An unprecedented potent inhibitor of MV4-11 cells: investigations into the mechanism of action beyond FLT3 inhibition

Abstract

Activating mutations in FLT3 occur in 30% of acute myeloid leukemia (AML) cases. The AML patient-derived MV4–11 cell line contains a genetic alteration in FLT3 (“FLT3-ITD”), causing constitutive FLT3 activation. From screening, we identified compound 1 with unprecedently high anti-MV4–11 effects, with IC₅₀ = 0.0021 ± 0.0003 nM. From the dose response curve, the effects of compound 1 are gradual and may have biphasic characteristics. Further studies identified compound 1 as a type-I FLT3 inhibitor with comparable potency to quizartinib and gilteritinib; however, compound 1 is much more potent against MV4–11 cells, indicating that it may have a second molecular mechanism of action independent of FLT3 inhibition. Interestingly, compound 1's high potency is uniquely toward MV4–11 cells, and distinct from other cell lines either with or without FLT3 mutations. Preliminary efforts to unravel this mechanism were undertaken. The results of apoptosis assay and cell cycle analysis showed that the effects of compound 1 on MV4–11 may be biphasic, with an immediate cell cycle stabilizing effect at low picomolar concentrations, and a stronger effect to arrest cell cycle and induce apoptosis at low nanomolar concentrations. However, kinase selectivity profiling demonstrates that other than FLT3, compound 1 lacks strong binding affinities with other kinases. Therefore, the high inhibitory potency of compound 1 on MV4–11 cells appear unlikely to be due to synergism of co-inhibition of FLT3 and any other kinases. Altogether, compound 1 may serve as a promising lead compound for further optimization and research on a potentially new molecular antiproliferative mechanism.