The KIT D816V-Targeting Drug PKC412 Induces Re-Expression of Bim and Synergizes with Mcl-1 Antisense Oligonucleotides in Producing Growth Inhibition in Neoplastic Human Mast Cells.

Systemic mastocytosis (SM) is a myeloid neoplasm involving uncommitted and mast cell (MC) committed hematopoietic progenitors. In most SM patients, the transforming c-KIT point mutation D816V is detectable. However, so far, little is known about the molecular mechanisms underlying KIT D816V-induced transformation. We examined the expression of two Bcl-2 family members, i.e. anti-apoptotic Mcl-1 and the pro-apoptotic tumor suppressor Bim, in the KIT D816V+ human mast cell line HMC-1. As assessed by RT-PCR, immunocytochemistry, and Western blotting, these cells were found to express Mcl-1 mRNA and the Mcl-1 protein, whereas only trace amounts of Bim were detectable in HMC-1 cells. Exposure of HMC-1 cells to the KIT D816V-targeting drug PKC412 resulted in a substantial increase in expression of Bim and in a marked decrease in expression of Mcl-1. Correspondingly, in Ba/F3 cells with doxycycline-inducible expression of KIT D816V (Ton.Kit.D816V), exposure to doxycycline (induction of KIT D816V) resulted in a significant decrease in expression of Bim, and PKC412 was found to counteract the KIT D816V-induced downregulation in Bim in these cells. Transfection of HMC-1 cells with Mcl-1-specific antisense oligonucleotides (ASO) or an Mcl-1-specific siRNA resulted in reduced survival and in an increased percentage of apoptotic cells compared to cells transfected with scramble control oligonucleotides. In consecutive experiments, combinations of PKC412 and Mcl-1 ASO were applied using varying drug concentrations. In these experiments, both compounds were found to synergize with each other in producing growth inhibition and apoptosis in HMC-1 cells. By contrast, no synergism was observed with combinations of Mcl-1 ASO and imatinib or with Mcl-1 ASO and 2CdA. In summary, our data show that the Bcl-2 family members Mcl-1 and Bim are differentially expressed in neoplastic MC, and that targeting of Mcl-1 is associated with reduced survival of HMC-1 cells. In addition, our data show that Mcl-1 ASO synergize with PKC412 in producing growth inhibition in neoplastic MC. These data support the concept that optimal growth inhibition of neoplastic MC in aggressive variants of SM may be achieved with combinations of targeted drugs.