Background: Oncogene fusions involving members of the TRK family of receptor tyrosine kinases (encoded by NTRK1, NTRK2, and NTRK3) have been identified in multiple cancer types. The use of TRK inhibitors in vitro, in vivo and in patients has demonstrated activity against a number of different NTRK oncogene fusions in different tumor types. Thus, the use of a pan-TRK inhibitor presents a therapeutic opportunity for multiple patient populations. LOXO-101 is an inhibitor of the TRK kinase and is highly selective for the TRKA/B/C family of kinases. Kinase domain (KD) mutations are the most commonly identified mechanism of acquired drug resistance found in patients treated with kinase inhibitors. We therefore undertook an unbiased genetic approach to identify candidate resistance mutations in the TRK kinase domain.
Methods: We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-MPRIP-NTRK1 and Ba/F3-TRIM24-NTRK2 cells to generate mutations permitting growth of Ba/F3 cells in the absence of IL-3 despite the presence of 100, 250, or 500 nM LOXO-101. Mutations identified by genomic DNA sequencing in the initial screen were validated by cloning the mutation-bearing cDNAs back into Ba/F3 cells to evaluate their sensitivity to LOXO-101 using both proliferation assays and TRK phosphorylation by immunoblot analyses. Modeling of the mutations was performed by mapping of the amino acid substitutions onto a drug-bound TRK kinase domain crystal structure.
Results: We have identified 6 amino acid positions in the TRKA and 3 positions in the TRKB KD that induce resistance to LOXO-101. In the TRKA KD we identified V573M, and F589L/C, G595S, F600L, F646V, and G667S. In the TRKB KD, we identified Q596E/P, F617L/C/I, and G623S. The TRKA F589 and TRKB F617 position corresponds to the gatekeeper position, homologous to L1196 in ALK or T790 in EGFR. Several other TRKA and TRKB resistance mutations have homologous mutations in other kinases: TRKA G667 (ALK G1269), TRKA V573 (ALK V1180M), TRKA G595/TRKB G623 (ALK G1202 and ROS1 G2202), and TRKA F646 (ALK F1245). The TRKA F600 has not been previously identified as a resistance mutation in other kinases, but is adjacent to a homologous ALK S1206 resistance position. TRKB Q596 position has also not been previously but is adjacent to the homologous ALK F1174 resistance position. The TRK mutations identified here reduce target inhibition by LOXO-101 as measured by TRK tyrosine phosphorylation and resultant downstream signaling through the MAPK or other critical pathways.
Conclusion: This genetic screen identified and validated several novel mutations in the TRKA and TRKB KD that may confer drug resistance to LOXO-101 in patients. Many, although not all, of the resistance mutations identified here are homologous to resistance mutations identified in other oncogenic kinases treated with kinase inhibitors. These data can be used to screen tumor samples from TRK+ patients who develop acquired resistance to TRK inhibitors and may assist in the design of next generation TRK inhibitors to potentially overcome acquired resistance in patients treated with LOXO-101 or structurally similar TRK inhibitors.
Citation Format: Adriana Estrada-Bernal, Anh T. Le, Brian Tuch, Tatiana G. Kutateladze, Robert C. Doebele. Identification of TRKA and TRKB kinase domain mutations that induce resistance to a pan-TRK inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-118.