NTRK2 and NTRK3 mutants are constitutively phosphorylated and sensitive to Trk inhibition. (A-B) Expression of total and phosphorylated TrkB and TrkC is increased in mutant-transformed Ba/F3 cells relative to WT cells. All mutants phosphorylate canonical downstream effectors, AKT and extracellular signal-regulated kinase (ERK). STAT3 phosphorylation was not evident in NTRK2^R458G. Both NTRK2 mutants result in increased SRC phosphorylation. GAPDH served as a loading control. Prior to lysis, WT cells were grown in IL-3–supplemented media and all lines were starved overnight in 0.1% bovine serum albumin (BSA) RPMI. (C-D) NTRK mutants are sensitive to entrectinib and larotrectinib. Six replicates of WT and mutant NTRK2 and NTRK3 Ba/F3 cells were plated with varying concentrations of entrectinib and larotrectinib for 72 hours. NTRK2^WT and NTRK3^WT cells were plated in media supplemented with IL-3. Cell viability was determined using a tetrazolamine-based viability assay. Viability is represented as a percentage of the untreated control. The average mean plus or minus SEM is shown. Additional validation studies are found in supplemental Figure 6B. (E) Mutant-transformed Ba/F3 cells were starved overnight in 0.1% BSA RPMI and then treated with increasing concentrations of entrectinib (ENT) for ∼16 hours and immunoblotted for Trk and downstream effectors. (F) ENT induces apoptosis in mutant-transformed Ba/F3 cells following 48 and 72 hours. Annexin V staining was performed in triplicate. The average mean plus or minus SEM is shown. Validation studies in supplemental Figure 6C. p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; p-SRC, phosphorylated SRC; p-STAT, phosphorylated STAT.