In vitro activity: DCC-2618 is a potent, orally bioactive, and spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. DCC-2618 inhibits the proliferation and survival of various human mast cell lines (e.g. HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC50<1 μM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. DCC-2618 is effective in vivo in patients with advanced systemic mastocytosis and is currently under investigation in clinical trials.

Kinase Assay: For evaluation of KIT and BTK signaling, HMC-1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were incubated in control medium or in DCC-2618 (0.5–5 μM) for 4 h at 37°C. Western blotting was performed essentially as described elsewhere. For evaluation of downstream signaling pathways of KIT, HMC-1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were first pre- incubated overnight in Iscove modified Dulbecco medium devoid of fetal calf serum and of stem cell factor. Cells (106) from each line were then treated with DCC-2618 (0.001–10 μM) for 90 min at 37°C. At the end of the treatment, ROSAKIT WT cells were stimulated with stem cell factor-containing supernatants (10%) of Chinese hamster ovary cells transfected with the murine scf (kl) gene (CHO-KL) at room temperature for 10 min. Thereafter, Western blotting was performed essentially as described previously.

Cell Assay: Drug-exposed cells (cell lines or primary cells) were analyzed for proliferation and survival. The bioassays employed are described in the Online Supplementary Methods