Fig. 7. Activation of FBXL2 overcomes osimertinib resistance of NSCLC.
a HEK293T cells were co-transfected with indicated expressing plasmids for 36 h, followed by Western blot analyses. Data are representative immunoblots of three independent assays. b, c HEK293T cells were co-transfected with Flag-EGFRT790M/C797S in the presence of HA-FBXL2 or a vector control for 36 h, and were then treated with 50 µg/mL cycloheximide (CHX) for an indicated time interval prior to Western blot analyses. The EGFR protein levels were quantified and a plot representing protein half-life was presented. PC-9-Flag-EGFRT790M/C797S cells stably expressing HA-FBXL2 or HA-FBXL2C420S were subjected to d Western blot analyses or e−g xenograft tumor growth assays (n = 6/group). Photos of tumor, growth curve, and tumor weight were presented. Paraffin-embedded tumors were subjected to IHC analyses. Data were quantified by AOD. Data were presented as means ± SEM. h PC-9 cells stably expressing EGFRT790M/C797S (PC-9/AZDR) were infected with lentivirus encoding HA-FBXL2. Cells were treated with an indicated dose of osimertinib for 72 h prior to MTS analyses. Data derived from three independent experiments in triplicates were presented as means ± SD. ***p < 0.001. i PC-9/AZDR cells stably expressing shFBXL2 were treated with an indicated dose of osimertinib in the absence or presence of 5 μM nebivolol for 48 h followed by MTS assays. Data from three independent experiments in triplicates were presented as means ± SD. j, k PC-9/AZDR stable cells (2 × 105) were subjected to xenograft tumor growth assays (n = 5/group). Mice were administrated with nebivolol alone or in combination with osimertinib. Photos of tumors and tumor growth curves were shown. Data were presented as means ± SEM. l A working model. The E3 ubiquitin ligase FBXL2 targets EGFR and EGFR mutants for proteasomal degradation, resulting in inhibition of tumor growth and TKI resistance of NSCLC. Grp94 binds to and protects EGFR from FBXL2-mediated degradation. The FDA-approved drug nebivolol can disrupt FBXO3−FBXL2 interaction to stabilize FBXL2 and degrade EGFR, thereby inhibiting both NSCLC growth and TKI resistance.*p < 0.05, **p < 0.01, ***p < 0.001; all by two-tailed Student’s t-test. Source data are provided as a Source data file.