Abstract Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have improved overall survival in non-small-cell lung cancer (NSCLC) patients whose tumors have EGFR activating mutations. After treatment with EGFR-TKIs, NSCLC tumors with EGFR activating mutations become resistant to EGFR-TKIs, and development of novel treatment strategies is needed to improve survival in such patients.Next-generation EGFR-TKIs have been developed, and a clinical trial showed clinical activity of 3rd generation EGFR-TKIs in patients with acquired resistance, especially secondary mutation T790M to EGFR-TKIs. Moreover, clinical trials demonstrated survival benefit of combination with cytotoxic agents and EGFR-TKIs in patients with EGFR-mutated NSCLC. In contrast, the IMPRESS study showed that continuation of gefitinib in addition to cisplatin and pemetrexed didn't show survival benefit in patients with acquired resistance to gefitinib.Several basic studies reported that efficacy of targeted therapy combined with chemotherapy is schedule dependent. We, therefore, conducted an in vitro study to develop a novel therapeutic strategy for NSCLC with EGFR mutations. Methods: In vitro effects of docetaxel and an EGFR-TKI, gefitinib (AstraZeneca) or AZD9291 (Selleckchem), were examined in five lung cancer cell lines (PC9, PC9GR, HCC827, HCC827GR, and H1975) using a 3D culture model (PrimeSurface 96, SUMITOMO BAKELITE CO.). The NSCLC cells were treated with the combinationeither concurrently or sequentially (EGFR-TKIs after docetaxel or docetaxel after EGFR-TKIs). Combination Index (CI) values were calculated at various levels of cytotoxicity (the Chou-Talalay method). We evaluated changes in MAPK pathway activation and in expressions of Bcl2 family proteins and cleaved PARP (Cell Signaling) by western blot analysis. Results: Docetaxel followed by EGFR-TKIs weremore effective at CI, compared with EGFR-TKIs followed by docetaxel in two of the five cell lines with EGFR activating mutations, PC9GR (EGFR19del+T790M) and HCC827GR (EGFR19del+METamp) cells. The HCC827GR cells were likely to be more sensitive than PC9GR to the combination with docetaxel and gefitinib. The combo led to inactivation of the MAPK pathway and alteration of the Bcl2 family proteins. d) Conclusion: In conclusion, our results suggest that the sequential combination of docetaxel followed by EGFR-TKIs was effective against NSCLC with acquired resistance to EGFR-TKIs. Citation Format: Ryohei Yoshida, Synsuke Okumura, Takaaki Sasaki, Yoshinobu Ohsaki. EGFR tyrosine kinase inhibitors combined with cytotoxic drugs for treatment of NSCLC with EGFR gene mutations: Efficacy and mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3501. doi:10.1158/1538-7445.AM2015-3501