Characterization of Transforming NTRK2 and NTRK3 Mutations Identified in Leukemia Patient Samples

Overview

The neurotrophin receptor tyrosine kinases (NTRK) play a seminal role in central nervous system development, but are expressed in various peripheral tissues as well. NTRKs regulate cellular function via activation of canonical cell growth and survival pathways via regulation of MAPK and PI3K respectively. Genetic aberrations that activate NTRK function have been recently shown to be promising therapeutic targets in lung cancer, sarcoma, neuroblastoma, and other carcinomas. However, their role as potential oncogenic drivers in blood cancers such as acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and acute lymphoblastic leukemia (ALL) have not yet been reported. In this study, we describe somatic mutations in NTRK2 and NTRK3 that are potential drivers of these hematological malignancies and demonstrate sensitivity to the NTRK inhibitor, entrectinib, in model systems. These mutations could serve as novel, actionable molecular targets in hematological malignancies.

Methods and Results

Over a dozen unique mutations in NTRK2 and NTRK3 were identified and then prioritized using deep sequencing data obtained from primary leukemia samples and small molecule inhibitor sensitivity data obtained on the freshly isolated patient sample material. All mutations were created using mutagenesis PCR and stably expressed in BaF3 cells, an IL-3 dependent murine pro-B cell line. IL-3 withdrawal transformation assays were performed and two point mutations in NTRK2 and two point mutations in NTRK3 were found to be transforming. Western blotting performed on the lysates obtained from NTRK mutant transformed BaF3 cells demonstrated increased phosphorylation of the receptor as well as signaling pathways known to be downstream of NTRKs, including PLCγ, AKT, and ERK1/2. Each mutation was verified in the patient genomic DNA using Sanger sequencing, and for samples with available material, NTRK protein expression was verified via capillary-based immunoblotting. Small molecule inhibitor or siRNA screens performed on the freshly isolated leukemia samples also identified NTRK as a key target further underscoring a dependency on NTRK activation in the patient samples. Results from small molecule inhibitor sensitivity assays performed on the NTRK mutant-transformed BaF3 cells demonstrated that all four transforming NTRK mutations were sensitive at low nanomolar concentrations (IC50: 0.73-17.7 nM) to entrectinib (RXDX-101, Ignyta), a selective and potent inhibitor of NTRKs, ROS1, and ALK.

Conclusions

Taken together, we have identified novel activating mutations in NTRK2 and NTRK3 with oncogenic potential in blood cancers that are specifically sensitive to the NTRK inhibitor entrectinib. Our results suggest that NTRK mutations may play a role in leukemogenesis by activating downstream pathways and could serve as a potential therapeutic target in leukemia patients harboring NTRK mutations. Further studies are ongoing to fully assess the molecular characteristics of these mutations and to demonstrate the effects of small molecule inhibitors targeting NTRKs in vivo.