H. Alachkar,1 M. Mutonga,1 G. Malnassy,1 J. Park,1 A. Woods,1 G. Raca,1 O. M. Odenike,1 Y. Matsuo,2 W. Stock,1 Y. Nakamura1; 1University of Chicago, Chicago, IL, 2OncoTherapy Science, Inc., Kanagawa, Japan
BACKGROUND: Gain-of-function mutations of FLT3 (FLT3-ITD) occur frequently (~30%) in cytogenetically normal acute myeloid leukemia (AML) and are associated with poor outcome. The use of FLT3 kinase inhibitors has not yet translated into an improved overall survival. Thus, novel therapeutic approaches are needed. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is highly expressed and is associated with aggressive cancer, but is hardly detectable in normal tissues.
METHODS: Protein levels were measured by western blot. Cell viability, apoptosis and differentiation were assessed by MTT and Annexin/PI staining and CD11b staining assays, respectively.
RESULTS: TOPK is highly expressed in AML cell lines and 50% AML blasts, but is not detected in CD34+ cells from healthy donors. AML cells transfected with TOPK-siRNA had significant decrease in cell viability (~70%, P<0.001) and a significant increase in apoptosis. Treatment of 10 AML cell lines with a small molecule TOPK inhibitor (TI) resulted in dose and time dependent decrease in cell viability. FLT3-mut cell lines were significantly more sensitive to TI treatment than FLT3-wt cells. The IC50 values were 20nM for FLT3-wt cells. TI activity was validated in blasts from AML patients relapsed after FLT3-inhibitor treatment as demonstrated by TI induced decrease in cell viability, and increase in cell differentiation and apoptosis. Importantly, TI decreased FLT3 expression and CEBPA phosphorylation, suggesting a different mechanism of action than that of FLT3-kinase inhibitors.
CONCLUSION: TI exhibits preferential cytotoxicity to FLT3-ITD mutated AML cells, possibly through inhibition of FLT3 expression, and may represent a new targeted therapy for this adverse risk subset of AML.