Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and accumulation of neoplastic mast cells (MC) in one or more internal organs. In most patients, the D816V-mutated variant of KIT is detectable. This mutant supposedly confers resistance against several tyrosine kinase inhibitors including imatinib and masitinib. In aggressive SM (ASM) or mast cell leukemia (MCL) the response to conventional drugs is poor and the prognosis is grave. In these patients, additional KIT-independent signalling pathways and molecules, such as BTK and LYN may play an important role in disease evolution and MC proliferation. R763/AS703569 is a multikinase inhibitor that blocks the kinase activity of KIT, BTK, LYN, Aurora-Kinase-A, Aurora-Kinase-B, ABL, AKT, and FLT3. We analyzed the effects of R763/AS703569 on growth and survival of the human mast cell leukemia cell line HMC-1 and the canine mastocytoma cell line C2. Two subclones of HMC-1 were used, one expressing KIT D816V (HMC-1.2) and one lacking KIT D816V (HMC-1.1). Both HMC-1 subclones were found to express Aurora-Kinase-A mRNA and Aurora-Kinase-B mRNA in RT-PCR experiments. As assessed by 3H-thymidine uptake, R763/AS703569 was found to inhibit proliferation of HMC-1 cells in a dose-dependent manner, with lower IC50 values obtained in HMC-1.2 cells (1-5 nM) compared to HMC-1.1 cells (10-10-50 nM). Moreover, R763/AS703569 produced growth inhibition in C2 cells (IC50: 1–5 nM). As assessed by light microscopy and Tunel assay, the growth-inhibitory effects of R763/AS703569 were found to be accompanied by apoptosis in all three cell lines. Correspondingly, R763/AS703569 was found to induce cleavage of caspase-3, caspase-8, and caspase-9 in HMC-1 cells. Moreover, R763/AS703569 was found to induce a G2/M cell cycle arrest in HMC-1 cells and C2 cells after 24 hours. In order to define the target spectrum for R763/AS703569 in HMC-1 cells, Western blot experiments were performed. In these experiments, R763/AS703569 was found to inhibit the phosphorylation of KIT, Aurora-Kinase-A, and BTK in HMC-1.1 cells, whereas no effects of R763/AS703569 on phosphorylation of LYN were seen. We then combined R763/AS703569 with dasatinib, a drug known to block LYN activation in HMC-1 cells. In these experiments, we were able to show that both drugs cooperate with each other in inducing apoptosis in HMC-1.1 cells and HMC-1.2 cells. In summary, our data suggest that R763/AS703569 is a novel promising drug that should be tested for its anti-neoplastic effects in patients with ASM and MCL in clinical trials. Complete inhibition of growth of neoplastic MC may require drug combinations employing R763/AS703569 and other targeted or cytotoxic drugs.
Sarno:Merck-Serono: Employment. Valent:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.
Asterisk with author names denotes non-ASH members.