In vitro activity: LOXO-195 is a 2nd generation, potent and selective TRK TKI (tyrosine kinase inhibitor) designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to other TRK TKIs such as larotrectinib (LOXO-101). It has IC50s of 0.6±0.1 nM,<2.5 nM for TRKA and TRKC respectively. Activity against the acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first 2 patients with TRK fusion-positive cancers who developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients. LOXO-195 abrogated resistance in TRK fusion-positive cancers that acquired kinase domain mutations, a shared liability with all existing TRK TKIs. This establishes a role for sequential treatment by demonstrating continued TRK dependence and validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets.

Kinase Assay: Binding affinities for each purified TRK kinase domain were measured using LanthaScreen(TM) Eu Kinase Binding technology (Invitrogen). Briefly, each donor europium antibody--labelled recombinant TRK kinase domain was incubated with the Fluor 236 probe and a serial dilution of each inhibitor, and the effect of added inhibitor on probe binding was monitored by TR-FRET. Enzyme activity was determined by monitoring the incorporation of [33P]PO4 from [γ-33P]-ATP into poly-EAY peptide substrate in the presence of a serial dilution of each compound. Kinase profiling was performed using KinaseProfiler(TM) (Millipore, Inc.). See Supplementary Methods for additional details.

Cell Assay: KM12 cells were obtained from the NCI-Frederick Cancer DCTD Tumor Cell Repository in 2011. CUTO-3 cells were obtained from Dr. Robert Doebele, University of Colorado Cancer Center in 2015. MO-91 cells were obtained from Dr. Stephen Nimer, University of Miami Sylvester Cancer Center in 2015. 84 NTRK gene rearrangement-negative human cancer cell lines were selected from the Eurofins OncoPanel collection of cell lines (Eurofins) in 2015. NIH 3T3 cells were obtained by Array BioPharma from 2007–2016. Eurofins cell lines were authenticated by short-tandem repeat (STR) analysis (Genetica DNA Laboratories, Inc., last July 2015). KM12, CUTO-3 and MO-91 cells were authenticated by confirmation of the presence of each fusion (e.g. TPM3-NTRK1, ETV6-NTRK3 or MPRIP-NTRK1, respectively) by the Oncomine Focus Assay NGS assay (Thermo Fisher Scientific, Inc., within 12 months of experiments). NIH 3T3 cells expressing ΔTRKA and ETV6-TRKC variants were engineered as described in Supplementary Methods. For cellular phospho-TRK measurement, cells were seeded into 96-well plates and incubated overnight at 37C, incubated for one hour with a dilution series of each inhibitor, followed by lysis of each well monolayer in situ and quantitation of phospho-TRKA levels by ELISA assay (Cell Signaling Technologies) per the manufacturer’s instructions. For cellular phospho-ERK assessment, cells expressing each FLAG-tagged ETV6-TRKC variant were seeded into 96-well plates were treated with each inhibitor for one hour, followed by formaldehyde fixation, saponin-based suspension/ permeabilization, incubation with phosphorylated ERK antibody (Cell Signaling Technology), APC-anti-FLAG and PE anti-rabbit secondary antibodies and analysis by flow cytometry.