Abstract Tropomyosin receptor kinases (TRK or NTRK) are the transmembrane tyrosine kinases activated by soluble growth factors named neurotrophins (NT). TRK family includes three different members, namely TRKA, TRKB and TRKC. Extracellular recognition of NT to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signal transduction pathways via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRKs are expressed primarily in neuronal tissues and regulate differentiation and survival of neuronal cells. Alteration of TRK pathway caused by gene fusion of TRK is frequently associated with the poor prognosis of a wide range of solid malignancies, such as neuroblastoma, breast cancer, pancreatic cancer, melanoma, thyroid cancer, glioblastoma, colorectal cancer, sarcomas, cholangiocarcinoma, and non-small cell lung cancer. The efficacy of TRK inhibition in TRK fusion-positive cancers has been demonstrated by the high response rate and lasting effect of the first-generation TRK inhibitors larotrectinib and entrectinib. However, acquired resistance mutations of TRK, such as solvent front mutations, gatekeeper mutations and DFG motif mutations, often emerge and abrogate the inhibitory effect of first-generation inhibitors. Thus, developing novel TRK inhibitors that can effectively overcome resistance mutations can provide great clinical benefits to patients. Here we introduce FCN-098, a potent second-generation TRK inhibitor. FCN-098 demonstrated marked kinase inhibition activity against wild-type (WT) and mutant TRK with IC50 at sub-nano or single nanomolar range. FCN-098 significantly and dose-dependently inhibited phosphorylation of TRK and its downstream effector ERK in TRK fusion-positive KM12 human colon cancer cells and BaF3 cells ectopically overexpressing WT or mutant TRK fusions. FCN-098 potently inhibited proliferation of KM12 cells and BaF3 cells overexpressing WT or various mutant TRK fusions while sparing TRK-negative cell lines. Consistent with in vitro results, FCN-098 showed remarkable anti-tumor activity in xenograft models driven by WT or mutant TRK fusions in a dose-dependent manner. In particular, the anti-tumor activity of FCN-098 was superior to another second-generation TRK inhibitor LOXO-195 at the same dose in xenografts driven by TPM3-TRKA G595R or ETV6-TRKC G623R mutation, and exhibited comparable potency to LOXO-195 in LMNA-TRKA G667C-driven xenografts. In non-clinical studies, FCN-098 exhibited excellent pharmacokinetic (PK) and safety properties, demonstrating longer T1/2 in both dogs and rats along with higher Cmax and larger dose-normalized AUC in dogs compared to LOXO-195. Overall, FCN-098 overcomes TRK resistance mutations in vitro and in vivo and exhibits favorable PK and safety profiles. FCN-098 has the potential to become an effective and safe therapeutic choice for a wide range of solid tumors harboring WT or mutant TRK fusions. Citation Format: Shu Lin, Tongshuang Li, Xianlong Wang, Lijun Yang, Zhifang Chen, Yue Rong, Xingdong Zhao, Yanxin Liu, Zongyao Zou, Min Lin, Yuwei Gao, Jiashu Zhou, Weibo Wang. FCN-098, a novel second-generation TRK inhibitor, overcomes TRK resistance mutations in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3087.