Abstract Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases and FLT3-ITD mutations are associated with a particularly unfavorable prognosis, with an increased risk of relapse and shorter overall survival (OS) compared with AML patients without the mutation. Even though multiple FLT3 inhibitors are in various stages of clinical evaluation, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments. Among them, a “gatekeeper” mutation (F691L) is resistant to most available FLT3 inhibitors. Here, we show the efficacy and mechanism of PLM-102, developed an orally available and highly selective small molecule, in overcoming drug-resistant FLT3-ITD-TKD mutations as a strong candidate of the 3rd generation of FLT3 inhibitor. PLM-102 displays extraordinary potency of enzyme inhibitory activities of all FLT3 WT/mutants including F691L with IC50 values of <1 nM. In FLT3-ITD-positive cell lines (MV4-11, MOLM-13, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), PLM-102 shows about 18~25-fold higher anti-proliferative activities than Gilteritinib. Also, PLM-102 potently inhibited the phosphorylation of FLT3 and its downstream signaling pathways, and induced apoptosis as evidenced by PARP-cleavage and caspase-3 activation. Moreover, in AML-related xenograft mouse models (MV4-11, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), once-daily oral dose of PLM-102 significantly suppressed the tumor growth and achieved complete tumor regression. In addition, the outstanding efficacy of our compound has been confirmed in large tumor xenograft models. These results are very encouraging because large tumors have a limited blood supply and high interstitial fluid pressure, leading to a poor absorption of anticancer drugs. Pharmacokinetics of PLM-102 displayed higher bone-marrow exposure indicating better target engagement and benefits in the clinical translation in AML. In conclusion, PLM-102 is a promising therapeutic candidate for the FLT3-ITD-mutated AML as well as the acquired resistance to current FLT3 inhibitors. Citation Format: Jin-Hee Park, Jae-Seon Lee, Su-jin Oh, So-Deok Lee, Yong June Choi, Keon Wook Kang, Miran Moon, Soo Yeon Jang, Myung Jin Kim, Yong-Chul Kim. Discovery of PLM-102, a highly potent 3rd generation FLT3 inhibitor, in drug-resistant FLT3-ITD-TKD mutated acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4009.