The Plk-1 Inhibitor BI 2536 Counteracts the Growth of Neoplastic Mast Cells and Synergizes with the KIT D816V-Targeting Drug Midostaurin (PKC412) in Producing Growth-Inhibition.

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in various internal organs. In most patients, the D816V-mutated variant of c-KIT, which mediates resistance against several tyrosine kinase (TK) inhibitors like imatinib, is found. In advanced SM, the response of neoplastic MC to conventional drugs is poor and the prognosis is grave. Therefore current research is attempting to identify novel targets in neoplastic MC. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias. In the present study, we analyzed expression and function of Plk-1 in neoplastic human MC, and asked whether Plk-1 can serve as a target of therapy in SM. As determined by immunohistochemistry, primary neoplastic MC were found to display activated/phosphorylated Plk-1 in all patients examined (n=5). The human MC leukemia cell line HMC-1 was also found to exhibit activated Plk-1. In addition, we found that primary neoplastic MC as well as HMC-1 cells express Plk-1 mRNA in RT-PCR experiments. As assessed by 3H-thymidine-uptake experiments, the Plk-1-targeting drug BI 2536 (Boehringer Ingelheim GmbH, Germany) was found to inhibit the proliferation of HMC-1 cells in a dose-dependent manner (IC50 5–15 nM). The effect of BI 2536 was seen in both subclones of HMC-1, i.e. in HMC-1.1 cells displaying KIT G560V (but not KIT D816V), and HMC-1.2 cells exhibiting both KIT G560V and KIT D816V, with comparable IC50 values. Moreover, BI 2536 was found to inhibit the proliferation of primary neoplastic cells, with IC50 values ranging between 5 and 50 nM. The growth-inhibitory effects of BI 2536 on HMC-1 cells were found to be associated with mitotic arrest and G2-M cell cycle arrest as well as consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after treatment with BI 2536. In a consecutive phase of the study, we asked whether combined targeting of KIT D816V and Plk-1 would lead to synergistic drug-interactions. For this purpose, HMC-1 cells and primary neoplastic MC were coincubated with BI 2536 and midostaurin (PKC412), a multitargeted kinase inhibitor that blocks KIT D816V TK activity. In these experiments, BI 2536 was found to synergize with midostaurin in counteracting the proliferation of HMC-1 cells and primary neoplastic MC. In conclusion, our data show that activated Plk-1 is detectable in MC neoplasms and plays a role in cell cycle progression and viability of neoplastic MC. Targeting of Plk-1 with BI 2536 leads to growth inhibition and apoptosis in neoplastic MC. Furthermore, BI 2536 synergizes with midostaurin in counteracting growth of neoplastic MC. Targeting of Plk-1 may be an attractive new pharmacologic concept in advanced SM.