APCK110, a Novel and Potent Inhibitor of c-Kit, Blocks Phosphorylation of AKT and STAT3, Induces Apoptosis, and Inhibits Proliferation of Acute Myeloid Leukemia (AML) Cells.

Tight control of protein tyrosine kinase (TK) activity is crucial for the regulation and maintenance of vital cellular functions such as proliferation, differentiation, and apoptosis. c-KIT is a TK and transmembrane receptor for stem cell factor (SCF). Binding of SCF to c-KIT results in activation of marrow precursors and other blood cells. Activating mutations of c-KIT associated with amino acid Asp-816 (D816) have been identified in leukemic cells of patients with AML and are thought to play an important pathophysiologic role in leukemogenesis. Identification of activating c-KIT mutations and development of novel compounds targeting these mutations may therefore be of therapeutic benefit in AML. Based on the 3-dimensional structure of c-KIT we have generated a number of compounds with activity against c-KIT mutated cells. Here we present initial results of the activity and mechanism of action of the novel c-KIT inhibitor APCK110 in AML cell lines and primary samples from patients with AML. Using an MTT assay, we first studied the antiproliferative effect of APCK110 in the AML cell lines OCI/AML3 and the SCF-responsive cell line OCIM2. Cells were incubated for 72 hours without or with APCK110 at concentrations of 50, 100, 250, and 500 nM, then harvested and their metabolic activity and viability determined as optical density. Next we determined expression of phospho-AKT and -STAT3 in the mastocytosis cell line HMC1.1 and phospho-c-KIT in the AML cell line OCI/AML3 by Western Immunoblotting. We further analyzed induction of caspase 3 and PARP cleavage in OCI/AML3 cells at APCK110 concentrations of 50, 100, 250, and 500 nM using Western Immunoblotting. To demonstrate the effect of APCK110 on primary AML cells, we incubated diagnostic marrow cells from 3 patients with AML with increasing concentrations of APCK110 and used the blast colony assay to measure inhibition of proliferation. We then compared the antiproliferative effect of APCK110 with that of dasatinib and imatinib in OCI/AML3 cells. We show that 1) APCK110 strongly inhibits proliferation of AML cells with 80% inhibition at 500 nM; 2) similar to cell lines, APCK110 also inhibits AML colony growth of primary samples in a dose-dependent manner of up to 80% at 500 nM concentration; 3) APCK110 blocks activation of phospho-AKT, phospho-STAT3, and phospho-c-KIT; 4) APCK110 induces apoptosis by cleavage of caspase 3 and PARP; and 5) APCK110 demonstrates more potent inhibition (up to 100% at 500 nM) of AML cell proliferation than dasatinib (60% at 500 nM) and dasatinib (none at 500 nM). In summary, APCK110 is a novel and potent inhibitor of mutated c-KIT that inhibits AML cell proliferation, blocks activation of intracellular signaling molecules, and induces caspase-dependent apoptosis. Further development of APCK110 for clinical trials of patients with AML should be pursued.