Abstract 4785: Signaling adaptation to FLT3 inhibition in FLT3/ITD leukemia results in a reactivation of ERK signaling that can be abrogated with MEK inhibition

The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3 mutant acute myeloid leukemia (AML) has been explored as a promising strategy for over a decade. However, FLT3 TKIs have thus far shown limited clinical benefit in patients with FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response. If true, combined targeted therapeutic approaches that overcome the adaptive resistance to FLT3 inhibition could be used to maximize the efficacy of anti-leukemia treatment for those expressing this mutation. To evaluate this hypothesis, the FLT3/ITD AML cell lines Molm14 and MV4;11 were treated with FLT3 TKIs for up to 48 hours at concentrations sufficient for maximal FLT3 inhibition and downstream signaling was analyzed by immunoblotting. We identified a rebound in ERK phosphorylation discernible by six hours and continuing for the duration of treatment, despite continued drug presence. This rebound resulted in near baseline levels of phosphorylated ERK (pERK) and was coupled to a rebound in phosphorylation of elements both upstream and downstream of ERK as well as in the expression levels of ERK target genes, suggesting a global reactivation of the signaling cascade. Rebound was sensitive to a reduction in serum, suggesting a growth factor-dependent mechanism. To explore the consequences of this adaptation, the impact of combinatory targeting of ERK activity using selective small molecule inhibitors of MEK was evaluated. When Molm14 and MV4;11 cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed. Additionally, the anti-leukemia effects were more pronounced for the combination compared to either drug alone, both in vitro and in vivo. In vitro, the addition of a MEK inhibitor (PD0325901 or trametinib) to FLT3 TKI (sorafenib) treatment synergistically increased cell death and decreased cell viability. This effect was most pronounced at mid-range sorafenib combined with low dose MEK inhibitor. In vivo, the addition of low-dose PD0325901 to sorafenib treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts in a transplant model (p < 0.05). Together, these studies reveal that FLT3/ITD leukemia cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to FLT3 inhibition. This adaptation li