In vitro and in vivo sensitivity of ETV6-NTRK3 to TRK inhibition. (A) Ba/F3 cells and (B) bone marrow cells harvested from Etv6-NTRK3/⁺;CD19-Cre mice were incubated in increasing concentrations of drug. Cell viability was measured after 48 hours using CellTiter-Blue viability assay. (C) Seventy-nine human cell lines were incubated in crizotinib or larotrectinib, and cell viability was measured after 72 hours using a cell count microscopy cytotoxicity assay. NTRK fusion cell lines: M0-91 (ETV6-NTRK3, acute myeloid leukemia), CUTO-3 (MPRIP-NTRK1, lung cancer), and KM12 (TPM3-NTRK1, colorectal carcinoma). (D-E) NSG mice (n = 10 per group) were injected with primary leukemic cells. Upon engraftment (10⁸ photons per second), mice were treated with vehicle, PLX7486 (provided as chow), or larotrectinib (200 mg/kg/d once daily gavage). Treatment length is indicated by the shaded area. Five mice from each group were sacrificed when vehicle-treated mice became moribund to evaluate leukemic infiltration. The remaining 5 mice treated with PLX7486 or larotrectinib were assessed for disease growth. Two mice from the larotrectinib-treated group with active disease were retreated at week 25 for 4 weeks. Splenic weights were recorded at the time of euthanasia. Error bars represent mean ± standard deviation. ***P < .001; ****P < .0001 by Student t test. (F) Representative slides of bone marrow showing restoration of normal architecture, hematopoiesis, and vasculature with PLX7486 or larotrectinib treatment (original magnification ×40; hematoxylin and eosin stain). Scale bar, 50 μm. Transverse section of the spinal cord showing infiltration of leukemic blasts in vehicle-treated mice, with clearance of leukemic blasts and restoration of normal architecture and vasculature with PLX7486 or larotrectinib treatment. Scale bar, 500 μm.