Background: APG-2449 is a novel, orally active FAK inhibitor and an ALK/ROS1 tyrosine kinase inhibitor (TKI) that has shown potent activity in preclinical models. It had been demonstrated that APG-2449 was well tolerated, and preliminary efficacy was observed in pts who were resistant to second-generation ALK/ROS1⁺ inhibitors (Zhao H et al. J Clin Oncol 2022; 40:9071). We provide updated safety and efficacy results and potential mechanisms of action(s) of this therapy. Methods: After the RP2D was determined as 1,200 mg daily (QD), NSCLC pts were enrolled into 2 dose expansion cohorts. Cohort 1 included pts who were resistant to second-generation ALK/ROS1⁺ TKIs. Cohort 2 included those who were ALK or ROS1⁺ TKI naïve. Results: As of December 9, 2022, 130 pts enrolled (median [range] age 53 [21-78] years; 53.8% female) with NSCLC, mesothelioma, or ovarian cancer were treated with APG-2449 at doses ranging from 900 to 1,500 mg. A total of 117 (90%) pts experienced treatment-related adverse events (TRAEs). The most frequent TRAEs included elevated blood creatinine (43.8%), ALT (40.8%), and AST (33.1%) levels, as well as gastrointestinal disorders: nausea (25.4%), vomiting (21.5%), and diarrhea (21.5%). A total of 17 (13.1%) TRAEs were grade ≥ 3. In a subgroup of pts with TKI-naïve NSCLC (n = 33; 31 with efficacy evaluable), the overall response rate (ORR) and the disease control rate (DCR = CR + PR + SD) were 70.6% (12/17) and 88.2% (15/17), respectively, in ROS1⁺ treatment naïve pts; the ORR and DCR were 78.6% (11/14) and 100% (14/14) in ALK⁺ treatment naïve pts. Of 27 NSCLC pts resistant to second-generation ALK inhibitors, 7 were observed with PR (7/27; 25.9%) during APG-2449 treatment at RP2D. In this subgroup, compared to baseline, pts who experienced PR showed lower phosphorylated FAK (pFAK) levels in peripheral blood mononuclear cells (PBMCs) by Day 28 (24 hours after dosing on D28) than pts who experienced SD. Furthermore, pts with progressive disease showed an increase of PBMC pFAK levels on D28 compared to baseline, indicating that APG-2449 could inhibit FAK phosphorylation. Pts with higher pFAK expression in tumor tissues at baseline tended to achieve better clinical responses than those with lower pFAK expression post APG-2449 treatment. Conclusions: APG-2449 showed a favorable preliminary safety profile and antitumor efficacy in pts with NSCLC. Preliminary efficacy was observed in those whose disease was TKI naïve and resistant to second-generation ALK inhibitors. FAK inhibition could be a novel approach to overcome ALK resistance in pts with NSCLC who are resistant to second-generation ALK inhibitors. Internal study identifier: APG2449XC101. Clinical trial information: NCT03917043.