The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukaemia

In approximately 23% of AML patients, Fms-like tyrosine kinase 3 (FLT3) contains a gain-of-function, internal tandem duplication (ITD) mutation that is associated with an unfavourable prognosis. FLT3 is therefore seen as a promising therapeutic avenue for AML and as a result, a more complete understanding of its function and signalling may lead to additional targets and resistance mechanisms being identified. To date, several FLT3 inhibitors including Quizartinib (AC220), a second-generation ITD-selective tyrosine kinase inhibitor, have been developed, but single-agent clinical trials have not been overwhelmingly successful. In most preclinical studies, the inhibitory effects of FLT3 inhibitors are mainly evaluated using mutant-expressing models, however, most AML patients harbour both a wild-type and mutant FLT3 allele as well as presenting with high plasma levels of FLT3 ligand (FLT3L). We hypothesized that FLT3L could act through WT-FLT3 to influence the efficacy of FLT3 inhibitors in cells with heterozygous mutations. In this study, we have examined the role of FLT3 inhibition and FLT3L activation on the cellular localisation and downstream signalling in several AML cell lines, some of which express ITD mutations. We also looked at mechanisms by which FLT3L could impair the efficacy of FLT3 inhibitors. Our results revealed that the majority of FLT3 in the MV4-11 and MOLM-13 cells was intracellular. Inhibition of ITD-FLT3 with quizartinib led to a dramatic relocalisation of FLT3 to the cell surface. This effect was more pronounced in MV4-11 (FLT3ITD/ITD) than the heterozygous MOLM-13 (FLT3ITD/WT) cells that also express WT. This was accompanied by inhibition of ERK, AKT and STAT5 signalling pathways and resulted in cell death. Quizartinib induced cell death only in AML lines expressing FLT3-ITD mutations (MOLM-13 and MV4-11), and this could be antagonised by FLT3 ligand (FLT3L). The largest inhibitory effects were seen in heterozygous cells expressing a mutated and wild-type allele. FLT3 inhibition was associated with downregulation of the anti-apoptotic protein Mcl-1 and upregulation of the pro-apoptotic BH3-only protein, Bim. Our experiments indicated that both of these proteins were regulated by both WT and ITD-FLT3 through the MAPK pathway. These results suggest that activation of FLT3 signalling by FLT3L confers resistance to quizartinib through upregulation of Mcl-1 and suppression of Bim expression. Taken together, our data suggest that FLT3 cell surface localisation and expression are controlled by ITD mutations and are key components of drug resistance. The data also suggests a novel therapeutic approach in patients with high plasma FLT3L levels, especially when using type II inhibitors such as quizartinib, is to co-target the MAPK/ERK pathway to abrogate the FLT3L-mediated resistance in FLT3-ITD AML.