Extended Data Fig. 2: Effect of the pan-Trk inhibitor entrectinib on glioma proliferation is mediated by TrkB inhibition.

a, Western blot of whole brain protein lysate collected from NSG mice that were either treated with one PO dose of 120 mg/kg of entrectinib or one dose of vehicle control (PO). The mouse brains were harvested after transcardial perfusion and mice were collected at either 30 min, 2 h and 4 h after vehicle or entrectinib dosing. The protein lysate was probed for the indicated antibodies to demonstrate inhibition of BDNF-TrkB signaling as an indication of effective drug penetration into brain tissue. b, Quantification of TrkB phosphorylation by comparing the ratio of the normalized phospho-TrkB (Tyr515) levels to corresponding total TrkB protein levels between the entrectinib treated and vehicle control mice (y axis is in arbitrary units, n = 4 technical replicates, pTrkB vehicle vs entrectinib 30 min P = 0.036, 2 h P = 0.0082, 4 h P < 0.0001). c, Quantification of MAPK pathway activation by comparing the ratio of the normalized phospho-ERK (T202/Y204) to corresponding total protein levels between the entrectinib treated and vehicle control treated mice (y axis is in arbitrary units, n = 3 technical replicates, pErk vehicle vs entrectinib 2 h P = 0.0220, 4 h P = 0.0010). d, Representative image of mouse brain (sagittal section) from SU-DIPG-XIII-P* xenografted to the pons treated with entrectinib (120 mg/kg PO) at endpoint in survival analyses (presented in Fig. 1g). White denotes HNA (tumor cells); DAPI nuclei are shown in blue (scale bar = 2000 µm). e, Experimental model of pontine xenografted WT and NTRK2 KO glioma (SU-DIPG-VI) treated with the Pan-Trk inhibitor, entrectinib, or vehicle control. f, Proliferation index of wild-type and NTRK2 KO SU-DIPG-VI glioma xenografted to the pons of NSG mice and treated with vehicle or entrectinib (120 mg/kg PO). Quantification by confocal microscopy analysis of EdU + /HNA+ co-positive tumor cells, as in representative Fig. 1c, n = 4 wild-type glioma xenografted, vehicle-treated mice, 5 wild-type glioma xenografted, entrectinib-treated mice, 5 NTRK2 KO glioma xenografted, vehicle-treated mice, 3 NTRK2 KO glioma xenografted, entrectinib-treated mice, WT vehicle vs WT entrectinib P = 0.0002, WT vehicle vs NTRK2 KO vehicle P < 0.0001). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Two-tailed one sample t test for b and c, one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis for f.