Purposes Mutations in the feline McDonough sarcoma–like tyrosine kinase 3 (FLT3) gene represent one of the most common genetic alterations in acute myeloid leukemia and are associated with an increased risk of relapse and poor prognosis. FLT3 inhibitors have significantly improved clinical outcomes in acute myeloid leukemia patients with FLT3 internal tandem duplication mutations. However, currently available FLT3 inhibitors often exhibit off-target effects, leading to numerous adverse reactions. Therefore, identifying more specific FLT3 inhibitors has important clinical significance. Methods We conducted high-throughput virtual screening for identifying small molecules that bind to the autoinhibited conformation of the FLT3 protein using the National Cancer Institute Developmental Therapeutics Program library. Then, we performed molecular screening of 159 small-molecule compounds targeting the autoinhibited conformation of FLT3. Following in vitro screening in 32D and 32D cells with FLT3 internal tandem duplication overexpression, TIP-20 was identified as the lead compound. TIP-20 was further evaluated in FLT3-internal tandem duplication mutation and FLT3 wild-type acute myeloid leukemia cell lines to assess its effects on apoptosis, cell cycle progression, downstream FLT3 signaling pathways, and kinase activity inhibition. The in vivo survival benefit of TIP-20 was evaluated using MV4-11 xenograft mouse models. RNA sequencing and Gene Set Enrichment Analysis were performed to further elucidate the antileukemic mechanisms of TIP-20. Results Cellular thermal shift assays confirmed the direct binding of TIP-20 to the FLT3 protein. TIP-20 inhibited FLT3 kinase activity, as demonstrated by kinase activity assays. In FLT3 internal tandem duplication mutation acute myeloid leukemia cell lines, TIP-20 suppressed cell proliferation, induced apoptosis, and caused G0/G1 cell cycle arrest. In addition, TIP-20 inhibited the phosphorylation of FLT3 and its downstream signaling components, including signal transducer and activator of transcription 5. Furthermore, TIP-20 reduced leukemia burden in the MV4-11 xenograft mouse model, with no significant differences observed in liver or kidney function between the treatment and control groups. Gene Set Enrichment Analysis indicated that TIP-20 enhanced or restored antigen presentation in MV4-11 cells. Conclusions TIP-20 is a novel FLT3 inhibitor with potent antileukemic activity. These findings suggest that TIP-20 may provide a new therapeutic option for leukemia patients.
Pang et al. (Sun,) studied this question.