Abstract

Systemic mastoyctosis is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC). Most patients express the D816V-mutated variant of c-KIT, which mediates resistance against most available tyrosine kinase inhibitors. Therefore, current research is focusing on novel targets in MC. We analyzed expression and function of the survival factor heme oxygenase-1 (HO-1) in neoplastic MC. As assessed by Northern blotting and RT-PCR, the human MC line HMC-1 that exhibits KIT D816V, was found to express HO-1 mRNA. Expression of the HO-1 protein was demonstrable by immunocytochemistry and Western blotting. To examine the role of mutated KIT in expression of HO-1, Ba/F3 cells with doxycycline-inducible expression of c-KIT D816V were employed. In these experiments, KIT D816V was found to induce HO-1 promoter activity and expression of HO-1 mRNA and HO-1 protein in these cells. Moreover, the KIT-D816V-targeting compound PKC412 was found to downregulate expression of HO-1 in HMC-1 cells. The KIT D816V-induced upregulation of HO-1 in Ba/F3 cells was completely blocked by a combination of LY294002 (PI3-kinase inhibitor) and PD89059 (MEK inhibitor), but not by single agents, suggesting involvement of multiple signaling pathways. We next examined whether targeting of HO-1 is associated with decreased survival. As assessed by 3H-thymidine uptake, the pegylated HO-1 inhibitor zinc-protoporphyrin (PEG-ZnPP) reduced proliferation of HMC-1 cells in a dose-dependent manner (IC50: 5 μM). The PEG-ZnPP-induced inhibition of growth was found to be associated with induction of apoptosis (control: 1±0.6 vs PEG-ZnPP, 5 μM: 55±5% apoptotic cells, p<0.05). The HO-1 inductor hemin (10 μM) increased the expression of HO-1 in HMC-1 cells and partly rescued these cells from PKC412-induced growth-inhibition (PKC412, 1 μM: 39±8% vs PKC412 + hemin: 23±11%). Finally, PKC412 and PEG-ZnPP were found to produce cooperative (additive) growth-inhibitory effects on HMC-1 cells. In summary, our data show that HO-1 is a novel survival factor and interesting target in neoplastic human MC exhibiting the D816V-mutated variant of KIT.

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