FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase. Activating mutations in FLT3 gene occur in approximately 30% of acute myeloid leukemia (AML) cases, leading to FLT3+-AML. Current FLT3 inhibitors, including midostaurin, quizartinib, and gilteritinib, face resistance issues, often due to mutations such as FLT3 F691L, which affect drug efficacy. Gilteritinib has the most promising resistance pattern but remains susceptible to this gatekeeper variant. Newer inhibitors like pexidartinib and FF-10101 show improved efficacy against resistance mutations, with FF-10101 demonstrating synergy with azacitidine.
This study utilized various reagents and cell lines to investigate resistance mechanisms in FLT3-mutated AML. Drug-resistant cell sublines were generated by exposing parental cells to escalating drug concentrations. Genomic investigation, conducted using the Geneyx Analysis Platform, identified key mutations associated with resistance.
Fig. 1. Establishment of drug-resistant cell sub-lines.
AML cell lines acquired cross resistance to FF-10101 and gilteritinib via repeated drug exposure, with drug-resistant cell lines showing significant resistance to both agents. Interestingly, the new pan-FLT3 inhibitor Chen-9u did not show resistance even after multiple growth cycles and remained effective against resistant strains. Resistant cells displayed altered cell cycles, decreased CDK4 level, and increased NRAS expression. DNA sequencing revealed several mutations linked to resistance: FLT3/N701K and MYCN/D31P for gilteritinib resistance, NRAS/G12C for FF-10101 resistance, and FLT3/C695F, which compromised FF-10101 binding.
In summary, this study highlights the development of FLT3+ inhibitor-resistant leukemia cell lines to better understand drug resistance mechanisms and identify novel treatment approaches. The researchers developed four distinct resistant cell lines, revealing genomic mutations, including FLT3/C695F and MYCN/D31P, which were not previously reported. Cross-resistance between gilteritinib and FF-10101 limits their effectiveness, but a novel inhibitor, Chen-9u, demonstrated potent inhibition without resistance emergence.
