2019

Abstract
Anaplastic lymphoma kinase-expressing (ALK+) T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer that frequently occurs in children and adolescents. Approximately 85% of the ALK+ ALCL patients harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. The expression of ALK is primarily identified in neural tissues at early stages of human development, which suggests that ALK contributes to neural tissue development probably through interactions with neurotrophic factors.
Nerve growth factor (NGF) is the first characterized neurotrophic factor. In addition to its canonical roles in neural tissue development, NGF has gained attention as a promoter of survival of cancer cells. Indeed, deregulation of NGF signaling has been attributed to cell proliferation, invasion, and metastasis. It is believed that NGF’s non-neural functions are essentially mediated through two cognate receptors: TrkA and p75NTR. The expression and role of NGF/TrkA signaling in NPM-ALK+ T-cell lymphoma have not been previously studied. Because of the biological similarities between ALK and NGF/TrkA signaling, we hypothesized that blockade of NGF/TrkA signaling may suppress NPM-ALK+ T-cell lymphoma cell survival, which can have important therapeutic implications.
Western blotting and immunohistochemical staining showed that TrkA and NGF proteins are highly expressed in 5 NPM-ALK+ T-cell lymphoma cell lines compared with normal human T lymphocytes. In addition, immunohistochemical staining demonstrated the expression of TrkA in 85% (11/13) of ALK+ T-cell lymphoma patient tumors. Immunoprecipitation showed physical association between TrkA and NPM-ALK. Specific downregulation of NPM-ALK using ALK siRNA was associated with a marked decrease in phosphorylated TrkA, suggesting functional interactions between the two proteins. Thereafter, we used a selective TrkA Inhibitor (catalogue number: 648450; Calbiochem, Billerica, MA) and performed in vitro assays to elucidate the contribution of TrkA signaling on the lymphoma cells. The TrkA Inhibitor decreased efficiently the phosphorylation and kinase activity of TrkA in NPM-ALK+ T-cell lymphoma cells. These effects were associated with decreased NPM-ALK+ T-cell lymphoma cellular proliferation. The TrkA Inhibitor also induced apoptosis and abrogated anchorage-independent colony formation of these cells. Moreover, the TrkA Inhibitor caused downregulation of pNPM-ALK, pIGF-IR, pSTAT3, and pAKT. Caspase-3, BCL-2 and BCL-XL were also reduced, supporting apoptosis occurrence. To rule out nonspecific effects that might have been caused by the TrkA Inhibitor, TrkA siRNA was used in some experiments. TrkA siRNA, but not scrambled siRNA, decreased the viability and proliferation of NPM-ALK+ T-cell lymphoma cells. In addition, TrkA siRNA caused biochemical effects similar to those observed with the TrkA Inhibitor. An ELISA assay demonstrated the presence of NGF in cell culture supernatant of serum-deprived NPM-ALK+ lymphoma cells. Importantly, NGF enhanced the viability and proliferation of NPM-ALK+ T-cell lymphoma cells, and TrkA blocking antibody abrogated these effects. These results suggest the presence of an autocrine NGF/TrkA loop that supports the survival of NPM-ALK+ T-cell lymphoma. We also examined the effects of TrkA Inhibitor in SCID-beige mice with NPM-ALK+ T-cell lymphoma xenografts implanted subcutaneously using SR-786 cells. The tumors were established after 8 days, and mice were randomly treated with vehicle or the TrkA Inhibitor for 21 days (10 mg/kg b.i.d; s.c.). Tumor volumes were remarkably smaller in the TrkA Inhibitor-treated mice compared with vehicle-treated mice.
Our data provide novel evidence that TrkA contributes to the survival of NPM-ALK+ T-cell lymphoma, and implicate that targeting TrkA may represent an effective approach to suppress this lymphoma.

Disclosures
No relevant conflicts of interest to declare.