Abstract B60: Therapeutic strategies for NTRK1-rearranged cancer
Authors: Fuse Miho J. , Fujita Naoya , Katayama Ryohei 
Published: May 2016

Abstract
The Neurotrophic Tyrosine Kinase, Receptor, Type 1 (NTRK1) gene encodes a single transmembrane receptor tyrosine kinase. The nerve growth factor binds to NTRK1 and plays an important role in differentiation and survival of neurons. NTRK1-rearranged cancer is reported in several cancer types and has recently been reported in glioblastoma, non-small cell lung cancer (NSCLC), colorectal cancer and so on. NTRK1 fuses to a partner gene which induces dimerization, leading to constitutive activation of the tyrosine kinase in the NTRK1 fusion protein ligand independently. Although there are currently no clinically available inhibitors that target NTRK1, several NTRK1 inhibitors are in clinical trials.
The purpose of this study is to identify novel potent inhibitors against NTRK1 and to reveal potential resistance mechanisms.
We used KM-12 cells (colon cancer) harboring TPM3-NTRK1 and established a Ba/F3 cell line transformed with TPM3-NTRK1. We conducted a drug screening containing FDA approved inhibitors against both KM-12 cells and TPM3-NTRK1 Ba/F3 cells. We identified several potent inhibitors including XL-184 (cabozantinib, FDA approved in medullary thyroid carcinoma) which is consistent with other studies, and BIBF-1120 (nintedanib, FDA approved for the treatment of idiopathic pulmonary fibrosis).
In order to identify amino acid mutations that confer resistance, we induced point mutations into TPM3-NTRK1 Ba/F3 cells with ENU (a DNA alkylating compound) and screened these cells under high concentrations of inhibitors including XL-184. We succeeded in identifying 5 novel resistant mutations in the NTRK1 kinase domain. Interestingly we did not obtain the gatekeeper mutation which is a common resistant mutation in EGFR-mutated or ALK-rearranged NSCLC treated with erlotinib, gefitinib or crizotinib respectively.
We also established a drug resistant KM-12 cell line. We treated cells with XL-184 at 1 μM and surprisingly a small fraction of viable cells were detected. We subsequently picked up these clones with innate resistance and further analyzed them. No mutation in the kinase domain of NTRK1 was found but we identified a bypass pathway activation using RTK array assay and drug screening. We further confirmed that co-treatment with XL-184 and the identified inhibitor completely reversed the resistance.
Our study provides potential therapeutic strategies and resistance mechanisms for NTRK1- rearranged cancers. Considering that the resistant mutations identified in ALK and ROS1 using ENU mutagenesis screening have been observed in crizotinib treated patients, there is a high possibility that post-treated patients with NTRK1-rearranged cancer will also resemble our results. Although we have shown that the resistance observed in the KM-12 cell line can be overcome by a combination of inhibitors, drugs that can overcome the resistant mutations are still required.
Citation Format: Miho J. Fuse, Naoya Fujita, Ryohei Katayama. Therapeutic strategies for NTRK1-rearranged cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B60.