Fig. 3: Identification of the EML4::NTRK3 fusion and in vitro cell modeling.

a Schematic representation of the EML4::NTRK3 fusion identified by RNA-sequencing in the relapse. b The selective expression of the EML4::NTRK3 fusion transcript in the relapse but not in the primary tumor was confirmed by RT-PCR. c Sequencing of the RT-PCR product of the relapse displaying the junction between EML4 and NTRK3. d Immunohistochemistry for pan-TRK shows strong and diffuse staining in the recurrence. e Negative pan-TRK immunostaining in the primary tumor (10X magnification). f Western blot analysis for KIT and the NTRK-fused protein product in the GIST-E4N3 cell model engineered to express the EML4::NTRK3 chimera. GIST-CTR cells served as a negative control. GAPDH was used as a loading control. A representative image of three biologically independent replicates is shown. g GIST-E4N3 cells show reduced sensitivity to imatinib relative to GIST-CTR. Relative cell count (RCC) was assessed by the SRB assay at 48 h post-treatment. *p-value ≤ 0.05, **p-value ≤ 0.01, ****p-value ≤ 0.0001. Data are presented as mean ± SEM (n = 6). h Colony growth assay demonstrating the capacity of GIST-E4N3 cells to survive a chronic, 3-week imatinib treatment (50 nM). For better visualization of individual colonies of vehicle-treated cells, images taken at 2-week DMSO treatment are shown. i Compared to GIST-CTR, imatinib-resistant derivatives of GIST-E4N3 cells (GIST-E4N3R) exhibit increased sensitivity to NTRK inhibitors (200 nM larotrectinib, dark green; 200 nM entrectinib, light green; 48 h-treatment). ****p-value ≤ 0.0001. Data are presented as mean ± SEM (n = 6).