Abstract 4381: High efficacy of T-LAK cell-originated protein kinase inhibitor in acute myeloid leukemia with FLT3-ITD mutation
Authors: Alachkar Houda , Mutonga Martin , Park Jae-Hyun , Malnassy Gregory , Woods Alex , Raca Gordana , Odenike Olatoyosi M. , Takamatsu Naofumi , Miyamoto Takashi , Hisada Shoji , Matsuo Yo , Stock Wendy , Nakamura Yusuke 
Published: August 2015

Abstract
The internal tandem duplication (FLT3-ITD) a gain-of-function mutation of FLT3, is associated with poor outcome in acute myeloid leukemia (AML). The use of FLT3 inhibitors currently undergoing clinical investigation has not yet improved overall survival. Therefore, novel therapies are needed. T-LAK cell-originated protein kinase (TOPK), a serine-threonine protein kinase, is highly expressed and associated with an aggressive cancer phenotype, but is hardly detectable in normal tissues. Here, we investigate TOPK in AML and demonstrate that it is highly expressed in most AML cell lines and some primary blasts, but is not detected in CD34+ cells of healthy donors. MV4-11 and U937 cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P&lt;0.001). We then examined the anti-leukemia activity of TOPK inhibitor OTS514 in AML. Primary blasts obtained from 3 patients (pts) with AML showed significant decrease in cell viability in a dose-dependent manner (IC50 values were 10-20nM). Moreover, OTS514 treatment resulted in significant decrease in number of colonies of CD34+ cells obtained from a pt with AML (41 vs 73, P = 0.01), but not of CD34+ cells obtained from healthy donors (39 vs 36, P = 0.67).
Treatment of 10 AML cell lines with OTS514 showed that FLT3 mutated cell lines were significantly more sensitive (IC5020nM). Annexin/PI staining showed 80% and 70% increase in apoptosis in FLT3-ITDmut cells (MV4-11 and MOLM13) treated with OTS514 (40nM; 48hrs). In contrast, only 40% and 10% apoptosis was observed in FLT3wt cells (U937 and KG1, respectively) treated under the same conditions. Additionally, cell cycle analysis in cells treated with OTS514 (20nM; 24 and 48 hrs) showed a dramatic decrease of S phase (∼98%; P = 0.003) in MV4-11 but only 70% (P&lt;0.001) and 30% (P = 0.02) in THP-1 (FLT3wt) cells compared with untreated cells.
Importantly, OTS514 treatment decreased cell viability and increased cell differentiation and apoptosis in primary blasts from pts relapsed after FLT3 inhibitor treatment (AC220). Furthermore, using a MV4-11-engrafted mouse model, we found that mice treated with 7.5mg/kg IV every day for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29, P&lt;0.001). Mechanistically, OTS514 or TOPK-siRNA significantly decreased FLT3 mRNA and protein levels and decreased CEBPA phosphorylation in FLT3-ITDmut cells. Furthermore, activating FLT3 in THP-1 cells via FLT3 ligand increased TOPK protein. Also, FLT3 knockdown or inhibition with MLN518 (50 and 100nM) decreased TOPK levels in MV4-11 and MOLM13 cells, but not in U937 cells, suggesting that TOPK is a downstream target of FLT3.
In conclusion, TOPK inhibitor exhibits preferential activity in FLT3-ITDmut AML, partially via inhibition of FLT3 expression. Thus, OTS514 represents new targeted therapy for this adverse risk subset of AML.
Citation Format: Houda Alachkar, Martin Mutonga, Jae-Hyun Park, Gregory Malnassy, Alex Woods, Gordana Raca, Olatoyosi M. Odenike, Naofumi Takamatsu, Takashi Miyamoto, Shoji Hisada, Yo Matsuo, Wendy Stock, Yusuke Nakamura. High efficacy of T-LAK cell-originated protein kinase inhibitor in acute myeloid leukemia with FLT3-ITD mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2015-4381