Background: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first-generation EGFR inhibitors; however, the efficacy of these drugs is limited by acquired resistance driven by the EGFR^T790Mmutation. The discovery of third-generation EGFR inhibitors overcoming EGFR^T790Mand their new resistance mechanisms have attracted much attention.Methods: We examined the antitumor activities and potential resistance mechanism of a novel EGFR third-generation inhibitor in vitro and in vivo using ELISA, SRB assay, immunoblotting, flow cytometric analysis, kinase array, qRT-PCR and tumor xenograft models. The clinical effect on a patient was evaluated by computed tomography scan.Results: We identified compound ASK120067 as a novel inhibitor of EGFR^T790M, with selectivity over EGFR^WT. ASK120067 exhibited potent anti-proliferation activity in tumor cells harboring EGFR^T790M(NCI-H1975) and sensitizing mutations (PC-9 and HCC827) while showed moderate or weak inhibition in cells expressing EGFR^WT. Oral administration of ASK120067 induced tumor regression in NSCLC xenograft models and in a PDX model harboring EGFR^T790M. The treatment of one patient with advanced EGFR T790M-positive NSCLC was described as proof of principle. Moreover, we found that hyperphosphorylation of Ack1 and the subsequent activation of antiapoptotic signaling via the AKT pathway contributed to ASK120067 resistance. Concomitant targeting of EGFR and Ack1 effectively overrode the acquired resistance of ASK120067 both in vitro and in vivo.Conclusions: Our results idenfity ASK120067 as a promising third-generation EGFR inhibitor and reveal for the first time that Ack1 activation as a novel resistance mechanism to EGFR inhibitors that guide to potential combination strategy.