Combination of bortezomib with a FLT3 inhibitor potentiates inhibition of proliferation and apoptosis of AML in vitro

e14551 Background: FLT3 receptor tyrosine kinase activating mutations contribute to leukemogenesis and poor prognosis in approximately 30% of acute myeloid leukemia (AML). An internal tandem duplication (ITD) mutation in the juxtamembrane domain of FLT3 results in loss of autoinhibition that leads to constitutive, ligand-independent activation of the receptor, with subsequent activation of multiple downstream signaling pathways, including RAS/MAPK and STAT5. Early phase clinical trials of FLT3 inhibitors show a lack of durability of responses, suggesting combination therapy may work better. Methods: In the course of conducting a synthetic lethality screen with a FLT3 inhibitor on the Ba/F3 murine cell line stably expressing human FLT3 or FLT3-ITD, we identified bortezomib, a proteasome inhibitor, as having potent activity against FLT3-ITD cells. The effects of drugs on proliferation, apoptosis, and phosphosignaling were quantified in Ba/F3 cells and in the HL60 (WT FLT3) and MV4–11 (FLT3-ITD) human cell lines, using an MTS- based colorimetric assay, caspase 3 and 7 activity assays, and immunoblotting, respectively. Results: A highly potent and specific FLT3 inhibitor was shown to have an IC50 of 1.9 nM, inhibiting proliferation, inducing apoptosis, and abrogating downstream STAT5 and ERK phosphorylation in FLT3-ITD cells. The IC50 of bortezomib for FLT3-ITD cells was 10 nM as compared to an IC50 of 40 nM for WT FLT3 cells. Surprisingly, bortezomib abrogated tyrosine phosphorylation of FLT3, STAT5, and ERK within 60 min of adding drug. When the FLT3 inhibitor and bortezomib were used at IC25 concentrations, a more pronounced inhibition of cell proliferation was observed when they were used in combination than with either alone. Conclusions: Bortezomib preferentially kills FLT3- ITD cells, showing a four-fold more potent inhibition of cell proliferation, induces apoptosis, and abrogates activation of FLT3 and its downstream effector pathways. The combination of proteasome inhibition with FLT3 inhibition results in enhanced cytotoxicity. The mechanism by which bortezomib affects the FLT3-ITD signaling axis and the possible synergy with FLT3 inhibitors remains to be elucidated. No significant financial relationships to disclose.