AbstractEGFR tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC) patients is inevitable. Identification of resistance mechanisms and corresponding targeting strategies can lead to more successful later-line treatment in many patients. Using spectrometry-based proteomics, we identified increased fibroblast growth factor receptor 1 (FGFR1) expression and Akt activation across erlotinib, gefitinib, and osimertinib EGFR-TKI-resistant cell line models. We show that while combined EGFR-TKI and FGFR inhibition showed some efficacy, simultaneous inhibition of FGFR and Akt or PI3K induced superior synergistic growth inhibition of FGFR1-overexpressing EGFR-TKI-resistant NSCLC cells. This effect was confirmed in vivo. Only dual FGFR and Akt inhibition completely blocked the resistance-mediating signaling pathways downstream of Akt. Further, increased FGFR1 expression was associated with significantly lower PFS in EGFR-TKI-treated NSCLC patients, and increased FGFR1 were demonstrated in a few post- vs. pre-EGFR-TKI treatment clinical biopsies. The superior therapeutic benefit of combining FGFR and Akt inhibitors provide the rationale for clinical trials of this strategy.