ALBERT

Description: Abstract 4146 Introduction KIT is a type III receptor tyrosine kinase together with FLT3, PDGFR and FMS. The interaction of KIT and its ligand stem cell factor (SCF) plays an important role in the cell survival, proliferation and differentiation. Activating mutations of KIT have been demonstrated in several kinds of human malignancies, such as mastocytoma, gastrointestinal stromal tumor, and acute myeloid leukemia (AML). Since KIT mutation seems a poor prognostic factor in CBF leukemia and the KIT expression is observed in most AML cells, KIT serves a molecular target for the treatment of AML. To date, several small molecules have been demonstrated to have a potency against KIT kinase, while KIT selective inhibitors are not yet developed for the clinical use. We recently developed a novel KIT selective inhibitor KI-328, and evaluated here its inhibitory effect on wild-type (Wt) and mutant KIT kinases. Methods We identified 5 types of KIT mutations (D816V, M541L, V540L, T417F/del418-419 and N822K) in AML cells, and established these mutant-KIT, as well as Wt-KIT, expressing IL-3-dependent mouse myeloid precursor 32D cells. Using these Wt- and mutant KIT expressing 32D cells, we examined the anti-leukemia activity of KI-328 in comparison with another potent KIT inhibitors. Results In Wt- and M541L-KIT expressing cells, KITs were phosphorylated by the SCF stimulation. In contrast, mutant KITs were constitutively phosphorylated in D816V-, V540L-, T417F-, and N822K-KIT expressing cells. However, the autonomous proliferation was observed only in D816V-KIT expressing cells, and the other mutant KIT expressing cells required SCF for their proliferations like Wt-KIT expressing cells. These results were confirmed by the colony formation ability in the semi-liquid media, where only D816V-KIT expressing cells could form the colony without any growth factors. The growth inhibitory effect of KI-328 was, therefore, examined in the existence of 50 ng/ml of SCF. KI-328 inhibited the growth of Wt-, M541L-, V540L-, T417F- and N822K-KIT expressing cells with the GI50 value 127 nM, 229 nM, 575 nM, 445 nM and 997 nM, respectively. The cell cycle analysis showed the KI-328 increased sub-G1 populations in these cells at each GI50 value. In consistent with the growth inhibitory effects, KI-328 potently inhibited the phosphorylations of Wt- and mutant KITs except D816V as well as their downstream molecules STAT3, AKT, MAPK at the concentration of over the GI50 value, indicating the proof of concept that KI-328 inhibits the growth of these cells by the KIT kinase inhibition. However, the significant growth inhibition was not observed in D816V-KIT expressing cells up to the 5 μM, and more than 2 μM of KI-328 were required for the de-phosphorylation of D816V-KIT. We further examined whether another potent KIT inhibitors showed the different sensitivities between D816V-KIT and Wt-KIT. Multi-kinase inhibitors such as dasatinib and sunitinib showed the same growth inhibitory effects on D816V- and Wt-KIT expressing cells: each GI50 value against D816V- and Wt-KIT was 43 nM and 72 nM, and 116 nM and 206 nM, respectively. In contrast, imatinib, which is relatively selective against KIT kinase, did not inhibit the growth of the D816V-KIT expressing cells like KI-328. Conclusions We demonstrated that KI-328 is a potent and selective KIT inhibitor. Although KI-328 did not show the significant growth inhibitory effect on the D816V-KIT expressing 32D cells up to the 5 μM, G-CSF mediating neutrophil maturation was observed when those were treated with less than 1 μM of KI-328, indicating that KI-328 has a weak potency against the D816V-KIT kinase. Therefore, the combination therapy with another potent KIT inhibitors, such as HSP90 inhibitor, might conquer the resistance against the D816V-KIT kinase. Since the kinase inhibitory profile seemed to be associated with the resistance against the D816V-KIT kinase, the structural analysis of the D816V-KIT is required for developing more potent inhibitors against all mutant KIT kinases. Disclosures: Shiotsu: Kyowa Hakko Kirin Co., Ltd.: Employment. Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Consultancy; Novartis Pharma Co. Ltd.: Research Funding. Ishida:Kyowa Hakko Kirin Co., Ltd.: Employment. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Description: Abstract 2597 Poster Board II-573 Introduction: Activating mutations of the Fms-like tyrosine kinase-3 gene (FLT3) occur in approximately 30–40% of acute myeloid leukemia (AML) patients. FLT3 mutations confer numerous oncogenic properties, including dysregulated proliferation, resistance to apoptosis and a block in differentiation. FLT3 mutations result in abnormal activation of the downstream pathways, including signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase kinase (Mek)/extracellular signal–regulated kinase (Erk) and phosphatidylinositol-3 kinase (PI3K)/Akt. Activation of these downstream effectors has been thought to allow leukemia cells to evade apoptosis. Targeting of FLT3 mutations is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. Results: FI-700 induced G1-phase cell cycle arrest and apoptosis as evidenced by increased sub-G1 DNA content and phosphatidylserine externalization in FLT3/ITD MOLM-13 (FLT3-ITD, wild-type (wt)-p53) and MV4-11NR (FLT3-ITD, mutated-p53) AML cells. FI-700 did not affect cell cycle distribution patterns nor did it induce apoptosis in FLT3/WT OCI-AML-3 (FLT3/WT, wt-p53) and HL-60 (FLT3/WT, del (del)-p53). Wt-p53 MOLM-13 and OCI-AML-3 cells were susceptible to Nutlin-induced apoptosis. FI-700 augmented Nutlin-induced Bax activation, mitochondrial membrane potential (MMP) loss, caspase-3 activation and phosphatidylserine externalization in MOLM-13 cells. FI-700 rapidly reduced Mcl-1 levels in FLT3/ITD cells, mainly by enhancing proteasomal Mcl-1 degradation. Levels of other Bcl-2 family proteins examined did not change significantly. Mcl-1 levels were only modestly reduced upon Nutlin treatment. The FI-700/Nutlin-3 combination profoundly reduced Mcl-1 levels. Immunoprecipitation/ immunoblotting results suggested that the drug combination results in a profound decrease in Mcl-1-bound Bim. FI-700 enhanced doxorubicin-induced apoptosis in FLT3/ITD MOLM-13 and MV4-11NR cells, suggesting that FI-700 can enhance both the p53-dependent and the p53-independent apoptotic effects of doxorubicin. Finally, cooperative apoptotic effects of FI-700/Nutlin-3 were seen in primary AML cells with FLT3/ITD. Conclusion: FLT3 inhibition by FI-700 immediately reduces anti-apoptotic Mcl-1 levels and enhances Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/ITD-expressing AML cells via the Mcl-1/Noxa axis. FLT3 inhibition, in combination with p53-inducing agents, might represent a potential therapeutic approach in AML with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.