Abstract
The majority of patients with systemic mastocytosis exhibit a D816V mutation in the activating loop of the Kit receptor expressed on mast cells. The Kit ligand regulates mast cell survival by transcriptional repression of the proapoptotic BH3-only protein Bim and by promoting Bim phosphorylation that makes it vulnerable for proteasomal-dependent degradation. We investigated here whether prevention of Bim degradation by a proteasomal inhibitor, MG132, would induce apoptosis in mast cells with the D816V mutation. Human umbilical cord blood-derived mast cells (CBMCs) with wild-type (wt) Kit and two different subclones of the human mast cell line-1 (HMC-1) were used for the study: HMC-1.1 with the V560G mutation in the juxtamembrane domain and HMC-1.2 carrying the V560G mutation together with the D816V mutation. MG132 at 1 μM induced apoptosis in all cell types, an effect accompanied by increased BH3-only proapoptotic protein Bim. The raise of Bim was accompanied by caspase-3 activation, and a caspase-3 inhibitor reduced MG132-induced apoptosis. Further, MG132 caused a reduction of activated Erk, a negative regulator of Bim expression, and thus Bim upregulation. We conclude that decreased phosphorylation and increased levels of Bim overcome the prosurvival effect of the D816V mutation and that the results warrant further investigations of the clinical effects of proteasomal inhibition in systemic mastocytosis.
Highlights
In view of the fact that the gain-of-function mutations in Kit is so common in systemic mastocytosis (SM) and has a key role in the pathogenesis, the D816V mutation represents an attractive drug target for SM
We found that the cell number dropped dramatically both in human mast cell line-1 (HMC-1).1 and HMC-1.2 cells, as well as in SCFtreated cord bloodderived mast cells (CBMCs) with wt Kit (Figure 1)
We could not detect the release of lactate dehydrogenase (LDH) from MG132 (1 and 10 mM)-treated HMC-1.1, HMC-1.2 or CBMCs, suggesting that MG132 is not cytotoxic to the cells
Summary
In view of the fact that the gain-of-function mutations in Kit is so common in SM and has a key role in the pathogenesis, the D816V mutation represents an attractive drug target for SM. Proteasome inhibition-mediated apoptosis in neoplastic mast cells C Moller Westerberg et al mast cells with the D816V mutation and thereby induce apoptosis in the cells For these studies, we used cord bloodderived mast cells (CBMCs) with wt Kit, and two variants of the human mast cell line-1 (HMC-1):[22] HMC-1.1 that has a mutation in the juxtamembrane region (V560G) and HMC-1.2 with the V560G mutation together with the D816V mutation in the catalytic domain.[5,23] Besides Bim we investigated the expression levels of Puma, another BH3-only protein that we recently demonstrated to be involved in mast cell apoptosis.[24] Here we provide evidence that the proteasome inhibitor MG132 increases the expression of Bim, reduces Erk and Kit activation and causes a caspase-3-dependent apoptosis, even in mast cells with D816V mutations
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