Targeting Dual Kinase Pathways: Overcoming AML Proliferation with Flt3 and Fes Inhibition

Acute myelogenous leukemia (AML) is a aggressive blood cancer with a particularly poor prognosis in older patients. The complexity of AML is highlighted by its diverse genetic mutations, with a significant number involving the Flt3 receptor tyrosine kinase. While Flt3 inhibitors have shown promise, their efficacy as single agents has been limited, suggesting the involvement of additional signaling pathways.

This study investigates the compound TL02-59, a phenoxyquinazoline inhibitor of the Fes protein-tyrosine kinase, which has been implicated in Flt3-related AML. TL02-59 demonstrated potent growth-suppressive activity against MV4-11 myeloid leukemia cells, which harbor an activating Flt3 mutation. The compound was found to be as effective as the clinical Flt3 inhibitors lestaurtinib and tandutinib, with an IC50 value of 25 nM.

However, TL02-59 showed a weaker inhibition of recombinant Fes (IC50 of 1.5 μM) and also blocked Flt3 kinase activity (IC50 of 0.6 μM), indicating that its potency in MV4-11 cells might be due to the simultaneous inhibition of both Fes and Flt3-ITD kinase activities. This was confirmed through anti-phosphotyrosine immunoblotting of Fes and Flt3 immunoprecipitates from TL02-59-treated cells.

To explore the downstream targets of Fes, mutants of Flt3-ITD resistant to TL02-59 were engineered, based on clinical cases of drug resistance to Flt3 inhibitors. These mutants were capable of transforming human TF-1 myeloid cells, leading to constitutive activation of endogenous Fes and sensitivity to TL02-59. Notably, TF-1 cells with TL02-59-resistant Flt3-ITD mutants showed reduced sensitivity to the growth-suppressive effects of the inhibitor, highlighting its dual specificity.

Preliminary experiments with one TL02-59-resistant Flt3-ITD mutant in TF-1 cells revealed that TL02-59 treatment still resulted in the inhibition of STAT5 phosphorylation, supporting a critical role for Fes in Flt3-ITD+ AML signaling. These findings suggest that the simultaneous targeting of both Flt3-ITD and Fes by TL02-59 may offer therapeutic benefits for this subtype of AML.