Background: Gene fusions involving members of the NTRK family have been identified in several types of cancer. The use of TRK inhibitors in vitro and in vivo have demonstrated activity against a number of different NTRK fusions in different tumor types and most recently in a patient with an NTRK1 gene fusion (Doebele et al., Cancer Discovery 2015). 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 common mechanism of acquired drug resistance found in patients. We therefore undertook a 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, Ba/F3-PAN3-NTRK2 and Ba/F3-ETV6-NTRK3 cells to generate mutations that could allow growth of Ba/F3 cells despite the presence of LOXO-101. Mutations identified 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 predicting the consequences of the amino acid substitutions within the context of a drug-bound TRK kinase domain crystal structure.

Results: We have identified three KD mutations within the TRKA kinase domain: V573M, and F589L, G667S. These mutations induce significant drug resistance in BA/F3 cells to LOXO-101 in vitro compared to the unmutated MPRIP-NTRK1. Furthermore, these mutations do not show inhibition of phosphorylation of TRKA at doses that inhibit the native MPRIP-NTRK1. The TRKA F589 position corresponds to the gatekeeper position, homologous to L1196 in ALK or T790 in EGFR, and a common position for drug resistance mutations. The TRKA G667 lies adjacent to the F589 position in the ATP/drug-bind pocket and similar resistance mutations have been observed in the ALK kinase domain following resistance to crizotinib. V573 is also positioned adjacent to both the G667 and F589 residues in the ATP-/drug-binding pocket. Although a similar mutation has been observed in vitro in ALK (V1180M), it has not yet been reported in patient tumor samples. Analysis of additional candidate resistance clones is ongoing.

Conclusion: This genetic screen identified several novel mutations in the TRK KD that may confer clinical resistance to LOXO-101. All three mutations identified to date appear to hinder binding by altering the ATP binding site of the kinase. These data should be confirmed in patients who develop clinical resistance, 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 Kutateladze, Robert C. Doebele. TRK kinase domain mutations that induce resistance to a pan-TRK inhibitor. [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 C65.